Reviewer #3 (Public Review):

Summary:<br /> This paper by Martin et al. describes the contribution of a Kv channel subunit (Kv1.8, KCNA10) to voltage-dependent K+ conductances and membrane properties of type I and type II hair cells of the mouse utricle. Previous work has documented striking differences in K+ conductances between vestibular hair cell types. In particular amniote type I hair cells are known to express a non-typical low-voltage-activated K+ conductance (GK,L) whose molecular identity has been elusive. K+ conductances in hair cells from 3 different mouse genotypes (wildtype, Kv1.8 homozygous knockouts, and heterozygotes) are examined here and whole-cell patch-clamp recordings indicate a prominent role for Kv1.8 subunits in generating GK,L. Results also interestingly support a role for Kv1.8 subunits in type II hair cell K+ conductances; inactivating conductances in null mice are reduced in type II hair cells from striola and extrastriola regions of the utricle. Kv1.8 is therefore proposed to contribute as a pore-forming subunit for 3 different K+ conductances in vestibular hair cells. The impact of these conductances on membrane responses to current steps is studied in the current clamp. Pharmacological experiments use XE991 to block some residual Kv7-mediated current in both hair cell types, but no other pharmacological blockers are used. In addition, immunostaining data are presented and raise some questions about Kv7 and Kv1.8 channel localization. Overall, the data present compelling evidence that the removal of Kv1.8 produces profound changes in hair cell membrane conductances and sensory capabilities. These changes at hair cell level suggest vestibular function would be compromised and further assessment in terms of balance behavior in the different mice would be interesting.

Strengths:

This study provides strong evidence that Kv1.8 subunits are major contributors to the unusual K+ conductance in type I hair cells of the utricle. It also indicates that Kv1.8 subunits are important for type II hair cell K+ conductances because Kv1.8-/- mice lacked an inactivating A conductance and had reduced delayed rectifier conductance compared to controls. A comprehensive and careful analysis of biophysical profiles is presented of expressed K+ conductances in 3 different mouse genotypes. Voltage-dependent K+ currents are rigorously characterized at a range of different ages and their impact on membrane voltage responses to current input is studied. Some pharmacological experiments are performed in addition to immunostaining to bolster the conclusions from the biophysical studies. The paper has a significant impact in showing the role of Kv1.8 in determining utricular hair cell electrophysiological phenotypes.

Weaknesses:

1. From previous work it is known that GK,L in type I hair cells have unusual ion permeation and pharmacological properties that differ greatly from type II hair cell conductances. Notably GK,L is highly permeable to Cs+ as well as K+ ions and is slightly permeable to Na+. It is blocked by 4-aminopyridine and divalent cations (Ba2+, Ca2+, Ni2+), enhanced by external K+, and modulated by cyclic GMP. The question arises, if Kv1.8 is a major player and pore-forming subunit in type I and type II cells (and cochlear inner hair cells as shown by Dierich et al. 2020) how are subunits modified to produce channels with very different properties? A role for Kv1.4 channels (gA) is proposed in type II hair cells based on previous findings in bird hair cells and immunostaining for Kv1.4 channels in rat utricle presented here in Fig. 6. However, hair cell-specific partner interactions with Kv1.8 that result in GK,L in type I hair cells and Cs+ impermeable, inactivating currents in type II hair cells remain for the most part unexplored.

2. Data from patch-clamp and immunocytochemistry experiments are not in close alignment. XE991 (Kv7 channel blocker) decreases remaining K+ conductance in type I and type II hair cells from null mice supporting the presence of Kv7 channels in hair cells (Fig. 7). Also, Holt et al. (2007) previously showed inhibition of GK,L in type I hair cells (but not delayed rectifier conductance in type II hair cells) using a dominant negative construct of Kv7.4 channels. However, immunolabelling indicates Kv7.4 channels on the inner face of calyx terminals adjacent to hair cells (Fig. 5). Some reconciliation of these findings is needed.

3. Strong immunosignal appears in the cuticle plates of hair cells in addition to signal in basal regions of hair cells and supporting cells. Please provide a possible explanation for this.

4. A previous paper reported that a vestibular evoked potential was abnormal in Kv1.8-/- mice (Lee et al. 2013) as briefly mentioned (lines 94-95). It would be very interesting to know if any vestibular-associated behaviors and/or hearing loss were observed in the mice populations. If responses are compromised at the sensory hair cell level across different zones, degradation of balance function would be anticipated and should be elucidated.

Reviewer #4 (Public Review):

The manuscript brings convincing results regarding genes involved in the radio-resistance of tardigrades. It is nicely written and the authors used different techniques to study these genes. There are sometimes problems with the structure of the manuscript but these could be easily solved. According to me, there are also some points which should be clarified in the result sections. The discussion section is clear but could be more detailed, although some results were actually discussed in the results section. I wish that the authors would go deeper in the comparison with other IR-resistant eucaryotes. Overall, this is a very nice study and of interest to researchers studying molecular mechanisms of ionizing radiation resistance.

I have two small suggestions regarding the content of the study itself.

1) I think the study would benefit from the analyses of a gene tree (if feasible) in order to verify if TDR1 is indeed tardigrade-specific.<br /> 2) It would be appreciated to indicate the expression level of the different genes discussed in the study, using, for example, transcript per millions (TPMs).

Reviewer #3 (Public Review):

Cells can oxidize diverse substrates in the mitochondria to sustain cellular energy metabolism. However, all of these substrates require covalent thioester linkage to coenzyme A (CoA). Thus, multiple energy metabolism substrates could potentially compete for a limited pool of mitochondrial CoA. Cells encode a set of mitochondrial acyl-CoA thioesterases (ACOTs) that free CoA up by removing attached substrates. The authors hypothesized that ACOT2, a mitochondrial ACOT with a preference for long-chain acyl-CoA substrates that arise during the oxidation of lipids as a fuel source, could regulate the balance of substrates used in the mitochondria by reducing the oxidation of lipids by removing them from CoA and freeing the mitochondrial pool of CoA for use by other substrates.

To test this hypothesis, the authors generated mice with loss of ACOT2 in the skeletal muscle, where this is most expressed, and assayed the CoA composition of muscle and their glucose/fatty acid catabolism in mice that were challenged with different diets, fasting or exercise to expose the muscle to different substrates conditions. These experiments were complemented with biochemical analysis of mitochondria isolated from the muscle of control and ACOT2 animals exposed to a variety of substrates and challenged with different simulated energy demands.

On the basis of these convincing experiments, the authors argue that loss of ACOT2 both in vivo and in vitro interestingly increases glucose oxidation, while not increasing oxidation of lipids. This is particularly surprising as the CoA competition model would predict that ACOT2 loss would increase lipid oxidation while hindering glucose oxidation. The authors argue that ACOT2 facilitates lipid oxidation due to ACOT2 reversal of lipid ligation to CoA preventing feedback inhibition of the lipid oxidation pathway that occurs when lipid supply outstrips the ability of the lipid oxidation pathway to metabolize the lipids. These findings will be valuable for the field of metabolism providing insight into how ACOTs regulate substrate catabolism in cells and tissues.

Reviewer #3 (Public Review):

Summary:<br /> The authors aimed to investigate how genetic and environmental factors influence the muscle insulin signaling network and its impact on metabolism. They utilized mass spectrometry-based phosphoproteomics to quantify phosphosites in skeletal muscle of genetically distinct mouse strains in different dietary environments, with and without insulin stimulation. The results showed that genetic background and diet both affected insulin signaling, with almost half of the insulin-regulated phosphoproteome being modified by genetic background on an ordinary diet, and high-fat high-sugar feeding affecting insulin signaling in a strain-dependent manner.

Strengths:<br /> Study uses state-of-the-art phosphoproteomics workflow allowing quantification of a large number of phosphosites in skeletal muscle, providing a comprehensive view of the muscle insulin signaling network. The study examined five genetically distinct mouse strains in two dietary environments, allowing for the investigation of the impact of genetic and environmental factors on insulin signaling. The identification of coregulated subnetworks within the insulin signaling pathway expanded our understanding of its organization and provided insights into potential regulatory mechanisms. The study associated diverse signaling responses with insulin-stimulated glucose uptake, uncovering regulators of muscle insulin responsiveness.

Weaknesses:<br /> The limitations acknowledged by the authors, such as the need for larger cohorts and the inclusion of female mice. Moreover as acknowledged by authors, they are unable to dissect to what extent the obesity and different life span cycle for different strain affects insulin signaling. This suggest that further research is needed to validate and expand upon the findings.

Reviewer #3 (Public Review):

Summary:<br /> The authors use docking and molecular dynamics (MD) simulations to investigate transient conformations that are otherwise difficult to resolve experimentally. The docking and simulations suggest an interesting series of events whereby agonists initially bind to the low-affinity site and then flip 180 degrees as the site contracts to its high-affinity conformation. This work will be of interest to the ion channel community and to biophysical studies of pentameric ligand-gated channels.

Strengths:<br /> I find the premise for the simulations to be good, starting with an antagonist-bound structure as an estimate of the low affinity binding site conformation, then docking agonists into the site and using MD to allow the site to relax to a higher affinity conformation that is similar to structures in complex with agonists. I cannot speak to the details of the simulation methods, but the predictions are interesting and provide a view into what a transient conformation that is difficult to observe experimentally might be like.

Weaknesses:<br /> Although the match in simulated vs experimental energies for two ligands was very good, the calculated energies for two other ligands were significantly different than the experiment. It is unclear to what extent the choice of method for the energy calculations influenced the results.

A control simulation, such as for an apo site, is lacking.

Reviewer #3 (Public Review):

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

Reviewer #3 (Public Review):

Summary:<br /> Navratna et al. have solved the first structure of a transmembrane N-acetyltransferase (TNAT), resolving the architecture of human heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) in the acetyl-CoA bound state using single particle cryo-electron microscopy (cryoEM). They show that the protein is a dimer, and define the architecture of the alpha- and beta- GSNAT fragments, as well as convincingly characterizing the binding site of acetyl-CoA.

Strengths:<br /> This is the first structure of any member of the transmembrane acyl transferase superfamily, and as such it provides important insights into the architecture and acetyl-CoA binding site of this class of enzymes.

The structural data is of a high quality, with an isotropic cryoEM density map at 3.3Å facilitating the building of a high-confidence atomic model. Importantly, the density of the acetyl-CoA ligand is particularly well-defined, as are the contacting residues within the transmembrane domain.

The open-to-lumen structure of HSGNAT presented here will undoubtedly lay the groundwork for future structural and functional characterization of the reaction cycle of this class of enzymes.

Weaknesses:<br /> While the structural data for the open-to-lumen state presented in this work is very convincing, and clearly defines the binding site of acetyl-CoA, to get a complete picture of the enzymatic mechanism of this family, additional structures of other states will be required.

A potentially significant weakness of the study is the lack of functional validation. The enzymatic activity of the enzyme characterized was not measured, and the enzyme lacks native proteolytic processing, so it is a little unclear whether the structure represents an active enzyme.

Reviewer #3 (Public Review):

Summary:<br /> The study attempts to shed light on the mechanisms underlying information-seeking in infants by investigating whether infants distinguish between informative and uninformative stimuli to resourcefully allocate their attention. The authors show that 8-month-old infants can learn whether a visual stimulus is informative or uninformative about the location of a later appearing rewarding stimulus by employing statistical regularities from the input. Specifically, infants showed decreased pupil dilation for informative over uninformative cues, which developed over the course of trials as more and more information was gathered from the input. The pattern of learning was in line with a reinforcement learning model which employed a steep learning curve in the beginning followed by a more shallow but steady learning growth over trials. After 17 trials, the authors presented novel cues that shared certain visual features with the previous stimuli and showed that pupil dilation was reduced for novel cues that shared features with the previous informative stimuli, suggesting that infants were able to generalize their acquired knowledge about the informativeness of certain features to novel stimuli. The present study adds to the existing literature about the underlying mechanisms of learning by showing that infants cannot only predict an upcoming stimulus based on statistical regularities of a preceding cue but also the informativeness of the cue itself.

Strengths:<br /> The authors use a suitable method to test the highly relevant question of whether and how infants infer the informativeness of stimuli from experience and whether they can generalize this knowledge to new stimuli. Their experiment is carefully designed and well controlled with conditions closely matched (e.g., the shape and color of objects and the structure of each trial). Their measure of interest (i.e., pupil dilation) is also examined at a time point in each trial when the conditions are the most similar, which further points to a thought-through and careful design. This empirical data is backed up with a computational approach (using a Bayesian model and training a reinforcement learning algorithm) to elucidate the learning mechanisms at play. This approach is explained concisely to readers not familiar with the models.

The results are convincing showing a clear difference between informative and uninformative condition and development over trials. Specifically, this difference is not apparent in the first trial (Fig. 2c) but develops over time which supports a learning trajectory. The data support the authors' conclusion that infants learn about the informativeness of the object cue from the input, and the employed learning algorithms give further insights into the learning trajectory of the infants. Overall, the statistical analyses seem solid and the priors for the Bayesian models are well reported.

Data and scripts are openly available fostering transparency.

Overall, the manuscript is very well and concisely written.

Weaknesses:<br /> The authors' conclusion that infants can generalize the acquired knowledge to similar but novel stimuli is weakened by methodological concerns regarding the analysis. It is not fully clear which trials the authors excluded and analyzed as they do not consistently report the trials in the manuscript (e.g., it is stated that after trial 17 the first generalization trial started, but also that trial 17 was excluded as the first trial of the generalization phase). As there are only a few novel trials and novel and familiar trials alternated, the inclusion or exclusion of trial analyses might have a significant impact on the results. Thus, this needs further clarification. The authors also mentioned that the novel stimuli shared relevant as well as irrelevant features, but it was not clear to me whether the authors could establish that only the relevant features contributed to the observed generalization effect.

Some methodological decisions were not explained and need justification, in particular, as the study is not preregistered. This includes, for example, the exclusion criteria and the choice not to analyze all generalization trials. Further, the authors did not perform model comparison (e.g., their model against a null model) and therefore do not report the strength of evidence for a difference in conditions.

Another weakness is that the sample sizes of 30 infants for the initial part and 19 infants for the generalization part of the experiment are rather small (especially with regard to the chosen weakly informative priors).

Reviewer #3 (Public Review):

Summary:<br /> The publication presents unique in-vivo images of the upper layer of the epidermis of the glabrous skin when a flat object compresses or slides on the fingertip. The images are captured using OCT, and are the process of recovering the strain that fingerprints experience during the mechanical stimulation.

The most important finding is, in my opinion, that fingerprints undergo pure compression/tension without horizontal shear, hinting at the fact that the shear stress caused by the tangential load is transferred to the deeper tissues and ultimately to the mechanoreceptors (SA-I / RA-I).

Strengths:<br /> - Fascinating new insights into the mechanics of glabrous skin. To the best of my knowledge, this is the first experimental evidence of the mechanical deformation of fingerprints when subjected to dynamic mechanical stimulation. The OCT measurement allows an unprecedented measurement of the depth of the skin whereas previous works were limited to tracking the surface deformation.<br /> - The robust data analysis reveals the continuum mechanics underlying the deformation of the fingerprint ridges.

Weaknesses:<br /> I do not see any major weaknesses. The work is mainly experimental and is rigorously executed. Two points pique my curiosity, however:

1. How do the results presented in this study compare with previous finite element analysis? I am curious to know if the claim that the horizontal shear strain is transferred to the previous layer is also captured by these models. The reason is that the FEA models typically use homogeneous materials and whether or not the behavior in-silico and in-vivo matches would offer an idea of the nature of the stratum corneum.<br /> 2. Was there a specific reason why the authors chose to track only one fingerprint? From the method section, it seems that nothing would have prevented tracking a denser point cloud and reconstructing the stain on a section of the skin rather than just one ridge. With such data, the author could extend their analysis to multiple ridges interaction and get a better sense of the behavior of the entire strip of skin.

Reviewer #3 (Public Review):

Rovira, et al., aim to characterize immune cells in the brain parenchyma and identify a novel macrophage population referred to as "dendritic-like cells". They use a combination of single-cell transcriptomics, immunohistochemistry, and genetic mutants to conclude the presence of this "dendritic-like cell" population in the brain. The strength of this manuscript is the identification of dendritic cells in the brain, which are typically found in the meningeal layers and choroid plexus. A weakness is the lack of specific reporters or labeling of this dendritic cell population using specific genes found in their single-cell dataset. Additionally, it is difficult to remove the meningeal layers from the brain samples and thus can lead to confounding conclusions. Overall, I believe this study should be accepted contingent on sufficient labeling of this population and addressing comments.

Reviewer #3 (Public Review):

Summary:<br /> The present manuscript by Shore et al. entitled Reduced GABAergic Neuron Excitability, Altered Synaptic Connectivity, and Seizures in a KCNT1 Gain-of-Function Mouse Model of Childhood Epilepsy" describes in vitro and in silico results obtained in cortical neurons from mice carrying the KCNT1-Y777H gain-of-function (GOF) variant in the KCNT1 gene encoding for a subunit of the Na+-activated K+ (KNa) channel. This variant corresponds to the human Y796H variant found in a family with Autosomal Dominant Nocturnal Frontal lobe epilepsy. The occurrence of GOF variants in potassium channel encoding genes is well known, and among potential pathophysiological mechanisms, impaired inhibition has been documented as responsible for KCNT1-related DEEs. Therefore, building on a previous study by the same group performed in homozygous KI animals, and considering that the largest majority of pathogenic KCNT1 variants in humans occur in heterozygosis, the Authors have investigated the effects of heterozygous Kcnt1-Y777H expression on KNa currents and neuronal physiology among cortical glutamatergic and the 3 main classes of GABAergic neurons, namely those expressing vasoactive intestinal polypeptide (VIP), somatostatin (SST), and parvalbumin (PV), crossing KCNT1-Y777H mice with PV-, SST- and PV-cre mouse lines, and recording from GABAergic neurons identified by their expression of mCherry (but negative for GFP used to mark excitatory neurons).

The results obtained revealed heterogeneous effects of the variant on KNa and action potential firing rates in distinct neuronal subpopulations, ranging from no change (glutamatergic and VIP GABAergic) to decreased excitability (SST GABAergic) to increased excitability (PV GABAergic). In particular, modelling and in vitro data revealed that an increase in persistent Na current occurring in PV neurons was sufficient to overcome the effects of KCNT1 GOF and cause an overall increase in AP generation.

Strengths:<br /> The paper is very well written, the results clearly presented and interpreted, and the discussion focuses on the most relevant points.

The recordings performed in distinct neuronal subpopulations are a clear strength of the paper. The finding that the same variant can cause opposite effects and trigger specific homeostatic mechanisms in distinct neuronal populations is very relevant for the field, as it narrows the existing gap between experimental models and clinical evidence.

Weaknesses:<br /> My main concern is in the epileptic phenotype of the heterozygous mice investigated. In fact, in their previous paper the Authors state that "...Kcnt1-Y777H heterozygous mice did not exhibit any detectable epileptiform activity" (first sentence on page 4). However, in the present manuscript, they indicate twice in the discussion section that these mice exhibit "infrequent seizures". This relevant difference needs to be clarified to correctly attribute to the novel pathophysiological mechanism a role in seizure occurrence. Were such infrequent seizures clearly identified on the EEG, or were behavioral seizures? Could the authors quantify this "infrequent" value? This is crucial also to place in the proper perspective the Discussion statement regarding "... the increased INaP contribution to ... network hyperexcitability and seizures".

Also, some statistical analysis seems to be missing. For example, I could not find any for the data shown in Fig. 6. Thus, the following statement: "the model PV neurons responded to KCNT1 GOF with decreased AP firing and an increased rheobase" requires proper statistical evaluation.

Reviewer #3 (Public Review):

Summary:

The fruit fly visual system has provided a powerful context in which to investigate fundamental questions in neural development, phototransduction, and systems neuroscience. Of recent interest is motion processing, particularly how visual motion cues are estimated locally, and then pooled to derive behaviorally meaningful signals. Many of these pooling operations have been shown to take place in the wide-field neurons in the lobula plate, cell types that have been explored using electrophysiological recordings for more than 50 years in a variety of Diptera. However, our understanding of the diversity and connectivity of these cells remains incompletely understood, and is of interest to many.

In this context, Reiser and colleagues describe the anatomy and connectivity of the complete set of Lobula Plate Tangential neurons in Drosophila, using a careful and systematic reconstruction of the FAFB dataset. Leveraging a previous study of retinal geometry, combined with their characterization of the anatomical inputs to the elementary motion detectors, T4 and T5, they then predict the motion sensitivities of each cell, their neurotransmitter identities, and map the connections of many of these cells into the central brain and contralateral optic lobe.

Strengths:

The quality of the connectomic analysis is exceptional, and the quantitative analysis that links connectivity to function is rigorous and impressive. This paper will be an important resource for the community.

Weaknesses:

Some of the findings could be better linked to previously published work in this field, and there may be a minor limitation to the predicted optimal motion axes, given one of the simplifying assumptions made.

Reviewer #3 (Public Review):

Summary:<br /> The work by Masala and colleagues highlights a striking artifact that can result from a particular viral method for expressing genetically encoded calcium indicators (GECIs) in neurons. In a cross-institutional collaboration, the authors find that viral transduction of GECIs in the hippocampus can result in aberrant slow-traveling calcium (Ca2+) micro-waves. These Ca2+ micro-waves are distinct from previously described ictal activity but nevertheless are likely a pathological consequence of overexpression of virally transduced proteins. Ca2+ micro-waves will most likely obscure the physiology that most researchers are interested in studying with GECIs, and their presence indicates that the neural circuit is in an unintended pathological state. Interestingly this pathology was not observed using the same viral transduction methods in the visual cortex. The authors recommend several approaches that may help other experimenters avoid this confound in their own data such as reducing the titer of viral injections or using recombinase-dependent expression. The intent of this manuscript is to raise awareness of the potential unintended consequences of viral overexpression, particularly for GECIs. A rigorous investigation into the exact causes of Ca2+ micro-waves or the mech

Strengths:

The authors clearly demonstrate that Ca2+ micro-waves occur in the CA1 and CA3 regions of the hippocampus following large volume, high titer injections of adeno-associated viruses (AAV1 and AAV9) encoding GECIs. The supplementary videos provide undeniable proof of their existence.

By forming an inter-institutional collaboration, the authors demonstrate that this phenomenon is robust to changes in surgical techniques or imaging conditions.

Weaknesses:

I believe that the weaknesses of the manuscript are appropriately highlighted by the authors themselves in the discussion. I would, however, like to emphasize several additional points.

As the authors state, the exact conditions that lead to Ca2+ micro-waves are unclear from this manuscript. It is also unclear if Ca2+ micro-waves are specific to GECI expression or if high-titer viral transduction of other proteins such as genetically encoded voltage indicators, static fluorescent proteins, recombinases, etc could also cause Ca2+ micro-waves.

The authors almost exclusively tested high titer (>5x10^12 vg/mL) large volume (500-1000 nL) injections using the synapsin promoter and AAV1 serotypes. It is possible that Ca2+ micro-waves are dramatically less frequent when titers are lowered further but still kept high enough to be useful for in vivo imaging (e.g. 1x10^12 vg/mL) or smaller injection volumes are used. It is also possible that Ca2+ micro-waves occur with high titer injections using other viral promoter sequences such as EF1α or CaMKIIα. There may additionally be effects of viral serotype on micro-wave occurrence.

The number of animals in any particular condition are fairly low (Table 1) with the exception of V1 imaging and thy1-GCaMP6 imaging. This prohibits rigorous comparison of the frequency of pathological calcium activity across conditions.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors explore the roles of dact1 and dact2 during zebrafish gastrulation and craniofacial development. Previous studies used morpholino (MO) knockdowns to show that these scaffolding proteins, which interact with disheveled (Dsh), are expressed during zebrafish gastrulation and suggested that dact1 promotes canonical Wnt/B-catenin signaling, while dact2 promotes non-canonical Wnt/PCP-dependent convergent-extension (Waxman et al 2004). This study goes beyond this work by creating loss-of-function mutant alleles for each gene and unlike the MO studies finds little (dact2) to no (dact1) phenotypic defects in the homozygous mutants. Interestingly, dact1/2 double mutants have a more severe phenotype, which resembles those reported with MOs as well as homozygous wnt11/silberblick (wnt11/slb) mutants that disrupt non-canonical Wnt signaling (Heisenberg et al., 1997; 2000). Further analyses in this paper try to connect gastrulation and craniofacial defects in dact1/2 mutants with wnt11/slb and other wnt-pathway mutants. scRNAseq conducted in mutants identifies calpain 8 as a potential new target of dact1/2 and Wnt signaling.

Strengths:<br /> When considered separately the new mutants are an improvement over the MOs and the paper contains a lot of new data.

Weaknesses:<br /> The hypotheses are very poorly defined and misinterpret key previous findings surrounding the roles of wnt11 and gpc4, which results in a very confusing manuscript. Many of the results are not novel and focus on secondary defects. The most novel result of overexpressing calpain8 in dact1/2 mutants is preliminary and not convincing.

Major Comments:<br /> 1) One major problem throughout the paper is that the authors misrepresent the fact that wnt11f2 and gpc4 act in different cell populations at different times. Gastrulation defects in these mutants are not similar: wnt11 is required for anterior mesoderm CE during gastrulation but not during subsequent craniofacial development while gpc4 is required for posterior mesoderm CE and later craniofacial cartilage morphogenesis (LeClair et al., 2009). Overall, the non-overlapping functions of wnt11 and gpc4, both temporally and spatially, suggest that they are not part of the same pathway.

2) There are also serious problems surrounding attempts to relate single-cell data with the other data in the manuscript and many claims that lack validation. For example, in Fig 1 it is entirely unclear how the Daniocell scRNA-seq data have been used to compare dact1/2 with wnt11f2 or gpc4. With no labeling in panel 1E of this figure these comparisons are impossible to follow. Similarly, the comparisons between dact1/2 and gpc4 in scRNA-seq data in Fig. 6 as well as the choices of DEGs in dact1/2 or gpc4 mutants in Fig. 7 seem arbitrary and do not make a convincing case for any specific developmental hypothesis. Are dact1 and gpc4 or dact2 and wnt11 co-expressed in individual cells? Eyeballing similarity is not acceptable.

3) Many of the results in the paper are not novel and either confirm previous findings, particularly Waxman et al (2004), or even contradict them without good evidence. The authors should make sure that dact2 loss-of-function is not compensated for by an increase in dact1 transcription or vice versa. Testing genetic interactions, including investigating the expression of wnt11f2 in dact1/2 mutants, dact1/2 expression in wnt11f2 mutants, or the ability of dact1/2 to rescue wnt11f2 loss of function would give this work a more novel, mechanistic angle.

4) The identification of calpain 8 overexpression in Dact1/2 mutants is interesting, but getting 1/142 phenotypes from mRNA injections does not meet reproducibility standards.

Reviewer #3 (Public Review):

Summary:

This is a really interesting study, looking at the efficacy of AAV-mediated delivery of wt HSPE2 gene into mouse mutants with the goal of rescuing lower urinary tract defects.

Strengths: Nice analysis of muscle physiology ex vivo, interesting approach.

Weaknesses: lack of rigor (see below). This is an awesome opportunity to learn much more about the disease, its affects on neurons, muscle, etc.

* Single-cell analysis of mutants versus control bladder, urethra including sphincter. This would be great also for the community.

* Detailed tables showing data from each mouse examined.

* Survival curves.

* Use of measurements that are done in vivo (spot assay for example). This sounds relatively simple.

* Assessment of viral integration in tissues besides the liver (could be done by QPCR).

* Discuss subtypes of neurons that are present and targeted in the context of mutants and controls.

Reviewer #3 (Public Review):

Summary of the findings:

The authors explore an important question concerning the underlying mechanism of representational drift, which despite intense recent interest remains obscure. The paper explores the intriguing hypothesis that drift may reflect changes in the intrinsic excitability of neurons. The authors set out to provide theoretical insight into this potential mechanism.

They construct a rate model with all-to-all recurrent connectivity, in which recurrent synapses are governed by a standard Hebbian plasticity rule. This network receives a global input, constant across all neurons, which can be varied with time. Each neuron also is driven by an "intrinsic excitability" bias term, which does vary across cells. The authors study how activity in the network evolves as this intrinsic excitability term is changed.

They find that after initial stimulation of the network, those neurons where the excitability term is set high become more strongly connected and are in turn more responsive to the input. Each day the subset of neurons with high intrinsic excitability is changed, and the network's recurrent synaptic connectivity and responsiveness gradually shift, such that the new high intrinsic excitability subset becomes both more strongly activated by the global input and also more strongly recurrently connected. These changes result in drift, reflected by a gradual decrease across time in the correlation of the neuronal population vector response to the stimulus.

The authors are able to build a classifier that decodes the "day" (i.e. which subset of neurons had high intrinsic excitability) with perfect accuracy. This is despite the fact that the excitability bias during decoding is set to 0 for all neurons, and so the decoder is really detecting those neurons with strong recurrent connectivity, and in turn strong responses to the input. The authors show that it is also possible to decode the order in which different subsets of neurons were given high intrinsic excitability on previous "days". This second result depends on the extent by which intrinsic excitability was increased: if the increase in intrinsic excitability was either too high or too low, it was not possible to read out any information about the past ordering of excitability changes.

Finally, using another Hebbian learning rule, the authors show that an output neuron, whose activity is a weighted sum of the activity of all neurons in the network, is able to read out the activity of the network. What this means specifically, is that although the set of neurons most active in the network changes, the output neuron always maintains a higher firing rate than a neuron with randomly shuffled synaptic weights, because the output neuron continuously updates its weights to sample from the highly active population at any given moment. Thus, the output neuron can read out a stable memory despite drift.

Strengths:

The authors are clear in their description of the network they construct and in their results. They convincingly show that when they change their "intrinsic excitability term", upon stimulation, the Hebbian synapses in their network gradually evolve, and the combined synaptic connectivity and altered excitability result in drifting patterns of activity in response to an unchanging input (Fig. 1, Fig. 2a). Furthermore, their classification analyses (Fig. 2) show that information is preserved in the network, and their readout neuron successfully tracks the active cells (Fig. 3). Finally, the observation that only a specific range of excitability bias values permits decoding of the temporal structure of the history of intrinsic excitability (Fig. 2f and Figure S1) is interesting, and as the authors point out, not trivial.

Weaknesses:

1) The way the network is constructed, there is no formal difference between what the authors call "input", Δ(t), and what they call "intrinsic excitability" Ɛ_i(t) (see Equation 3). These are two separate terms that are summed (Eq. 3) to define the rate dynamics of the network. The authors could have switched the names of these terms: Δ(t) could have been considered a global "intrinsic excitability term" that varied with time and Ɛ_i(t) could have been the external input received by each neuron in the network. In that case, the paper would have considered the consequence of "slow fluctuations of external input" rather than "slow fluctuations of intrinsic excitability", but the results would have been the same. The difference is therefore semantic. The consequence is that this paper is not necessarily about "intrinsic excitability", rather it considers how a Hebbian network responds to changes in excitatory drive, regardless of whether those drives are labeled "input" or "intrinsic excitability".

A revised version of the manuscript models "slope-based" excitability changes in addition to "threshold-based" changes. This serves to address the above concern that as constructed here changes in excitability threshold are not distinguishable from changes in input. However, it remains unclear what the model would do should only a subset of neurons receive a given, fixed input. In that case, are excitability changes sufficient to induce drift? This remains an important question that is not addressed by the paper in its current form.

2) Given how the learning rule that defines the input to the readout neuron is constructed, it is trivial that this unit responds to the most active neurons in the network, more so than a neuron assigned random weights. What would happen if the network included more than one "memory"? Would it be possible to construct a readout neuron that could classify two distinct patterns? Along these lines, what if there were multiple, distinct stimuli used to drive this network, rather than the global input the authors employ here? Does the system, as constructed, have the capacity to provide two distinct patterns of activity in response to two distinct inputs?

A revised version of the manuscript addresses this question, demonstrating that the network is capable of maintaining two distinct memories.

Impact:

Defining the potential role of changes in intrinsic excitability in drift is fundamental. Thus, this paper represents an important contribution. What we see here is that changes in intrinsic excitability are sufficient to induce drift. This raises the question for future work of the specific contributions of changing excitability from changing input to representational drift.

Reviewer #3 (Public Review):

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

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

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

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

Additionally, the study assumes that whoever disappeared from the dataset is attrition in academia. While in reality, those attritions could be researchers who moved to another country or another institution that is not included in the AARC (Academic Analytics Research Centre) dataset.

Reviewer #3 (Public Review):

Summary:<br /> This paper focuses on the roles of a toxoplasma protein (SPARKEL) with homology to an elongin C and the kinase SPARK that it interacts with. They demonstrate that the two proteins regulate the abundance of PKA and PKG, and that depletion of SPARKEL reduces invasion and egress (previously shown with SPARK), and that their loss also triggers spontaneous bradyzoite differentiation. The data are overall very convincing and will be of high interest to those who study Toxoplasma and related apicomplexan parasites.

Strengths:<br /> The study is very well executed with appropriate controls. The manuscript is also very well and clearly written. Overall, the work clearly demonstrates that SPARK/SPARKEL regulate invasion and egress and that their loss triggers differentiation.

Weaknesses:<br /> 1. The authors fail to discriminate between SPARK/SPARKEL acting as negative regulators of differentiation as a result of an active role in regulating stage-specific transcription/translation or as a consequence of a stress response activated when either is depleted.

2. The function of SPARKEL has not been addressed. In mammalian cells, Elongin C is part of an E3 ubiquitin ligase complex that regulates transcription and other processes. From what I can tell from the proteomic data, homologs of the Elongin B/C complex were not identified. This is an important issue as the authors find that PKG and PKA protein levels are reduced in the knockdown strains

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors conducted a comprehensive study of the X-linked miR-506 family miRNAs in mice on its origin, evolution, expression, and function. They demonstrate that the X-linked miR-506 family, predominantly expressed in the testis, may be derived from MER91C DNA transposons and further expanded by retrotransposition. By genetic deletion of different combinations of 5 major clusters of this miRNA family in mice, they found these miRNAs are not required for spermatogenesis. However, by further examination, the mutant mice show mild fertility problem and inferior sperm competitiveness. The authors conclude that the X-linked miR-506 miRNAs finetune spermatogenesis to enhance sperm competition.

Strengths:

This is a comprehensive study with extensive computational and genetic dissection of the X-linked miR-506 family providing a holistic view of its evolution and function in mice. The finding that this family miRNAs could enhance sperm competition is interesting and could explain their roles in finetuning germ cell gene expression to regulate reproductive fitness.

Weaknesses:

The authors specifically addressed the function of 5 clusters of X-link miR-506 family containing 19 miRNAs. There is another small cluster containing 3 miRNAs close to the Fmr1 locus. Would this small cluster act in concert with the 5 clusters to regulate spermatogenesis? In addition, any autosomal miR-506 like miRNAs may compensate for the loss of X-linked miR-506 family. These possibilities should be discussed.

Direct molecular link to sperm competitiveness defect remains unclear but is difficult to address.

Reviewer #3 (Public Review):

Summary:

The manuscript by Zhou and colleagues describes the potential of a two-compound combination (2C), CHIR99021 and A-485, which can generate regenerative cardiac cells (RCCs) from human embryonic stem cell-derived TNNT2+ cardiomyocytes. The authors have also demonstrated this phenomenon in neonatal rats CMs in vitro. Further, the administration of 2C can generate RCCs in adult mouse hearts and significantly improve survival and cardiac function in mice subjected to myocardial infarction. Interestingly, 2C treatment induces global changes in transcription and epigenetic modifications.

Strengths of the study:

1. This study describes the potential of 2C in improving the regeneration of the heart post-MI. The findings may have a translation potential. The idea of promoting the regenerative capacity of the heart by reprogramming CMs into RCCs is interesting.

2. The authors have validated the effect of 2C independently in hESCs, rat CMs, and a model of MI.

3. The authors explored the mechanism by Single-cell RNA-seq and Chip-Seq, which points to the transcriptional and epigenetic activation of genes essential for RCC.

Weaknesses of the study:

1. The mechanism involved in the 2C-mediated generation of RCCs is still unclear. The leads found in the RNA-seq and ChIP-seq were not validated experimentally.

2. Considering the very low number of RCCs (0.6%-1.5% of cells) generated, I cannot comprehend how the heart is protected from MI. Did the author believe 2C would affect the survival or metabolism of existing CM under hypoxia? What percentage of cells were regenerated by 2C treatment post-MI?

3. I would like to know about administering 2C in mice, which could have generated RCCs- dedifferentiated CMs in the heart. Does 2C affect the cardiac functions in mice under basal conditions? Also, does 2C administration affect any physiology in mice? The cardiac structural and functional parameters are required post-2C administration.

4. It is also not tested whether 2C would affect other cell types of the heart, including fibroblasts and endothelial cells, in vitro and in vivo. Assuming the level of protection by 2C in mice, it would affect other cell types.

5. It is still being determined how the authors chose the dose of 2C for in vivo and in vitro studies, although the concentration used for screening is different. Assessing the effect of 2C in a dose-dependent manner is essential.

6. A-485 affects H3K27Ac but not H3K9Ac. However, data show that both H3K27Ac and H3K9Ac are affected. An explanation is required.

7. The authors use "regeneration" even at the screening stage. I am wondering if regeneration could be assessed by the experimental approach they adopted.

Reviewer #3 (Public Review):

Summary:<br /> Günther and colleagues leverage ancient DNA data to track the genomic history of one of the most important farm animals (cattle) in Iberia, a region showing peculiarities both in terms of cultural practices as well as a climatic refugium during the LGM, the latter of which could have allowed the survival of endemic lineages. They document interesting trends of hybridisation with wild aurochs over the last 8-9 millennia, including a stabilisation of auroch ancestry ~4000 years ago, at ~20%, a time coincidental with the arrival of domestic horses from the Pontic steppe. Modern breeds such as the iconic Lidia used in bullfighting or bull running retain a comparable level of auroch ancestry.

Strengths:<br /> The generation of ancient DNA data has been proven crucial to unravel the domestication history of traditional livestock, and this is challenging due to the environmental conditions of the Iberian peninsula, less favourable to DNA preservation. The authors leverage samples unearthed from key archaeological sites in Spain, including the karstic system of Atapuerca. Their results provide fresher insights into past management practices, and permit characterisation of significant shifts in hybridization with wild aurochs.

Weaknesses:<br /> - Treatment of post-mortem damage: the base quality of nucleotide transitions was recalibrated down to a quality score of 2, but for 5bp from the read termini only. In some specimens (e.g. moo022), the damage seems to extend further. Why not use dedicated tools (e.g. mapDamage), or check the robustness by conditioning on nucleotide transversions?

- Their more solid analyses are based on qpAdm, but rely on two single-sample donor populations. As the authors openly discuss, it is unclear whether CPC98 is a good proxy for Iberian aurochs despite possibly forming a monophyletic clade (the number of analysed sites is simply too low to assess this monophyly; Supplementary Table S2). Additionally, it is also unclear whether Sub1 was a fully unadmixed domestic specimen, depleted of auroch ancestry. The authors seem to suggest themselves that sex-biased introgression may have already taken place in Anatolia ("suggesting that sex-biased processes already took place prior to the arrival of cattle to Iberia").

Alternatively, I recommend using Struct-f4 as it can model the ancestry of all individuals together based on their f4 permutations, including outgroups and modern data, and without the need to define pure "right" and "left" populations such as CPC98 and Sub1. It should work with low-coverage data, and allows us to do f4-based MDS plots as well as to estimate ancestry proportions (including from ghost populations).

- In the admixture graph analyses (supplementary results), the authors use population groups based on a single sample. If these samples are pseudohaploidised (or if coverage is insufficient to estimate heterozygosity - and it is at least for moo004 and moo014), f3 values are biased, implying that the fitted graph may be wrong. The graph shown in Fig S7 is in fact hard to interpret. For example, the auroch Gyu2 from Anatolia but not the auroch CPC98 also from Anatolia received 62% of ancestry from North Africa? The Neolithic samples moo004 and moo014 also show the same shocking disparity. I would consider re-doing this analysis with more than a sample per population group.

Reviewer #3 (Public Review):

Summary:

The researchers aim add to the literature on faculty career pathways with particular attention to how gender disparities persist in the career and funding opportunities of researchers. The researchers also examine aspects of institutional prestige that can further amplify funding and career disparities. While some factors about individuals' pathways to faculty lines are known, including the prospects of certain K award recipients, the current study provides the only known examination of the K99/R00 awardees and their pathways.

Strengths:

The authors establish a clear overview of the institutional locations of K99 and R00 awardees and the pathways for K99-to-R00 researchers and the gendered and institutional patterns of such pathways. For example, there's a clear institutional hierarchy of hiring for K99/R00 researchers that echo previous research on the rigid faculty hiring networks across fields, and a pivotal difference in the time between awards that can impact faculty careers. Moreover, there's regional clusters of hiring in certain parts of the US where multiple research universities are located. Moreover, documenting the pathways of HBCU faculty is an important extension of the study by Wapman et al. (2022: https://www.nature.com/articles/s41586-022-05222-x), and provides a more nuanced look at the pathways of faculty beyond the oft-discussed high status institutions. (However, there is a need for more refinement in this segment of the analyses). Also, the authors provide important caveats throughout the manuscript about the study's findings that show careful attention to the complexity of these patterns and attempting to limit misinterpretations of readers.

Weaknesses:

The authors have addressed my recommendations in the previous review round in a satisfactory way.

Reviewer #3 (Public Review):

Summary<br /> CLC-2 channels play an important role in cellular homeostasis and electrical excitability, and dysfunctions are associated with aldosteronism and leukodystrophy. Structural insights into the functioning of CLC-2 are just emerging. CLC-2 channels are distinct among the members of the CLC family in that they are activated by hyperpolarization. Earlier studies have implicated channel regulation by a "ball-and-chain" type of channel block mechanism which underlies its strong rectification and use-dependent "run-up" properties. Structural insights into these mechanisms are currently lacking. In this manuscript, Xu et al present CryoEM structures of CLC-2 in the apo and inhibitor-bound conformations in the 2.5-2.7 A resolution range. Several novel structural features are presented that lend support to the "ball-and chain" model, identify an interesting role for the c-terminal domain in gating, and establish the interaction pocket for AK-42. Electrophysiology and simulations nicely support the structural work. Overall, an elegant study, with high-quality data, and a well-presented manuscript.

Strengths<br /> 1. The cryoEM data presented reveals that the channel is in a closed conformation at depolarizing potential (0 mv). Structures for the closed state of CLCs were not previously available. A strong density for Glu205, which constitutes the Egate, allows for an unambiguous assignment of its position at the Scen Cl-binding site, thereby establishing the basis for the block in the closed channel.<br /> 2. The apo state particles were sorted into two classes that differ in the conformation of the CTD. A previously unobserved rearrangement of the CBS region in the CTD is reported wherein the CTD is positioned closer to the TM region in one of the subunits, breaking the C2 symmetry. The data implicates a role for the conformational flexibility of CTD in gating.<br /> 3. The most interesting finding of this work, is perhaps, the presence of an additional density, corresponding to a hairpin-like structure, that is seen only at the subunit where the CTD is positioned away from the TMD. The authors propose that the additional density corresponds to a 13 aa stretch in the N-terminal region. The position of the hairpin at the intracellular mouth of the CL-permeation pathway is likely to impede ion conduction, by a mechanism analogous to the "ball-and-chain" proposed in other voltage-gated channels.<br /> 4. The structure of CLC-2 in complex with a selective inhibitor AK-42 is in a conformation very similar to that of the apo state, with a clear additional density for the AK-42 molecule. Binding site interaction provides insights into AK-42 selectivity for CLC-2 vs CLC-1.

Reviewer #3 (Public Review):

Summary: The authors present a thought-provoking and comprehensive re-analysis of previously published human cell genomics data that seeks to understand the relationship between the sites where the Origin Recognition Complex (ORC) binds chromatin, where the replicative helicase (Mcm2-7) is loaded, and where DNA replication actually begins (origins). The view that these should coincide is influenced by studies in yeast where ORC binds site-specifically to dedicated nucleosome-free origins where Mcm2-7 can be loaded and remains stably positioned for subsequent replication initiation. However, this is most certainly not the case in metazoans where it has already been reported that chromatin bindings sites of ORC and Mcm2-7 do not necessarily overlap, nor do they always overlap with origins. This is likely due to Mcm2-7 possessing linear mobility on DNA (i.e., it can slide) such that other chromatin-contextualized processes can displace it from the site in which it was originally loaded. Additionally, Mcm2-7 is loaded in excess and thus only a fraction of Mcm2-7 would be predicted to coincide with replication start sites. This study reaches a very similar conclusion of these previous studies: they find a high degree of discordance between ORC, Mcm2-7, and origin positions in human cells.

Strengths: The strength of this work is its comprehensive and unbiased analysis of all relevant genomics datasets. To my knowledge, this is the first attempt to integrate these observations. It also is an important cautionary tale to not confuse replication factor binding sites with the genomic loci where replication actually begins, although this point is already widely appreciated in the field.

Weaknesses: The major weakness of this paper is the lack of novel biological insight and that the comprehensive approach taken failed to provide any additional mechanistic insight regarding how and why ORC, Mcm2-7, and origin sites are selected or why they may not coincide.

Reviewer #3 (Public Review):

Sasani et al. develop and implement a new method for mutator allele discovery in the BXD mouse population. This new method, termed "aggregate mutation spectrum distance" or AMSD, carries several notable strengths, including the ability to aggregate de novo mutations across individuals to reduce data sparsity and to combine mutation rate frequencies across multiple nucleotide contexts into a single estimate. As demonstrated by simulations, this method is better suited to mutator discovery under certain scenarios, as compared to conventional QTL or association mapping. Overall, the theoretical premise of the AMSD method is judged to be both strong and innovative, and the methodology could be extended to other species and populations to enable discovery of additional mutator alleles.

The authors then apply their method to the BXD mouse recombinant inbred mapping population. As proof-of-principle, they first successfully re-identify a known mutator locus in this population on chr4. Next, to assess possible genetic interactions involving this known mutator, Sasani et al. condition on the chr4 mutator genotype and reimplement the AMSD scan. This strategy led them to identify a second locus on chr6 that interacts epistatically with the chr4 locus; mice with "D" alleles at both loci exhibit a significantly increased burden of C>A de novo mutations, even though mice with the D allele at the chr6 locus alone show no appreciable increase in the C>A mutation fraction. This exciting discovery not only adds to the catalog of known mutator alleles, but also reveals key aspects of mutator biology and reinforces the hypothesis that segregating variants in genes associated with DNA repair influence germline mutation spectra.

Despite a high level of overall enthusiasm for this work, there are some limitations to the AMSD method. However, it is my judgement that the authors present a balanced summary of the strengths and weaknesses of their method in the revised manuscript. I also think that the authors' conclusions may actually somewhat undersell the scientific impact of their findings. As the authors note, few mutation rate modifiers have been identified in mammals. This is potentially because large- and moderate-effect modifiers are rapidly selected against due to their deleterious effects, but could also be due to pervasive epistasis wherein modifiers are only expressed on certain "permissive" genetic backgrounds, such as the chr6 locus the authors discover in this paper. The potential background dependence of mutator expression could partially shelter it from the action of selection, allowing the allele persist in populations. This discovery has significant implications for our understanding of mutation rate evolution, but only earns a cursory mention in the paper.

Reviewer #3 (Public Review):

Summary:

In the submitted manuscript by Go et al, the authors evaluated the tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) and made a number of interesting observations, including the following: 1) CCL5 expression within the tumor microenvironment negatively correlated with clinical outcomes in human patients with PDAC; 2) there were both positive and negative correlations between CCL5 expression and the expression of specific genes (e.g. those encoding CD56 and CD16, respectively) included among gene signature lists for Treg, MDSC, TAM, and NK cells; 3) CCR5 inhibition with the inhibitor, maraviroc, reduced Treg infiltration but not that of other immune cell types in an orthotopic murine model of PDAC; 4) CCR5 inhibition augmented anti-PD1 immunotherapy when combined with ionizing radiation (IR) therapy in the murine model; 5) the above therapy resulted in increased infiltration of CD8+ cytotoxic T cells as well as of a subset of NKG2D-negative, tissue-residency (tr) marker expressing NK cells (deemed Cluster 1 NK in their data sets) that inversely correlated with the number of E-cadherin+ cells (i.e. tumor cells) and showed predicted interactions with cDC1 dendritic cells (including XCL1/XCL2 expressed by the NK and XCR1 expressed by the cDC1); 6) the authors identified a number of putative signals stemming from the trNK (e.g. IL-16, TNFSF14, FASLG, CSF, MIF) as well as incoming from cDC1s to NK (e.g. BAG6-NKp30); 7) these trNK cells positively correlated with good outcomes and with CD8+ T cell infiltrations in human PDAC as well as in many other solid tumor types; and 8) importantly, the benefit of IR therapy was specific to the subset of PDAC patients (represented in the TCGA dataset) that were predicted to have low amounts of trNK cells. The authors used murine experimental models, multiplexed imaging analyses, and a number of publicly available sequencing data sets from human tumor samples to perform their investigations. Based on their findings, the authors proposed that combining IR with CCR5 inhibition and anti-PD1 immunotherapy is a promising strategy to treat solid cancers.

Strengths:

Overall, the collective analyses and conclusions appear to be novel and could be of high and rapid impact on the field, particularly in terms of directing clinical trials to incorporate IR with CCR5 inhibition and immunotherapy. The manuscript is well written; the figures are for the most part clear; and the Discussion is very thoughtful.

Weaknesses:

There were a number of minor typographical errors, missing references, or minor issues with the figures. In general, while many of the observations provided strong suggestive evidence of relationships, phenotypes, and functions, the authors often used language to indicate that such things were confirmed, validated, or proven. In fact, there was a paucity of such functional/confirmatory experiments. This does not necessarily detract from the overall significance, excitement for, and potential impact of the study; but the language could likely be adjusted to be more in keeping with the true nature of the findings. The main title and running title are a bit different; consider making them more similar.

Reviewer #3 (Public Review):

This work by Fleck et al. and colleagues documented the auxin feeding-induced effects in adult flies, since auxin could be used in temporally control gene expression using a modified Gal4/Gal80 system. Overall, the experiments were well designed and carefully executed. The results were quantified with appropriate statistical analyses. The paper was also well written and the results were presented logically. Their findings demonstrate that auxin-fed flies have significantly lower triglyceride levels than the control flies using Ultra High-pressure Liquid Chromatography-Mass Spectrometry (UHPLC-MS)-based metabolomics assays. Further transcriptome analyses using the whole flies show changes of genes involved in fatty acid metabolism. However, female oogenesis and fecundity do not seem to be affected, at least using the current assays. These results indicate that auxin may not be used in experiments involving lipid-related metabolism, but could be appropriate to be applied for other biological processes. Researchers need to be careful when applying this strategy in their own experimental design and should perform proper controls.

Reviewer #3 (Public Review):

Summary of Work<br /> This paper conducts the largest GWAS study of A. thaliana in response to a viral infection. The paper identifies a 1.5 MB region in the chromosome associated with disease, including SNPs, structural variation, and transposon insertions. Studies further validate the association experimentally with a separate experimental infection procedure with several lines and specific T-DNA mutants. Finally, the paper presents a geographic analysis of the minor disease allele and the major association. The major take-home message of the paper is that structural variants and not only SNPs are important changes associated with disease susceptibility. The manuscript also makes a strong case for negative frequency-dependent selection maintaining a disease susceptibility locus at low frequency.

Strengths and Weaknesses<br /> A major strength of this manuscript is the large sample sizes, careful experimental design, and rigor in the follow-up experiments. For instance, mentioning non-infected controls and using methods to determine if geographic locus associations were due to chance. The strong result of a GWAS-detected locus is impressive given the complex interaction between plant genotypes and strains noted in the results. In addition to the follow-up experiments, the geographic analysis added important context and broadened the scope of the study beyond typical lab-based GWAS studies. I find very few weaknesses in this manuscript.

Support of Conclusions<br /> The support for the conclusions is exceptional. This is due to the massive amount of evidence for each statement and also due to the careful consideration of alternative explanations for the data.

Significance of Work<br /> This manuscript will be of great significance in plant disease research, both for its findings and its experimental approach. The study has very important implications for genetic associations with disease beyond plants.

Reviewer #3 (Public Review):

Summary:<br /> In this interesting study, the authors characterize the mechanisms whereby a C. elegans TGF-beta DAF-7 responds to various forms of food cues to regulate foraging.<br /> Building on their previous findings that characterized the functional role of daf-7 in the ASJ sensory neurons in response to a bacterial pathogen and in regulating searching behaviors, the authors of this manuscript show that ingestion of E. coli OP50, a common laboratory food for the worms, suppresses ASJ expression of daf-7 and secreted water-soluble cues of OP50 increase it. They further show that the level of daf-7 expression in ASJ is positively associated with a higher level of roaming/exploration. The authors identify that the function of a C. elegans ortholog of Anaplastic Lymphoma Kinase in the interneurons AIA regulates ASJ expression of daf-7 in response to food information and the related searching behavior.

Strengths:<br /> The study addresses an important question that appeals to a wide readership. The findings are demonstrated by strong results produced from well designed experiments.

Reviewer #3 (Public Review):

This study is well designed and executed and provides new and important insights into the role of two TFs during the maturation of female gametocytes and fertilization in the mosquito midgut. However, it but would benefit from a more thorough characterization of the phenotype to understand at which step of development these factors are required.

Overall the authors have shown only limited willingness to comprehensively address reviewer concerns and incorporate their suggestions.

Reviewer #3 (Public Review):

Summary & Strengths:

This study is useful in revealing the neural correlates of goal-directed navigation in the rodent parahippocampal regions, including the medial entorhinal cortex, presubiculum, and parasubiculum. It shows that task-relevant information represented by the parahippocampus is strongly related to task performance. It also reports the relationships of navigational factors (e.g., head direction signal) recorded during foraging in an open field with task variables.

Gonzalez and Giocomo investigated the neural activities in the parahippocampal cortex modulated by visual cues and error signals while the animal performed a goal-directed navigation task on the tree maze. They confirmed that the firing rates and spatial firing patterns in the parahippocampus were significantly correlated with the animal's task performance and the general navigational coding in the open field arena. The authors have concluded that the parahippocampal neurons encode mismatch-like signals, suggesting the functional role of the parahippocampus as a feedback system in a goal-directed task. However, a few major concerns should be addressed more closely to support the conclusion.

1) Due to the limitations of histological verification, the neural responses in the medial entorhinal cortex, presubiculum, and parasubiculum are analyzed together, and this limits the study from understanding the differential information processing across these regions. Because the medial entorhinal cortex and the pre/parasubiculum are believed to be located in very different positions in the information flow within the rodent medial temporal lobe with different anatomical connections, it would have been more convincing if the distinctive functions between the regions could be identified.

2) The authors should carefully differentiate rate remapping and global remapping in their analysis. Rate remapping generally indicates firing rate modulation with little or no shift of spatial firing fields (Leutgeb et al., 2005; Colgin et al., 2008). Therefore, the neurons exhibiting global remapping should not be included in the analysis suited for rate remapping (e.g., the encoding model that considers the cue-dependent rate-remapping effect).

3) One of the major findings in this study is that the parahippocampal neural responses to a visual cue or reward were correlated with task performance. One can expect that cue representation before the decision point is likely to have a greater impact on task performance. Although the Uz score between the left cue and right cue seemed not significantly different from zero on the stem, it would be beneficial if the authors verify whether the remapping score based on the firing rate maps will still be correlated with the task performance when examined only before the decision point, not for the entire maze.

4) There is a need to set the analytic epoch in more detail. The boundary between outbound and inbound journeys was set as 'last goal well visit.' However, even in a correct trial, if the reward was not received in the first goal well, an error signal could occur before the animal triggered the second goal well which was rewarding. This might have caused the rate remapping between two cue conditions, specifically on the arms. To eliminate this possibility, it is recommended to set the outbound journey from the home well trigger to the first goal well approach or to select only trials where the animal received rewards from the first goal well triggering.

Weaknesses:

Incomplete results could limit support for the arguments of the study and may require more rigorous analytical methods.

Reviewer #3 (Public Review):

The paper by Li et al. describes the role of the TOR pathway in Aspergillus flavus. The authors tested the effect of rapamycin in WT and different deletion strains. This paper is based on a lot of experiments and work but remains rather descriptive and confirms the results obtained in other fungi. It shows that the TOR pathway is involved in conidiation, aflatoxin production, pathogenicity, and hyphal growth. This is inferred from rapamycin treatment and TOR1/2 deletions. Rapamycin treatment also causes lipid accumulation in hyphae. The phenotypes are not surprising as they have been shown already for several fungi. In addition, one caveat is in my opinion that the strains grow very slowly and this could cause many downstream effects. Several kinases and phosphatases are involved in the TOR pathway. They were known from S. cerevisiae or filamentous fungi. The authors characterized them as well with knock-out approaches.

Reviewer #3 (Public Review):

In this manuscript, the authors explored the transcriptional heterogeneity of the periosteum with single nuclei RNA sequencing. Without prior enrichment of specific populations, this dataset serves as an unbiased representation of the cellular components potentially relevant to bone regeneration. By describing single-cell cluster profiles, the authors characterized over 10 different populations in combined steady state and post-fracture periosteum, including stem cells (SSPC), fibroblast, osteoblast, chondrocyte, immune cells, and so on. Specifically, a developmental trajectory was computationally inferred using the continuum of gene expression to connect SSPC, injury-induced fibrogenic cells (IIFC), chondrocyte, and osteoblast, showcasing the bipotentials of periosteal SSPCs during injury repair. Additional computational pipelines were performed to describe the possible gene regulatory network and the expected pathways involved in bone regeneration. Overall, the authors provided valuable insights into the cell state transitions during bone repair and proposed sets of genes with possible involvements in injury response.

While the highlights of the manuscript are the unbiased characterization of periosteal composition, and the trajectory of SSPC response in bone fracture response, many of the conclusions can be more strongly supported with additional clarifications or extensions of the analysis.

1. As described in the method section, both the steady-state data and full dataset underwent integration before dimensional reduction and clustering. It would be appreciated if the authors could compare the post-integration landscapes of uninjured cells between steady state and full dataset analysis. Specifically, fibroblasts were shown in Figure 1C and 1E, and such annotations did not exist in Figure 2B. Will it be possible that the original 'fibroblasts' were part of the IIFC population?

2. According to Figure 2, immune cells were taking a significant abundance within the dataset, specifically during days 3 & 5 post-fracture. It will be interesting to see the potential roles that immune cells play during bone repair. For example, what are the biological annotations of the immune clusters (B, T, NK, myeloid cells)? Are there any inflammatory genes or related signals unregulated in these immune cells? Do they interact with SSPC or IIFC during the transition?

3. The conclusion of Notch and Wnt signaling in IIFC transition was not sufficiently supported by the analysis presented in the manuscript, which was based on computational inferences. It will be great to add in references supporting these claims or provide experimental validations examining selected members of these pathways.

Reviewer #3 (Public Review):

Summary:<br /> Canelo et al. used a combination of mathematical modeling and behavioral experiments to ask whether flies use an all-or-none EC model or a graded EC model (in which the turn amplitude is modulated by wide-field optic flow). Particularly, the authors focus on the bar-ground discrimination problem, which has received significant attention in flies over the last 50-60 years. First, they use a model by Poggio and Reichardt to model flight response to moving small-field bars and spots and wide-field gratings. They then simulate this model and compare simulation results to flight responses in a yaw-free tether and find generally good agreement. They then ask how flies may do bar-background discrimination (i.e. complex visual environment) and invoke different EC models and an additive model (balancing torque production due to background and bar movement). Using behavioral experiments and simulation supports the notion that flies use an all-or-none EC since flight turns are not influenced by the background optic flow. While the study is interesting, there are major issues with the conceptual framework.

Strengths:<br /> - They ask a significant question related to efference copies during volitional movement.<br /> - The methods are well detailed and the data (and statistics) are presented clearly.<br /> - The integration of behavioral experiments and mathematical modeling of flight behavior.<br /> - The figures are overall very clear and salient.

Weaknesses:<br /> - Omission of saccades: While the authors ask a significant question related to the mechanism of bar-ground discrimination, they fail to integrate an essential component of the Drosophila visuomotor responses: saccades. Indeed, the Poggio and Reichardt model, which was developed almost 50 years ago, while appropriate to study body-fixed flight, has a severe limitation: it does not consider saccades. The authors identify this major issue in the Discussion by citing a recent switched, integrate-and-fire model (Mongeau & Frye, 2017). The authors admit that they "approximated" this model as a smooth pursuit movement. However, I disagree that it is an approximation; rather it is an omission of a motor program that is critical for volitional visuomotor behavior. Indeed, saccades are the main strategy by which Drosophila turn in free flight and prior to landing on an object (i.e. akin to a bar), as reported by the Dickinson group (Censi et al., van Breugel & Dickinson [not cited]). Flies appear to solve the bar-ground discrimination problem by switching between smooth movement and saccades (Mongeau & Frye, 2017; Mongeau et al., 2019 [not cited]). Thus, ignoring saccades is a major issue with the current study as it makes their model disconnected from flight behavior, which has been studied in a more natural context since the work of Poggio.

Critically, recent work showed that a group of columnar neurons (T3) appear specialized for saccadic bar tracking through integrate-and-fire computations, supporting the notion of parallel visual circuits for saccades and smooth movement (Frighetto & Frye, 2023 [not cited]).

A major theme of this work is bar fixation, yet recent work showed that in the presence of proprioceptive feedback, flies do not actually center a bar (Rimniceanu & Frye, 2023). Furthermore, the same study found that yaw-free flies do not smoothly track bars but instead generate saccades. Thus prior work is in direct conflict with the work here. This is a major issue that requires more engagement by the authors.

- Relevance of the EC model: EC-related studies by the authors linked cancellation signals to saccades (Kim et al, 2014 & 2017). Puzzlingly, the authors applied an EC model to smooth movement, when the authors' own work showed that smooth course stabilizing flight turns do not receive cancellation signals (Fenk et al., 2021). Thus, in Fig. 4C, based on the state of the field, the efference copy signal should originate from the torque commands to initiate saccades, and not from torque to generate smooth movement. As this group previously showed, cancellation signals are quantitatively tuned to that of the expected visual input during saccades. Importantly, this tuning would be to the anticipated saccadic turn optic flow. Thus the authors' results supporting an all-or-none model appear in direct conflict with the author's previous work. Further, the addition-only model is not particularly helpful as it has been already refuted by behavioral experiments (Rimneceanu & Frye, Mongeau & Frye).

- Behavioral evidence for all-or-none EC model: The authors state "unless the stability reflex is suppressed during the flies' object evoked turns, the turns should slow down more strongly with the dense background than the sparse one". This hypothesis is based on the fact that the optomotor response magnitude is larger with a denser background, as would be predicted by an EMD model (because there are more pixels projected onto the eye). However, based on the authors' previous work, the EC should be tuned to optic flow and thus the turning velocity (or amplitude). Thus the EC need not be directly tied to the background statistics, as they claim. For instance, I think it would be important to distinguish whether a mismatch in reafferent velocity (optic flow) links to distinct turn velocities (and thus position). This would require moving the background at different velocities (co- and anti-directionally) at the onset of bar motion. Overall, there are alternative hypotheses here that need to be discussed and more fully explored (as presented by Bender & Dickinson and in work by the Maimon group).

Reviewer #3 (Public Review):

In this study, the authors developed and tested a novel framework for extracting muscle synergies. The approach aims at removing some limitations and constrains typical of previous approaches used in the field. In particular, the authors propose a mathematical formulation that removes constrains of linearity and couple the synergies to their motor outcome, supporting the concept of functional synergies and distinguishing the task-related performance related to each synergy. While some concepts behind this work were already introduced in recent work in the field, the methodology provided here encapsulates all these features in an original formulation providing a step forward with respect to the currently available algorithms. The authors also successfully demonstrated the applicability of their method to previously available datasets of multi-joint movements.

Preliminary results positively support the scientific soundness of the presented approach and its potential. The added values of the method should be documented more in future work to understand how the presented formulation relates to previous approaches and what novel insights can be achieved in practical scenarios and confirm/exploit the potential of the theoretical findings.

Reviewer #3 (Public Review):

Summary:<br /> The mechanism underlying the well-documented CO2-regulated activity of connexin 26 (Cx26) remains poorly understood. This is largely due to the labile nature of CO2-mediated carbamylation, making it challenging to visualize the effects of this reversible posttranslational modification. This paper by Brotherton et al. aims to address this gap by providing structural insights through cryo-EM structures of a carbamylation-mimetic mutant of the gap junction protein.

Strengths:<br /> The combination of the mutation, elevated PCO2, and the use of LMNG detergent resulted in high-resolution maps that revealed, for the first time, the structure of the cytoplasmic loop between transmembrane helix (TM) 2 and 3.

Weaknesses:<br /> The presented maps merely reinforce their previous findings, wherein wildtype Cx26 favored a closed conformation in the presence of high PCO2. While the structure of the TM2-TM3 loop may suggest a mechanism for stabilizing the closed conformation, no experimental data was provided to support this mechanism. Additionally, the cryo-EM maps were not effectively presented, making it difficult for readers to grasp the message.

Reviewer #3 (Public Review):

Summary:<br /> Unlike most eukaryotes, Blastocystis has a branched glycolysis pathway, which is split between the cytoplasm and the mitochondrial matrix. An outstanding question was how the glycolytic intermediates generated in the 'preparatory' phase' are transported into the mitochondrial matrix for the 'pay off' phase. Here, the authors use bioinformatic analysis to identify two candidate solute carrier genes, bGIC-1, and bGIC-2, and use biochemical and biophysical methods to characterise their substrate specificity and transport properties. The authors demonstrate that bGIC-2 can transport dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate, establishing this protein as the 'missing link' connecting the two split branches of glycolysis in this branch of single-celled eukaryotes. The authors also present their data on bGIC-1, which suggests a role in anion transport and bOGC, which is a close functional homologue of the human oxoglutarate carrier (hOGC, SLC25A11) and human dicarboxylate carrier (hDIC, SLC25A10).

Strengths:<br /> The results are presented in a clear and logical arrangement, which nicely leads the reader through the process of gene identification and subsequent ligand screening and functional reconstitution. The results are compelling and well supported - the thermal stabilisation data is supported by the exchange studies. Caveats, where apparent, are discussed and rational explanations are given.

Weaknesses:<br /> The study does not contain any significant weaknesses in my view. I would like to see the authors include the initial rate plots used in the main figures (possibly as insets), so we can observe the data points used for these calculations. It would also have been interesting to include the AlphaFold models for bGIC-1 and bGIC-2 and a discussion/rationalisation for the substrate specificity discussed in the study.

Reviewer #3 (Public Review):

The authors describe the role, location, and function of the MTA and MTB mating type genes in the multi-mating-type species T. thermophila. The ciliate is an important group of organisms to study the evolution of mating types, as it is one of the few groups in which more than two mating types evolved independently. In the study, the authors use deletion strains of the species to show that both mating types genes located in each allele are required in both mating individuals for successful matings to occur. They show that the proteins are localized in the cell membrane, not the cilia, and that they interact in a complex (MTRC) with a set of 6 associated (non-mating type-allelic) genes. This complex is furthermore likely to interact with a cyclin-dependent kinase complex. It is intriguing that T. thermophila has two genes that are allelic and that are both required for successful mating. This coevolved double recognition has to my knowledge not been described for any other mating-type recognition system. I am not familiar with experimental research on ciliates, but as far as I can judge, the experiments appear well performed and mostly support the interpretation of the authors with appropriate controls and statistical analyses.

The results show clearly that the mating type genes regulate non-self-recognition, however, I am not convinced that self-recognition occurs leading to the suppression of mating. An alternative explanation could be that the MTA and MTB proteins form a complex and that the two extracellular regions together interact with the MTA+MTB proteins from different mating types. This alternative hypothesis fits with the coevolution of MTA and MTB genes observed in the phylogenetic subgroups as described by Yan et al. (2021 iScience). Adding MTAxc and/or MTBxc to the cells can lead to the occupation of the external parts of the full proteins thereby inhibiting the formation of the complex, which in turn reduces non-self interactions. Self-recognition as explained in Figure 2S1 suggests an active response, which should be measurable in expression data for example. This is in my opinion not essential, but a claim of self-recognition through the MTA and MTB should not be made.

The authors discuss that T. thermophila has special mating-type proteins that are large, while those of other groups are generally small (lines 157-160 and discussion). The complex formed is very large and in the discussion, they argue that this might be due to the "highly complex process, given that there are seven mating types in all". There is no argument given why large is more complex, if this is complex, and whether more mating types require more complexity. In basidiomycete fungi, many more mating types than 7 exist, and the homeodomain genes involved in mating types are relatively small but highly diverse (Luo et al. 1994 PMID: 7914671). The mating types associated with GPCR receptors in fungi are arguably larger, but again their function is not that complex, and mating-type specific variations appear to evolve easily (Fowler et al 2004 PMID: 14643262; Seike et al. 2015 PMID: 25831518). The large protein complex formed is reminiscent of the fusion patches that develop in budding or fission yeasts. In these species, the mating type receptors are activated by ligand pheromones from the opposite mating type that induce polarity patch formation (see Sieber et al. 2023 PMID: 35148940 for a recent review). At these patches, growth (shmooing) and fusion occur, which is reminiscent (in a different order) of the tip transformation in T. thermophilia. The fusion of two cells is in all taxa a dangerous and complex event that requires the evolution of very strict regulation and the existence of a system like the MTRC and cyclin-dependent complex to regulate this process is therefore not unexpected. The existence of multiple mating types should not greatly complicate the process, as most of the machinery (except for the MTA and MTB) is identical among all mating types.

The Tetrahymena/ciliate genetics and lifecycle could be better explained. For a general audience, the system is not easy to follow. For example, the ploidy of the somatic nucleus with regards to the mating type is not clear to me. The MAC is generally considered "polyploid", but how does this work for the mating type? I assume only a single copy of the mating type locus is available in the MAC to avoid self-recognition in the cells. Is it known how the diploid origin reduces to a single mating type? This does not become apparent from Cervantes et al. 2013. Also, the explanation of co-stimulation is not completely clear (lines 49-60). Initially, direct cell-cell contact is mentioned, but later it is mentioned that "all cells become fully stimulated", even when unequal ratios are used. Is physical contact necessary? Or is this due to the "secrete mating-essential factors" (line 601)? These details are essential, for interpretation of the results and need to be explained better.

Abstract and introduction: Sexes are not mating types. In general, mating types refer to systems in which there is no obvious asymmetry between the gametes, beyond the compatibility system. When there is a physiological difference such as size or motility, sexes are used. This distinction is of importance because in many species mating types and sexes can occur together, with each sex being able to have either (when two) or multiple mating types. An example are SI in angiosperms as used as an example by the authors or mating types in filamentous fungi. See Billiard et al. 2011 [PMID: 21489122] for a good explanation and argumentation for the importance of making this distinction.

Reviewer #3 (Public Review):

Summary: The authors investigate the effect of the lipid aldehyde trans-2-hexadecenal (t-2-hex) in yeast using multiple omic analyses that show that a large range of cellular functions across all compartments are affected, e.g. transcriptomic changes affect 1/3 of all genes. The authors provide additional analyses, from which they built a model that mitochondrial protein import caused by modification of Tom40 is blocked.

Strengths: Global analyses (transcriptomic and functional genomics approach) to obtain an unbiased overview of changes upon t-2-hex treatment.

Weaknesses: It is not clear why the authors decided to focus on mitochondria, as only 30 genes assigned to the GO term "mitochondria" are increasing, and also the follow-up analyses using SATAY is not showing a predominance for mitochondrial proteins (only 4 genes are identified as hits). The provided additional experimental data do not support the main claims as neither protein import is investigated nor is there experimental evidence that lipidation of Tom40 occurs in vivo and impacts on protein translocation.

Reviewer #3 (Public Review):

This is interesting biology. Vitamin B6 deficiency has been linked to cognitive impairment. It is not clear whether supplements are effective in restoring functional B6 levels. Vitamin B6 is composed of pyridoxal compounds and their phosphorylated forms, with pyridoxal 5-phosphate (PLP) being of particular importance. The levels of PLP are determined by the balance between pyridoxal kinase and phosphatase activities. The authors are testing the hypothesis that inhibition of pyridoxal phosphatase (PDXP) would arrest the age-dependent decline in PLP, offering an alternative therapeutic strategy to supplements. Published data illustrating that ablation of the Pdxp gene in mice led to increases in PLP levels and improvement in learning and memory trials are consistent with this hypothesis.

In this report, the authors conduct a screen of a library of ~40k small molecules and identify 7,8-dihydroxyflavone (DHF) as a candidate PDXP inhibitor. They present an initial characterization of this micromolar inhibitor, including a co-crystal structure of PDXP and 7,8-DHF. In addition, they demonstrate that treatment of cells with 7,8 DHP increases PLP levels. Overall, this study provides further validation of PDXP as a therapeutic target for the treatment of disorders associated with vitamin B6 deficiency and provides proof-of-concept for inhibition of the target with small-molecule drug candidates.

Strengths include the biological context, the focus on an interesting and under-studied class of protein phosphatases that includes several potential therapeutic targets, and the identification of a small molecule inhibitor that provides proof-of-concept for a new therapeutic strategy. Overall, the study has the potential to be an important development for the phosphatase field in general.

Weaknesses include the fact that the compound is very much an early-stage screening hit. It is an inhibitor with micromolar potency for which mechanisms of action other than inhibition of PDXP have been reported. Extensive further development will be required to demonstrate convincingly the extent to which its effects in cells are due to on-target inhibition of PDXP.

Reviewer #3 (Public Review):

Summary:

The authors set out to determine the extent to which the cation channel TRPV4 is expressed in secretory cells of sweat glands and the effect of blocking TRPV4 activity on sweat production, mediated via effects on the chloride channel anoctamin 1.

Strengths:

The study makes use of a diverse array of techniques, including super-resolution microscopy, live-cell calcium imaging, behavioral tests, and immunohistochemistry of human tissues in support of the claim that functional TRPV4 expression is detectable in sweat glands, and that TRPV4-deficient mice do not show respond to stimulation of sweat production (acetylcholine).

Weaknesses:

Figure 2: The calcium imaging-based approach shows average traces from 6 cells per genotype, but it was unclear if all acinar cells tested with this technique demonstrated TRPV4-mediated calcium influx, or if only a subset was presented.

Figure 4: The climbing behavioral test shows a significant reduction in climbing success rate in TRPV4-deficient mice. The authors ascribe this to a lack of hind paw 'traction' due to deficiencies in hind paw perspiration, but important controls and evidence that could rule out other potential confounds were not provided or cited.

In general, the results support the authors' claims that TRPV4 activity is a necessary component of sweat gland secretion, which may have important implications for controlling perspiration as well as secretion from other glands where TRPV4 may be expressed.

Reviewer #3 (Public Review):

Summary:

Sperm-egg fusion is a critical step in successful fertilization. Although several proteins have been identified in mammals that are required for sperm-egg adhesion and fusion, it is still unclear whether there are other proteins involved in this process and how the reported proteins complex and/or cooperate to complete the fusion process. In this study, the authors first identified TMEM81 as a structural homologue of IZUMO1 and SPACA6, and using AlphaFold-Multimer, a recent advance in protein complex structure prediction, predicted the interactions between human proteins associated with gamete fusion. While the prediction is compelling and well discussed, the experimental evidence to verify this interaction is lacking, so the prediction remains a hypothesis.

Strengths:

The authors present a pentameric complex formation of four previously reported proteins involved in egg/sperm interaction together with TMEM181 using a deep learning tool, AlphaFold-Multimer.

Weaknesses:

While it is intriguing to see that some of the proteins involved in sperm-egg interaction are successfully predicted to be assembled into a single multimeric structure by AlphaFold-Multimer, it is necessary to experimentally validate the interactions. As there are more candidate proteins in the process, it will be necessary to test other possible protein interactions to prove the adequacy of the candidates chosen by the authors, as similar analysis with some other proteins will provide more rationale for further 3D multi-protein modeling. In addition, the lack of biochemical data to support the predicted bindings between proteins limits the proposed complex to remain mainly hypothetical.

Reviewer #3 (Public Review):

Using theory, the authors study mechanisms for establishing planar cell polarity (PCP) through local and global modules. These modules refer to the interaction between neighbouring cells and tissue-wide gradients, respectively. Whereas local interactions alone can lead to tissue-wide alignment PCP, a global gradient can set the direction of PCP and maintain the pattern in presence of noise. In contrast, the authors argue that a global gradient can only generate PCP to an extent that is proportional to the gradient magnitude.

The authors formulate a discrete model in one and two spatial dimensions that describe the assembly dynamics of PCP proteins on membranes. The number of proteins per cell remains constant. Additive noise is introduced to account for stochasticity in the attachment/detachment kinetics of proteins. Furthermore, 'quenched' noise is introduced to account for variations of protein numbers between cells. The authors perform simulations of the stochastic discrete model in various situations. In addition, they derive a continuum description to perform some analytical computations.

The strength of this analysis relies clearly on showing that simple dynamics can lead to tissue-wide PCP even in absence of a gradient in protein expression. A number of phenomena observed in tissues are qualitatively reproduced. In two spatial dimensions, they find swirling patterns that resemble patterns found in tissues when a global gradient is absent. The model also captures qualitative effects due to the down-regulation of one of the PCP proteins in a certain region of the tissue.

The main weak point is that, from a physical point of view, the findings are not particularly surprising. Furthermore, some assumptions underlying the model, need some more justification. This holds notably for the question, of why additive noise is appropriate to account for the effect of stochasticity in the attachment-detachment dynamics of the proteins. Finally, the authors consider a situation that they consider to be one of the most interesting features of PCP, namely, the formation of PCP in presence of a region with a down-regulated PCP protein and in presence of a gradient. Unfortunately, the effect is not very clear and the data provided remains limited.

Reviewer #3 (Public Review):

This study aims to discover ethologically relevant feature selectivity of mouse retinal ganglion cells. The authors took an innovative approach that uses large-scale calcium imaging data from retinal ganglion cells stimulated with both artificial and natural visual stimuli to train a convolutional neural network (CNN) model. The resulting CNN model is able to predict stimuli that maximally excite individual ganglion cell types. The authors discovered that modeling suggests that the "transient suppressed-by-contrast" ganglion cells are selectively responsive to Green-Off, UV-On contrasts, a feature that signals the transition from the ground to the sky when the animal explores the visual environment. They tested this hypothesis by measuring the responses of these suppressed-by-contrast cells to natural movies, and showed that these cells are preferentially activated by frames containing ground-to-sky transitions and exhibit the highest selectivity of this feature among all ganglion cell types. They further verified this novel feature selectivity by single-cell patch clamp recording.

This work is of high impact because it establishes a new paradigm for studying feature selectivity in visual neurons. The data and analysis are of high quality and rigor, and the results are convincing. Overall, this is a timely study that leverages rapidly developing AI tools to tackle the complexity of both natural stimuli and neuronal responses and provides new insights into sensory processing.

Reviewer #3 (Public Review):

This manuscript uses ASO to inhibit the self-cleaving ribozyme within CPEB intron 3 and test its effect on CPEB3 expression and memory consolidation. The authors conclude that the intronic ribozyme negatively affects CPEB3 mRNA splicing and expression, and suggests its implications for experience-induced gene expression underlying learning and memory.<br /> The strength of the manuscript is in its exploration of a potentially novel mechanism of regulating CPEB3 expression in learning and memory, a combination of both biochemical and behavioral approaches to gain a wide perspective of this regulatory mechanism, and the application of ASO in this context. The introduction is sufficiently detailed. Statistics are thorough and appropriate. If the results could be more robust, the mechanism would provide a novel target and venue to modify learning and memory paradigm.<br /> The weakness of the manuscript is that the magnitude of the activity-dependent regulation of ribozyme, the effects of ASOs on CPEB3 expression (mRNA and protein) and downstream target gene expression, in vitro and in vivo, are generally weak, raising concerns about the robustness of the result. This may have caused some of the inconsistencies between the data presentation (see below). Also unclear is whether the ribozyme activity is physiologically regulated by experience without ASO interference.<br /> While the statistics tests support corresponding figure panels and their conclusions. The manuscript can be significantly strengthened by additional evidence, clarification of some methodologies, and reconciling some inconsistent results.<br /> The premise of a comparable timescale between transcription and ribozyme activity as the foundation of the whole thesis was based on in vitro measurement of self-scission half-life and a broadly generalized transcription rate (which actually varies significantly between genes). This premise is weak and needs direct experimental support.<br /> The physiological relevance of the proposed mechanism has yet to be demonstrated without ASO interference.<br /> Fig2b: how were total and uncleaved Ribozymes measured by qRT-PCR? Where are the primers' locations? If the two products were amplified using different primers, their subtraction to derive % cleavage would not be appropriate.<br /> Line 400-403: shouldn't ribozyme-blocking ASO prevent ribozyme self-cleavage, and as a result should further increase ribozyme levels? This would contradict the result in fig3a.

Reviewer #3 (Public Review):

The potential for sexual selection and the extent of sexual dimorphism in gene expression have been studied in great detail in animals, but hardly examined in plants so far. In this context, the study by Zhao, Zhou et al. al represents a welcome addition to the literature.

Relative to the previous studies in Angiosperms, the dataset is interesting in that it focuses on reproductive rather than somatic tissues (which makes sense to investigate sexual selection), and includes more than a single developmental stage (buds + mature flowers).

Reviewer #3 (Public Review):

This important study by Chen et al help in advancing our knowledge about the regulation of inner hair cell (IHC) development and revealed the role of Cldn9 in IHC embryonic and postnatal induction by transdifferentiation from the supporting cells. The authors developed an inducible doxycycline (dox)-tet-OFF-Cldn9 transgenic mice to regulate expression levels of Cldn9 and show that downregulation of Cldn9 resulted in additional, although incomplete row of IHCs immediately adjacent to the original IHC row. These induced extra IHCs had similar well developed hair bundles, able to mechanotransduce and were innervated by auditory neurons resembling wild-type IHCs. In addition, the authors knock down Cldn9 postnatally using shRNA injections in P1-7 mice with similar induction of extranumerary IHC next to the original row of IHCs. The conclusions of this paper are mostly well supported by the data, but some data analysis needed to be clarified and some crucial controls should be provided to improve the confidence in the presented results. There is a great potential for practical use of these valuable findings and new knowledge on IHC developmental regulation to design Cldn9 gene therapy in the future.<br /> The described by Chen et al mechanisms of extra hair cell generation by suppression of the tight junction protein Cldn9 expression level are very interesting and previously unknown. In particular, the generation of extra IHCs postnatally using downregulation of Cldn9 by shRNA could potentially be very useful as a replacement of HCs lost after noise-induced trauma, ototoxic agents, or other environmental trauma. On the other hand, the replacement of lost hair cells due to various genetic mutations by inducing a supernumerary IHCs with the same abnormalities would not be reasonable.<br /> The authors show that postnatally generated ectopic IHCs are viable and mechanotransducive, but it would be nice to show the maturation steps of ectopic IHC during this postnatal period. For example, stereocilia bundles of the ectopic hair cells should mature later than the original IHCs. A few days after viral delivery of shRNA, you should be able to observe immature IHC bundles that unequivocally will define newly generated IHCs. Unfortunately, the authors show only examples of already mature ectopic IHCs at P21 and in 5-6 weeks old mice and at relatively low resolution. Also, during maturation, IHCs usually have transient axo-somatic synapses that are not present in mature IHCs. It would be great to see if, in 5-6 weeks old mouse, the ectopic IHCs still have axo-somatic synapses or not, and if the majority of the ectopic IHCs have innervation. Some of the data in this study would benefit from showing corresponding controls and some - from higher resolution imaging.<br /> In the mammalian cochlea, each HC is separated from the next by intervening supporting cells, forming an invariant and alternating mosaic along the cochlea's length. Cochlear supporting cells in some conditions can divide and trans-differentiate into HCs, serving as a potential resource for HC differentiation, using transcription and other developmental signaling factors.<br /> However, when ectopic hair cells are generated from supporting cell trans-differentiation, the intricate mosaic of the organ of Corti is altered, which could by itself lead to hearing issues. In case of downregulation of Cldn9, the extra row of IHCs seems to be positioned immediately adjacent to the original IHC row. It is not clear if the newly formed unusual junctions between the ectopic and original IHCs are sufficiently tight to prevent leakage of the endolymph to the basolateral surface of IHCs. Also, it is not clear if the other organ of Corti tight junctions could lose their tightness due to the downregulation of Cldn9, which could over time affect the endocochlear potential as shown by this study and hearing abilities.<br /> Importantly, CLDN9 immunofluorescence staining data that show cytoplasmic staining of supporting cells should be revisited and the organ of Corti schematics showing CLDN9 expression should be corrected, considering that CLDN9 localizes to the tight junctions of the reticular lamina as was shown by immunoEM in this study and described in previous publications (Kitajiri et al., 2004; Nakano et al., 2009, Ramzan et al., 2021).<br /> While the current version of the manuscript will be of interest to scientists working in the inner ear development and regeneration field, it could be more valuable to the hearing researchers outside this immediate field and perhaps developmental biologists and cell biologists after proper revision.

Reviewer #3 (Public Review):

In this study, the authors were assessing the role of double global knockout of substance P and CGPRα on the transmission of acute and chronic pain. The authors first generated the double knockout (DKO) mice and validated their animal model. This is then followed by a series of acute and chronic pain assessments to evaluate if the global DKO of these neuropeptides are important in modulating acute and chronic pain behaviors. Authors found that these DKO mice Substance P and CGRPα are not required for the transmission of acute and chronic pain although both neuropeptides are strongly implicated in chronic pain. This study does provide more insight into the role of these neuropeptides on chronic pain processing, however, more work still needs to be done. (see the comments below).

1. In assessing the double KO (result #1), why are different regions of the brains shown for substance P and CGRPα (for example, midbrain for substance P and amygdala for CGRPα)? Since the authors mentioned that these peptides co-expressed in the brain (as in the introduction), shouldn't the same brain regions be shown for both IHC? It would be ideal if the authors could show both regions (midbrain and amygdala) in addition to the DRG and spinal cord for both peptides in their findings.<br /> In addition, since this is double KO, the authors should show more representative IHC-stained brain regions (spanning from the anterior to posterior).<br /> 2. It is also unclear as to why the authors only assessed the loss of substance P signaling in the double KO mice. Shouldn't the same be done for CGRPα signaling? Either the authors assess this, or the authors have to provide clear explanations as to why only substance P signaling was assessed.<br /> 3. Has these animal's naturalistic behavior been assessed after the double KO (food intake, sleep, locomotion for example)? I think this is important as changes to these naturalistic behaviors can affect pain processes or outcomes.<br /> 4. Figure 2H: The authors acknowledge that there is a trend to decrease with capsaicin-evoked coping-like responses. However, a close look at the graph suggests that the lack of significance could be driven by 1 mouse. Have the authors run an outlier test? Alternatively, the authors should consider adding more n to these experiments to verify their conclusions.<br /> 5. Similarly, the values for WT in the evoked cFos activity (Figure 2- Suppl Figure 1) are pretty variable. Considering that the n number is low (n = 5), authors should consider adding more n.<br /> Also, since the n number is low in this experiment (eg. 5 vs 4), does this pass the normality test to run a parametric unpaired t-test? Either the authors increase their n numbers or run the appropriate statistical test.<br /> 6. In most of the results, authors ran a parametric test despite the low n number. Authors have to ensure that they are carrying out the appropriate statistical test for their dataset and n number.<br /> 7. Along the same line of comment with the previous, authors should increase the n number for DKO for staining (Figure 4) as n number is only 3 and there is variability in the cFos quantification in the ipsilateral side.<br /> 8. Authors should provide references for statement made in Line 319-321 as authors mentioned that there are accumulating evidence indicating that secretion of these neuropeptides from nociceptor peripheral terminals modulates immune cells and the vasculature in diverse tissues.<br /> 9. Authors state that the sample size used was similar to those from previous studies, but no references were provided. Also, even though the sample sizes used were similar, I believe that the right statistic test should be used to analyze the data.<br /> 10. In the discussion, the authors noted that knocking out of a gene remains the strongest test of whether the molecule is essential for a biological phenomenon. At the same time, it was acknowledged that Substance P infusion into the spinal cord elicits pain, but it is analgesic in the brain. The authors might want to expand more on this discussion, including how we can selectively assess the role of these neuropeptides in areas of interest. For example, knocking out both Substance P and CGRPα in selected areas instead of the global KO since there are reported compensatory effects.

Reviewer #3 (Public Review):

Summary:<br /> Pesnot Lerousseau and Summerfield aimed to explore how humans generalize abstract patterns of sensory data (concepts), focusing on whether and how spatial representations may facilitate the generalization of abstract concepts (rotational invariance). Specifically, the authors investigated whether people can recognize rotated sequences of stimuli in both spatial and nonspatial domains and whether spatial pre-training and multi-modal mapping aid in this process.

Strengths:<br /> The study innovatively examines a relatively underexplored but interesting area of cognitive science, the potential role of spatial scaffolding in generalizing sequences. The experimental design is clever and covers different modalities (auditory, visual, spatial), utilizing a two-dimensional feature manifold. The findings are backed by strong empirical data, good data analysis, and excellent transparency (including preregistration) adding weight to the proposition that spatial cognition can aid abstract concept generalization.

Weaknesses:<br /> The examples used to motivate the study (such as "tree" = oak tree, family tree, taxonomic tree) may not effectively represent the phenomena being studied, possibly confusing linguistic labels with abstract concepts. This potential confusion may also extend to doubts about the real-life applicability of the generalizations observed in the study and raises questions about the nature of the underlying mechanism being proposed.

Next, the study does not explore whether scaffolding effects could be observed with other well-learned domains, leaving open the question of whether spatial representations are uniquely effective or simply one instance of a familiar 2D space, again questioning the underlying mechanism.

Further doubt on the underlying mechanism is cast by the possibility that the observed correlation between mapping task performance and the adoption of a 2D strategy may reflect general cognitive engagement rather than the spatial nature of the task. Similarly, the surprising finding that a significant number of participants benefited from spatial scaffolding without seeing spatial modalities may further raise questions about the interpretation of the scaffolding effect, pointing towards potential alternative interpretations, such as shifts in attention during learning induced by pre-training without changing underlying abstract conceptual representations.

Conclusions:<br /> The authors successfully demonstrate that spatial training can enhance the ability to generalize in nonspatial domains, particularly in recognizing rotated sequences. The results for the most part support their conclusions, showing that spatial representations can act as a scaffold for learning more abstract conceptual invariances. However, the study leaves room for further investigation into whether the observed effects are unique to spatial cognition or could be replicated with other forms of well-established knowledge, as well as further clarifications of the underlying mechanisms.

Impact:<br /> The study's findings are likely to have a valuable impact on cognitive science, particularly in understanding how abstract concepts are learned and generalized. The methods and data can be useful for further research, especially in exploring the relationship between spatial cognition and abstract conceptualization. The insights could also be valuable for AI research, particularly in improving models that involve abstract pattern recognition and conceptual generalization.

In summary, the paper contributes valuable insights into the role of spatial cognition in learning abstract concepts, though it invites further research to explore the boundaries and specifics of this scaffolding effect.

Reviewer #3 (Public Review):

Summary:<br /> Anderson at al utilize an array of orthogonal techniques to highlight the important of protein dynamics for the function and inhibition of the kinase ERK2. ERK2 is important for a large variety of biological functions.

Strengths:<br /> This is a thorough and detailed study that uses a variety of techniques to identify critical molecular/chemical parameters that drive ERK2 in specific states.

Weaknesses:<br /> No details rules were identified so that novel inhibitors could be designed. Nevertheless, the mode of action of these existing inhibitors are much better defined.

Reviewer #3 (Public Review):

The authors present the mechanism, validation, and modular application of LOVtag, a light-responsive protein degradation tag that is processed by the native degradosome of Escherichia coli. Upon exposure to blue light, the c-terminal alpha helix unfolds, essentially marking the protein for degradation. The authors demonstrate the engineered tag is modular across multiple complex regulatory systems, which shows its potential widespread use throughout the synthetic biology field. The step-by-step rational design of identifying the protein that was most dark-stabilized as well as most light-responsive for degradation, was useful in terms of understanding the key components of this system. The most compelling data shows that the engineered LOVTag can be fused to multiple proteins and achieve light-based degradation, without affecting the original function of the fused protein.

Reviewer #3 (Public Review):

Summary and Strength:

The manuscript by Amir et al. describes that Sertoli-specific inactivation of the mTORC1 and mTORC2 complex by KO of either Raptor or Rictor, respectively, resulted in progressive changes in blood-testis-barrier (BTB) function, testis weight, and sperm parameters, including counts, morphology, mtDNA content and sperm DNA methylation.

The described studies are based on the hypothesis that a decline of BTB function with increasing chronological age of a male contributes to the DNA methylation changes that are known to occur in sperm DNA of old males when compared to sperm DNA from isogenic young males. In order to demonstrate the relevance of a functioning BTB for the maintenance of sperm methylation patterns, the authors generated mice with genetically disrupted mTORC2 complex or mTORC1 complex in Sertoli cells and determined sperm methylation patterns in comparison to isogenic wild-type males. In line with previously published scientific literature (e.g. Mok et al., 2013; Dong et al, 2015; and others), the manuscript corroborates that a Sertoli-cell specific deletion of mTORC2 caused a loss of BTB function and a progressive spermatogenic defect. The authors further show that sperm DNA is differentially methylated (DMRs) as a consequence of either a mTORC2 disruption (associated with a loss of BTB function) or following a mTORC1 disruption (BTB function either increased or not leaky) when compared to their isogenic age-matched wt controls. Those DMRs overlap partially with changes in sperm DNA methylation that were found when comparing sperm from 8-week males with sperm isolated from 22-week-old male mice.

The authors interpret the observed changes as representative of the sperm DNA methylation changes that occur during normal chronological aging of the male. For an aged control group, the authors use sperm DNA of 22-week-old wild-type mates from the mTORC2 and mTORC2 KO breeding and compare the sperm methylation patterns found in sperm from those 22-week males to 8-week young males, that are intended to represent an old and a young cohort, respectively. DNA methylation analysis indicates that a disruption of mTORC2 (& decrease of BTB function) results in increased DNA methylation of sperm DNA, while a disruption of mTORC1 (and proposed increase of BTB tightness, not shown in the manuscript, though) resulted in increased hypomethylation.

Weaknesses:

While the hypothesis and experimental system are interesting and the data demonstrating the relevance of the mTORC2 complex for BTB function is convincing, several open questions limit the evidence that supports the hypothesis that the sperm DNA methylation changes seen in old males are caused by BTB failure following an imbalance of mTOR signaling complexes. The major critique points are the lack of a chronologically old group and the choice of 8 weeks & 22 weeks age of age:

- Data illustrating the degree of BTB decline and sperm DNA methylation changes from chronologically "old" male mice is missing. 22-week-old mice are not considered old but are of good and mature breeding age, equivalent to humans in their mid-late twenties. (In the manuscript, the 22-week-old wildtype mice show no evidence of BTB breakdown (Figure 3), so why are their sperm used to represent "aged" sperm?

- Adding a group of "old" wild-type mice of 12-14 months of age, which is closer to the end of effective reproduction in mice, more equivalent to 45-59 year-old humans) could be used to illustrate that (a) aging causes a marked decrease in BTB function at this time in mouse life, and that this BTB breakdown chronologically aligns with the age-associated DNA hypermethylation seen in old sperm. Age-matched "old" mTORC1 KO, with a (supposedly) tighter BTB barrier, could then be expected to have a sperm DMA methylation profile closer to that of younger wild-type animals. Such data are currently missing. While the progressive testicular decline observed in the mTORC1 KO (Fig.5) could make it difficult to obtain the appropriately aged mTORC1 KO tissues, it is completely feasible to obtain data from chronologically old wild-type males. (The progressive testicular decline further raises the question of what additional defects the KO causes, and how such additional defects would influence the sperm DNA methylation profile.) The addition of data from an old group to the currently included groups could strengthen the interpretation that the observations in the BTB-defective mTORC2 KO mice are modelling an age-related testicular decline, provided that the DMRs seen in the chronologically old group significantly overlap with the BTB-defective changes.

- In the current form, the described differences in sperm DNA methylation are based on comparisons between pubertal mice (8 weeks) and mature but not old adult males (22 weeks), while a chronologically "old" group is missing from the data sets and comparisons. Thus, it appears that the described sperm methylation changes reflect developmental changes associated with normal maturation and not necessarily declining sperm quality due to aging. (Sperm obtained from 8-week-old mice likely were generated, at least in part, during the 1st wave of spermatogenesis, which is known to differ from the continuously proceeding spermatogenesis during the remained of the mature life. During the 1st wave of spermatogenesis, Sertoli cells are known to undergo gene expression changes which could contribute to varying degrees of BTB function, and thus have effects on the sperm DNA methylation profiles of such 1st wave sperm.)

- It is unclear why the aging-related DMRs between the 8 and 22-week-old wild-type mice vary so dramatically between the two wild-type groups derived from the mTORC1 and the mTORC2 breeding (Fig. S4). If the main difference was due to mTORC1 or mTORC2 activity, both wildtype groups should behave very similarly. Changes seen in a truly "old" mouse (e.g. 20 weeks to 56 weeks), changes in "young mTORC1" and in "old mTORC2" are missing. How do those numbers and profiles compare to the shown samples?

Some general comments regarding the chosen age of animals:

- As mentioned, sperm from 8-week-old mice represent many sperm that were produced in the 1st wave of spermatogenesis; 22-week-old mice are not considered chronologically old mice, but mature and "relatively" young animals. 18-24 month-old mice are considered to be equivalent to 56-69 year-old humans, and might be more suitable to detect aging effects. "Old mice" for study purposes should be at least 12-14 months of age, ideally >18 months of age. 22 weeks (5 months of age) are mice at good breeding age, but still considered mature adults, not old males, and therefore are not expected to show typical aging health problems (like declining fertility).

Even the cited reference (Flurkey et al. 2007) defines that "... mice used a reference group for "young mice" should be at least 3 months of age (~ 13 weeks), i.e. fully sexually mature. The authors specifically state: " The young adult group should be at least 3 months old because, although mice are sexually mature by 35 days, relatively rapid maturational growth continues for most biologic processes and structures until about 3 months. The upper age range for the young adult group is typically about 6 months. ... For the middle-aged group, 10 months is typically the lower limit.... The upper age limit for the middle-aged group is typically 14-15 months, because at this age, most biomarkers still have not changed to their full extent, and some have not yet started changing. For the old group, the lower age limit is 18 months because age-related change for almost all biomarkers of aging can be detected by then. The upper limit is 22-26 months, depending on the genotype." According to this reference, mice up to 6 months of age are generally considered "mature adults" (equivalent to humans 20-30 yrs), mice of 10-14 month are "middle-aged adults" (equivalent to ~38-47 human years) and 18-24 month mice are "old" (equivalent to human of 56-69 yrs.).

Going on these commonly used age ranges, it is unclear why the authors used 8-week-old mice (generally considered pubertal to late adolescent age) as young mice and 5-month-old mice as "old mice".

Differences seen between these cohorts most likely do not reflect aging, but more likely reflect changes associated with normal developmental maturation, since testis and epididymides continue to grow until about 10-11 weeks of age.

- The DMRs identified between 8 and 22-week-old animals could represent DMRs that are dependent on developmental maturation more than being changed in an "age-dependent" manner (in the sense of increased chronological age). This interpretation is congruent with the fact that those DMRs are enriched for developmental categories.

Ron White recommends taking notes on 3 x 5 inch index cards. One should place the Dewey Decimal or Library of Congress catalog number in the upper left of their bibliography card and in the upper right corner one should number their cards consecutively (1, 2, 3, etc.). White indicates the importance of these numbers is primarily that they are unique, presumably so one can refer to them or reorder them if they are put out of order. (p46-7)

https://web.archive.org/web/20060422014759/http://that4.2ch.net/test/read.cgi/stationery/1021438965/l50

https://web.archive.org/web/20060422014759/http://that4.2ch.net/test/read.cgi/stationery/1021438965/l50

the 2ch site that inspired Hawk Sugano's Pile of Index Cards method

ᔥ[[hawkexpress]] in How to link between Cards

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Ma et al. describes a multi-model (pig, mouse, organoid) investigation into how fecal transplants protect against E. coli infection. The authors identify A. muciniphila and B. fragilis as two important strains and characterize how these organisms impact the epithelium by modulating host signaling pathways, namely the Wnt pathway in lgr5 intestinal stem cells.

Strengths:<br /> The strengths of this manuscript include the use of multiple model systems and follow-up mechanistic investigations to understand how A. muciniphila and B. fragilis interacted with the host to impact epithelial physiology.

Weaknesses:<br /> The major weakness is that, as presented, the manuscript is quite difficult to follow, even for someone familiar with the field. The lack of detail in figure legends, organization of the text, and frequent use of non-intuitive abbreviated group names without a clear key (ex. EP/EF, or C E A B) make comprehension challenging. The results section is perhaps too succinct and does not provide sufficient information to understand experimental design and interpretation without reading the methods section first or skipping to the discussion (as an example: WNT-c59 treatment). Extensive revisions could be encouraged to aid in communicating the potentially exciting findings.

The bioinformatics section of the methods requires revision and may indicate issues in the pipeline. Merging the forward and reverse reads may represent a problem for denoising. Also since these were sequenced on a NovaSeq, the error learning would have to be modified or the diversity estimates would be inappropriately multiplied. "Alpha diversity and beta diversity were calculated by normalized to the same sequence randomly." Not sure what this means, does this mean subsampled? "Blast was used for sequence alignment", does this mean the taxonomic alignment? This would need to be elaborated on and database versions should be included. The methods, including if any form of multiple testing was included, for LEFSE was also not included.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript coins a term "the census population size" which they define from the diversity of malaria parasites observed in the human community. They use it to explore changes in parasite diversity in more than 2000 people in Ghana following different control interventions.

Strengths:<br /> This is a good demonstration of how genetic information can be used to augment routinely recorded epidemiological and entomological data to understand the dynamics of malaria and how it is controlled. The genetic information does add to our understanding, though by how much is currently unclear (in this setting it says the same thing as age-stratified parasite prevalence), and its relevance moving forward will depend on the practicalities and cost of the data collection and analysis. Nevertheless, this is a great dataset with good analysis and a good attempt to understand more about what is going on in the parasite population.

Weaknesses:<br /> Overall the manuscript is well-written and generally comprehensively explained. Some terms could be clarified to help the reader and I had some issues with a section of the methods and some of the more definitive statements given the evidence supporting them.

Reviewer #3 (Public Review):

We commend the authors on addressing our points and do believe that the manuscript is much improved. Even with the added in vitro data (Figure 8, Supplementary Figure 6), however, a clear mechanistic explanation for how MCT1 is modulating/inhibiting fibrosis in hepatic stellate cells is lacking and this represents a key area for future exploration. The authors provide interesting follow up experiments that suggest lactate can potentiate TGF-β1 signaling, a phenomenon that has previously been described in pulmonary fibrosis. Additionally, MCT1 depletion decreased the pSMAD3/SMAD ratio, but this was overcome with higher doses of TGB- β1 ligand. It remains unclear how intracellular versus extracellular lactate is signaling to exert the observed effects, and how the altered metabolism/metabolic flux in NAFLD is contributing to organ level metabolic dysregulation. These will be keys questions to answer going forward potentially using the novel in vivo models that the authors have contributed here.

A major finding of this work is that loss of monocarboxylate transporter 1 (MCT1), specifically in stellate cells, can decrease fibrosis in the liver. However, the underlying mechanism whereby MCT1 influences stellate cells is not addressed. It is unclear if upstream/downstream metabolic flux within different cell types leads to fibrotic outcomes. Ultimately, the paper opens more questions than it answers: why does decreasing MCT1 expression in hepatocytes exacerbate disease, while silencing MCT1 in fibroblasts seems to alleviate collagen deposition? Mechanistic studies in isolated hepatocytes and stellate cells could enhance the work further to show the disparate pathways that mediate these opposing effects. The work highlights the complexity of cellular behavior and metabolism within a disease environment but does little to mechanistically explain it.

Reviewer #3 (Public Review):

Summary:<br /> In this study, McAllester and Pool develop a new simulation model to explain the maintenance of balanced inversion polymorphism, based on (sexually) antagonistic alleles and a trade-off between male reproduction and survival (in females or both sexes). In support of the plausibility of this model, the authors use laboratory experiments on four naturally occurring inversion polymorphisms in Drosophila melanogaster, finding evidence for the existence of the above-mentioned trade-off in two out of the four cases.

Strengths:<br /> 1. The study develops and analyzes a new (Drosophila melanogaster-inspired) model for the maintenance of balanced inversion polymorphism, combining elements of (sexually) antagonistically (pleiotropic) alleles, negative frequency-dependent selection, and synergistic epistasis. To this end, the authors developed and used a new simulator (although it was not 100% clear as to why SLiM could not have been used as SLiM has been used to study inversions).

2. The above-mentioned model assumes, as a specific example, a trade-off between male reproductive display and survival; in the second part of their study, the authors perform laboratory experiments on four common D. melanogaster inversions to study whether these polymorphisms may be subject to such a trade-off. The authors find that two of the four inversions show suggestive evidence that is consistent with a trade-off between male reproduction and survival. The new amplicon sequencing approach to track inversion frequencies used by the authors seems promising in terms of studying fitness effects/trade-offs associated with polymorphic inversions and how such effects play out dynamically.

Weaknesses:<br /> 1. Mechanisms of balancing selection maintaining balanced inversion polymorphism. In Section 1.1 a better and more accurate overview of the different selective mechanisms that might contribute to the maintenance of balanced inversion polymorphisms should be given (for a recent review see Berdan et al. 2023). For example, negative frequency-dependent selection (NFDS), spatially and temporally varying selection are not mentioned here and are brought up only later, which is not really ideal. While I agree that in most cases our understanding of balanced inversion polymorphism is very limited, there are many empirical examples of these and other mechanisms of balancing selection being at play (e.g., NFDS: Wright & Dobzhansky 1946; Nassar et al. 1973; Álvarez-Castro and Álvarez 2005; Jay et al. 2021; and many examples of evidence for other mechanisms as well). Thus, while the prevalence of inversion polymorphisms is indeed in many cases "enigmatic", the reader should not be given the impression that we do not have yet any empirical evidence for specific mechanisms in particular cases. Similarly, the authors mention the classical (essentially Dobzhansky's) scenario of epistatically interacting loci only in passing, even though this coadaptation scenario may be simpler than the local adaptation mechanism of Kirkpatrick & Barton (2016): in its simplest form, epistatic coadaptation does not require any migration load or locally adaptive alleles à la Kirkpatrick & Barton, but just the capture of 2 overdominant loci, with inversion protecting this fittest double heterozygote from recombination load (Charlesworth 1974; also see Charlesworth & Charlesworth 1974; Charlesworth & Flatt 2021; also see discussion in Charlesworth and Barton 2018). On the other hand, the plausibility of the mutational load/associative overdominance (AOD) mechanism (Sturtevant & Mather 1938; Nei et al. 1967; Ohta 1971) seems to be given too much weight: new work by Charlesworth (2023; https://www.biorxiv.org/content/10.1101/2023.10.16.562579v1) suggests that load likely contributes only very modestly to heterokaryotypic advantage of inversions at intermediate frequencies, and that is very unlikely to provide a sufficient selective (heterotic) advantage to new autosomal inversions in order to explain their establishment (also see Nei et al. 1967; Connallon and Olito 2021; Jay et al. 2022).

2. The general reduction principle and inversion polymorphism. In Section 1.2., the authors state that "there has not been a proposed mechanism whereby alleles at multiple linked loci would directly benefit from linkage and thereby maintain an associated inversion polymorphism under indirect selection." Perhaps I am misunderstanding something, but in my reading, this statement is factually incorrect. In fact, the simplest version of Dobzhansky's epistatic coadaptation model (see Charlesworth 1974; also see Charlesworth and Charlesworth 1973 and discussion in Charlesworth & Flatt 2021; Berdan et al. 2023) seems to be an example of exactly what the authors seem to have in mind here: two loci experiencing overdominance, with the double heterozygote possessing the highest fitness (i.,e., 2 loci under epistatic selection, inducing some degree of LD between these loci), with subsequent capture by an inversion; in such a situation, a new inversion might capture a haplotype that is present in excess of random expectation (and which is thus fitter than average). The selective benefits of recombination suppression in the inversion heterokaryotype will then confer a heterozygote advantage to the inversion and prevent it from going to fixation (see Charlesworth 1974). This is probably the simplest (or one of the simplest) models of multilocus balancing selection that can act on inversions. Incidentally, this model represents a prime example of the "reduction principle", which the authors mention on two occasions in their paper: generally, any multi-locus polymorphism held at equilibrium by any type of balancing selection involving fitness epistasis will cause selection for reduced recombination (e.g., Feldman & Liberman, 1986; Zhivotovsky et al., 1994); notably, the example of inversion polymorphism is explicitly discussed in Altenberg's and Feldman's (1987) paper on the reduction principle. It is also noteworthy in this context that the 2-locus epistatic model of Charlesworth (1974) assumes constant fitness values/selection coefficients but actually leads to what one could call "apparent" frequency-dependent selection with different equilibria.

3. Trade-offs and antagonistic pleiotropy involved in maintaining inversions. Throughout the manuscript, previous work implicating trade-offs and/or antagonistic pleiotropy (AP) in the maintenance of inversion polymorphisms should be more adequately acknowledged and discussed - from the text as it currently stands one gains the impression that barely anything is known about the connection between inversions and pleiotropy/AP/trade-offs (e.g., Betrán et al. 1998; Mérot et al. 2020, which is cited but not really in the context of AP/trade-offs; Pei et al. 2023, etc.). The paper by Pei and colleagues is particularly relevant in the context of the present study: the authors find that the inverted allele has beneficial effects on male siring success and female fecundity but negative effects on survival. Generally, numerous studies have found that inversion polymorphisms have "pleiotropic" (albeit not always antagonistically pleiotropic) effects upon multiple fitness components (e.g., Etges 1989; Betrán et al. 1998; Küpper et al. 2016; Durmaz et al. 2018; Mérot et al. 2020). More broadly, the general role of AP in maintaining (life-history) polymorphisms should be mentioned by referring to previous theories (e.g., Rose 1982, 1985; Curtsinger et al. 1994; Charlesworth & Hughes 2000 chapter in Lewontin Festschrift; Conallon & Chenoweth 2019 - this latter paper is particularly relevant in terms of AP effects in the context of sexual antagonism).

4. Sexually antagonistic selection and inversion polymorphism. The authors' model of sexual antagonism being involved in maintaining an inversion polymorphism is novel and interesting, but again I felt that the authors' ideas could be better connected to what has been done before. First, several papers have made connections between sexual antagonism and inversion polymorphisms: for example, an important study that deserves discussion in this context is the paper by Natri and colleagues (2019) where the authors study sexual antagonism as a source of balancing selection that maintains an inversion polymorphism in the ruff. Similarly, another relevant study in this context is Hearn et al. 2022 on Littorina saxatilis snails. Also see Giraldo-Deck et al. (2022). A very interesting paper that may be worth discussing is Connallon & Chenoweth (2019) about dominance reversals of antagonistically selected alleles (even though C&C do not discuss inversions): AP alleles (with dominance reversals) affecting two or more life-history traits provide one example of such antagonistically selected alleles (also see Rose 1982, 1985; Curtsinger et al. 1994) and sexually antagonistically selected alleles provide another. The two are of course not necessarily mutually exclusive, thus making a conceptual connection to what the authors model here.

5. The model. In general, the description of the model and of the simulation results was somewhat hard to follow and vague. There are several aspects that could be improved: (1) it would help the reader if the terminology and distinction of inverted vs. standard arrangements and of the three karyotypes would be used throughout, wherever appropriate. (2) The mention of haploid populations/situations and haploid loci (e.g., legend to Figure 1) is somewhat confusing: the mechanism modelled here, of course, requires suppressed recombination in the inversion/standard heterokaryotype; and thus, while it may make sense to speak of haplotypes, we're dealing with an inherently diploid situation. (3) The authors have a situation in mind where the 2 karyotypes (INV vs. STD) in the heterokaryotype carry distinct sets of loci in LD with each other, with one karyotype/haplotype carrying antagonistic variants favoring high male display success and with the other karyotype/haplotype carrying non-antagonistic alternative alleles at these loci and which favor survival. Thus, at each of the linked loci, we have antagonistic alleles and non-antagonistic alleles - however, the authors don't mention or discuss the degree of dominance of these alleles. The degree of dominance of the alleles could be an important consideration, and I found it curious that this was not mentioned (or, for that matter, examined). (4) In many cases, the authors do not provide sufficient detail (in the main text and the main figures) about which parameter values they used for simulations; the same is true for the Materials & Methods section that describes the simulations. Conversely, when the text does mention specific values (e.g., 20N generations, 0.22-0.25M, etc.), little or no clear context or justification is being provided. (5) The authors sometimes refer to "inversion mutation(s)" - the meaning of this terminology is rather ambiguous.

6. Throughout the manuscript, especially in the description and the discussion of the model and simulations, a clearer conceptual distinction between initial "capture" and subsequent accumulation / "gain" of variants by an inversion should be made. This distinction is important in terms of understanding the initial establishment of an inversion polymorphism and its subsequent short- as well as long-term fate. For example, it is clear from the model/simulations that an inversion accumulates (sexually) antagonistic variants over time - but barely anything is said about the initial capture of such loci by a new inversion.

Reviewer #3 (Public Review):

The manuscript by Li et al. presents an elegant application of sterile insect technology (pgSIT) utilizing a CRISPR-Cas9 system to suppress mosquito vector populations. The pgSIT technique outlined in this paper employs a binary system where Cas9 and gRNA are conjoined in experimental crosses to yield sterile male mosquitoes. Employing a multiplexed strategy, the authors combine multiple gRNA to concurrently target various genes within a single locus. This approach successfully showcases the disruption of three distinct genes at different genomic positions, resulting in the creation of highly effective sterile mosquitoes for population control. The pioneering work of the Akbari lab has been instrumental in developing this technology, previously demonstrating its efficacy in Drosophila and Aedes aegypti.

By targeting the female-specific splice isoform (exon-5) of doublesex in conjunction with intersex and β-tubulin, the researchers induce female lethality, leading to a predominance of sterile male mosquitoes. This innovation is particularly noteworthy as the deployment of sterile mosquitoes on a large scale typically requires substantial investment in sex sorting. However, this study circumvents this challenge through genetic manipulation.

Reviewer #3 (Public Review):

The authors use a set of reporter assays to probe the impact of TnpB on IS605 transposition. This is an innovative approach to studying transposition that will be of interest to other groups. The authors conclude that TnpB likely has two activities: (ii) promoting "homing", where the transposon is restored following excision, and (ii) promoting the transposition activity of the transposase TnpA. The paper provides an independent validation of the recently reported homing activity of TnpB, where the transposon is restored following excision, and suggests an additional function for TnpB in enhancing the transposase activity of the TnpA transposase.

Strengths<br /> - The innovative use of reporter assays is an excellent way to infer the details of transposition, making a convincing case that TnpB plays two distinct roles.

Weaknesses<br /> - The authors need to discuss their conclusions in light of a very relevant recent paper from the Sternberg group demonstrating a role for TnpB in homing.<br /> - There doesn't appear to be an assessment of how well the model fits the experimental data, or any attempt to test how accurately the model can predict the effects of perturbing the system.<br /> - The figures could be presented more clearly.

Reviewer #3 (Public Review):

George et al. present a convincing new Python toolbox that allows researchers to generate synthetic behavior and neural data specifically focusing on hippocampal functional cell types (place cells, grid cells, boundary vector cells, head direction cells). This is highly useful for theory-driven research where synthetic benchmarks should be used. Beyond just navigation, it can be highly useful for novel tool development that requires jointly modeling behavior and neural data. The code is well organized and written and it was easy for us to test.

We have a few constructive points that they might want to consider.

- Right now the code only supports X,Y movements, but Z is also critical and opens new questions in 3D coding of space (such as grid cells in bats, etc). Many animals effectively navigate in 2D, as a whole, but they certainly make a large number of 3D head movements, and modeling this will become increasingly important and the authors should consider how to support this.

- What about other environments that are not "Boxes" as in the name - can the environment only be a Box, what about a circular environment? Or Bat flight? This also has implications for the velocity of the agent, etc. What are the parameters for the motion model to simulate a bat, which likely has a higher velocity than a rat?

- Semi-related, the name suggests limitations: why Rat? Why Not Agent? (But its a personal choice)

- A future extension (or now) could be the ability to interface with common trajectory estimation tools; for example, taking in the (X, Y, (Z), time) outputs of animal pose estimation tools (like DeepLabCut or such) would also allow experimentalists to generate neural synthetic data from other sources of real-behavior.

- What if a place cell is not encoding place but is influenced by reward or encodes a more abstract concept? Should a PlaceCell class inherit from an AbstractPlaceCell class, which could be used for encoding more conceptual spaces? How could their tool support this?

- This a bit odd in the Discussion: "If there is a small contribution you would like to make, please open a pull request. If there is a larger contribution you are considering, please contact the corresponding author3" This should be left to the repo contribution guide, which ideally shows people how to contribute and your expectations (code formatting guide, how to use git, etc). Also this can be very off-putting to new contributors: what is small? What is big? we suggest use more inclusive language.

- Could you expand on the run time for BoundaryVectorCells, namely, for how long of an exploration period? We found it was on the order of 1 min to simulate 30 min of exploration (which is of course fast, but mentioning relative times would be useful).

- Regarding the Geometry and Boundary conditions, would supporting hyperbolic distance might be useful, given the interest in alternative geometry of representations (ie, https://www.nature.com/articles/s41593-022-01212-4)?

- In general, the set of default parameters might want to be included in the main text (vs in the supplement).

- It still says you can only simulate 4 velocity or head directions, which might be limiting.

- The code license should be mentioned in the Methods.

Reviewer #3 (Public Review):

Summary:<br /> The authors aim to demonstrate the effectiveness of their developed methodology, which utilizes super-resolution microscopy and single-molecule tracking in live cells on a high-throughput scale. Their study focuses on measuring the diffusion state of a molecule target, the estrogen receptor, in both ligand-bound and unbound forms in live cells. By showcasing the ability to screen 5067 compounds and measure the diffusive state of the estrogen receptor for each compound in live cells, they illustrate the capability and power of their methodology.

Strengths:<br /> Readers are well introduced to the principles in the initial stages of the manuscript with highly convincing video examples. The methods and metrics used (fbound) are robust. The authors demonstrate high reproducibility of their screening method (R2=0.92). They also showcase the great sensitivity of their method in predicting the proliferation/viability state of cells (R2=0.84). The outcome of the screen is sound, with multiple compounds clustering identified in line with known estrogen receptor biology.

Weaknesses:<br /> - Potential overstatement on the relationship of low diffusion state of ER bound to compound and chromatin state without any work on chromatin level.<br /> - Could the authors clarify if the identified lead compound effects are novel at any level?<br /> - More video example cases on the final lead compounds identified would be a good addition to the current data package.

Reviewer #3 (Public Review):

Summary of Author's Results/Intended Achievements<br /> The authors were trying to ascertain the underlying learning mechanisms and network structure that could explain their primary experimental finding: passive exposure to a stimulus (independent of when the exposure occurs) can lead to improvements in active (supervised) learning. They modeled their task with 5 progressively more complex shallow neural networks classifying vectors drawn from multi-variate Gaussian distributions.

Account of Major Strengths:<br /> Overall, the experimental findings were interesting. The modelling was also appropriate, with a solid attempt at matching the experimental condition to simplified network models.

Reviewer #3 (Public Review):

Complex behavior requires complex neural control involving multiple brain regions. The currently available tools to measure neural activity in multiple brain regions in small animals are limited and often involve obligatory head-fixation. The latter, obviously, impacts the behaviors under study. Hur and colleagues present a novel recording device, the E-Scope, that combines optical imaging of fluorescent calcium imaging in one brain region with high-density electrodes in another. Importantly, the E-Scope can be implanted and is, therefore, compatible with usage in freely moving mice. The authors used their new E-Scope to study neural activity during social interactions in mice. They demonstrate the presence of neural correlates of social interaction that happen simultaneously in the cerebellum and the anterior cingulate cortex.

The major accomplishment of this study is the development and introduction of the E-Scope. The evaluation of this part can be short: it works, so the authors succeeded.

The authors managed to reduce the weight of the implant to 4.5 g, which is - given all functionality - quite an accomplishment in my view. However, a mouse weighs between 20 and 40 g, so that an implant of 4.5 g is still quite considerable. It can be expected that this has an impact on the behavior and, possibly, the well-being of the animals. Whether this is the case or not, is not really addressed in this study. The authors suffice with the statement that "Recorded animals made more contact with the other mouse than with the object (Figure 2A), suggesting a normal preference for social contact with the E-Scope attached." A direct comparison between mice before and after implant, or between mice with and without an implant would provide more insight into the putative impact of the E-Scope on (social) behavior.

In Figure 1 D-G, the authors present raw data from the neurophysiological recordings. In panel D, we see events with vastly different amplitudes. It would be very insightful if the authors would describe which events they considered to be action potentials, and which not. Similarly, indicating the detected complex spikes in the raw traces of Figure 1E would provide more insight into the interpretation of the data. Although the authors mention to consider the occurrence of complex spikes and simple spikes, a clear definition of what is considered a single unit recording is lacking. As there is quite a wide range in reported firing rates in Figure 2 - figure supplement 3, more clarity on this aspect would be insightful. Furthermore, in their text, the authors state that the pause in simple spike firing following a complex spike normally lasts until around 40 ms, and for this statement they refer to Figure 1G that shows a pause of less than 10 ms.

The number of Purkinje cells recorded during social interactions is quite low: only 11 cells showed a modulation in their spiking activity (unclear whether in complex spikes, simple spikes or both. During object interaction, only 4 cells showed a significant modulation. Unclear is whether the latter 4 are a subset of the former 11, or whether "social cells" and "object cells" are different categories. Having so few cells, and with these having different types of modulation, the group of cells for each type of modulation is really small, going down to 2 cells/group. The small group sizes complicate the interpretation of the data - in particular also on the analysis of movement-related activity that is now very noisy (Figure 2 - figure supplement 4).

In conclusion, the authors present a novel method to record neural activity with single cell-resolution in two brain regions in freely moving mice. Given the challenges associated with understanding of complex behaviors, this approach can be useful for many neuroscientists. The authors demonstrate the potential of their approach by studying social interactions in mice. Clearly, there are correlations in activity of neurons in the anterior cingulate cortex and the cerebellum related to social interactions. To bring our understanding of these patterns to a higher level, more detailed analyses (and probably also larger group sizes of cerebellar neurons) are required, though.

Reviewer #3 (Public Review):

The authors report a study in which they use intracranial recordings to dissociate subjectively aware and subjectively unaware stimuli, focusing mainly on prefrontal cortex.

The authors have dealt successfully with some of my previous concerns, especially the more direct link to the Gaillard et al., (2009) paper, and the associated analyses, has improved the manuscript. Some of my other concerns regarding the theoretical embedding of the findings have only been partially mitigated and some interesting results derived from suggestions for additional analyses will be used for future papers.

Reviewer #3 (Public Review):

This study examines context-dependent stimulus selection by recording neural activity from several sensory and motor cortical areas along a sensorimotor pathway, including S1, S2, MM, and ALM. Mice are trained to either withhold licking or perform directional licking in response to visual or tactile stimulus. Depending on the task rule, the mice have to respond to one stimulus modality while ignoring the other. Neural activity to the same tactile stimulus is modulated by task in all the areas recorded, with significant activity changes in a subset of neurons and population activity occupying distinct activity subspaces. Recordings further reveal a contextual signal in the pre-stimulus baseline activity that differentiates task context. This signal is correlated with subsequent task modulation of stimulus activity. Comparison across brain areas shows that this contextual signal is stronger in frontal cortical regions than in sensory regions. Analyses link this signal to behavior by showing that it tracks the behavioral performance switch during task rule transitions. Silencing activity in frontal cortical regions during the baseline period impairs behavioral performance.

Overall, this is a superb study with solid results and thorough controls. The results are relevant for context-specific neural computation and provide a neural substrate that will surely inspire follow-up mechanistic investigations. We only have a couple of suggestions to help the authors further improve the paper.

1. We have a comment regarding the calculation of the choice CD in Fig S3. The text on page 7 concludes that "Choice coding dimensions change with task rule". However, the motor choice response is different across blocks, i.e. lick right vs. no lick for one task and lick left vs. no lick for the other task. Therefore, the differences in the choice CD may be simply due to the motor response being different across the tasks and not due to the task rule per se. The authors may consider adding this caveat in their interpretation. This should not affect their main conclusion.

2. We have a couple of questions about the effect size on single neurons vs. population dynamics. From Fig 1, about 20% of neurons in frontal cortical regions show task rule modulation in their stimulus activity. This seems like a small effect in terms of population dynamics. There is somewhat of a disconnect from Figs 4 and S3 (for stimulus CD), which show remarkably low subspace overlap in population activity across tasks. Can the authors help bridge this disconnect? Is this because the neurons showing a difference in Fig 1 are disproportionally stimulus selective neurons?

Reviewer #3 (Public Review):

Summary:<br /> This article aims to investigate the impact of neuroprosthesis (intracortical microstimulation) implanted unilaterally on the lesion side in the context of locomotor recovery following unilateral thoracic spinal cord injury.

Strength:<br /> The study reveals that stimulating the left motor cortex, on the same side as the lesion, not only activates the expected right (contralateral) muscle activity but also influences unexpected muscle activity on the left (ipsilateral) side. These muscle activities resulted in a substantial enhancement in lift during the swing phase of the contralateral limb and improved trunk-limb support for the ipsilateral limb. They used different experimental and stimulation conditions to show the ipsilateral limb control evoked by the stimulation. This outcome holds significance, shedding light on the engagement of the "contralateral projecting" corticospinal tract in activating not only the contralateral but also the ipsilateral spinal network.

The experimental design and findings align with the investigation of the stimulation effect of contralateral projecting corticospinal tracts. They carefully examined the recovery of ipsilateral limb control with motor maps. They also tested the effective sites of cortical stimulation. The study successfully demonstrates the impact of electrical stimulation on the contralateral projecting neurons on ipsilateral limb control during locomotion, as well as identifying important stimulation spots for such an effect. These results contribute to our understanding of how these neurons influence bilateral spinal circuitry. The study's findings contribute valuable insights to the broader neuroscience and rehabilitation communities.

Weakness:<br /> The term "ipsilateral" lacks a clear definition in the title, abstract, introduction, and discussion, potentially causing confusion for the reader.

The unexpected ipsilateral (left) muscle activity is most likely due to the left corticospinal neurons recruiting not only the right spinal network but also the left spinal network. This is probably due to the joint efforts of the neuroprosthesis and activation of spinal motor networks which work bilaterally at the spinal level.

However, in my opinion, readers can easily link the ipsilateral cortical network to the ipsilateral-projecting corticospinal tract, which is less likely to play a role in ipsilateral limb control in this study since this tract is disrupted by the thoracic spinal injury.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript explores the behavioral responses of C. elegans to hydrogen sulfide, which is known to exert remarkable effects on animal physiology in a range of contexts. The possibility of genetic and precise neuronal dissection of responses to H2S motivates the study of responses in C. elegans. The manuscript is well-written in communicating the complex physiology around C. elegans behavioral responses to H2S and in appropriately citing prior and related relevant work.

There are three parts to the manuscript.

In the first, an immediate behavioral response-increased locomotory rate-upon exposure to H2S is characterized. The experimental conditions are critical, and data are obtained from exposure of animals to 150ppm H2S at 7% O2. The authors provide evidence that this is a chemosensory response to H2S, showing a requirement for genes encoding components of the cilia apparatus and implicating a role for tax-4 and daf-11. Neuron-specific rescue in the ASJ neurons suggests the ASJ neurons contribute to the response to H2S. One caveat is that previous work has shown that the dauer-constitutive phenotype of daf-11 mutants can be suppressed by ASJ ablation, suggesting that there may be pervasive changes in animal nervous system signaling that are ASJ-dependent in daf-11 mutants, which may indirectly alter chemosensory responses to H2S. More direct methods to assess whether ASJ senses H2S, e.g. using calcium imaging, would better assess a direct role for the ASJ neurons in a behavioral response to H2S. The authors also point out interesting parallels between the response to H2S and CO2 though provide some genetic data separating the two responses. Importantly, the authors note that when aerotaxis (O2-sensing and movement) in the presence of bacterial food is intact, as in npr-1 215F animals, the response to H2S is abrogated. Mutation in gcy-35 in the npr-1 215F background restores the H2S chemosensory response.

There is a second part of the paper that conducts transcriptional profiling of the response to H2S that corroborates and extends prior work in this area.

The final part of the paper is the most intriguing, but for me, also the most problematic. The authors examine how H2S-evoked locomotory behavioral responses are affected in mutants defective in the stress and detoxification response to H2S, most notably hif-1. Prior genetic studies have established the pathways leading to HIF-1 activation/stabilization, as well as potential downstream mechanisms. The authors conduct logical genetic analysis to complement studies of the hif-1 mutant and in part motivated by their transcriptional profiling studies, examine the role of iron sequestration/free iron in the locomotory response to H2S, and further speculate on how the behavior of mutants defective in mitochondrial function might be affected by exposure to H2S.

In some regard, this part of the manuscript is interesting because the analysis begins to connect how the behavior of an animal to a toxic compound is affected by mutations that affect sensitivity to the toxic compound. However, what is unclear is what is being studied at this point. In the context, of noting that H2S at 150ppm is known to be lethal, its addition to mutants clearly sensitized to its effects would be anticipated to have pervasive effects on animal physiology and nervous system function. The authors note that the continued increased locomotion of wild-type animals upon H2S exposure might be due to the byproducts of detoxification or the detrimental effects of H2S. The latter explanation seems much more likely, in which case what one may be observing is the effects of general animal sickness, or even a bit more specifically, neuronal dysfunction in the presence of a toxic compound, on locomotion. As such, what is unclear is what conclusions can be taken away from this part of the work.

Strengths:<br /> 1. Characterization of a motor behavior response to H2S<br /> 2. Transcriptional profiling of the response to H2S corroborating prior work.

Weaknesses:<br /> Unclear significance and experimental challenges regarding the study of locomotory responses to animals sensitized to the toxic effects of H2S under exposure to H2S.

Reviewer #3 (Public Review):

Summary:<br /> There is a growing body of literature on the clustering of co-active synapses in adult mice, which has important implications for understanding dendritic integration and sensory processing more broadly. However, it has been unclear when this spatial organization of co-active synapses arises during development. In this manuscript, Leighton et al. investigate the emergence of spatially organized, co-active synapses on pyramidal dendrites in the mouse visual cortex before eye-opening. They find that some dendrite segments contain highly active synapses that are co-active with their neighbors as early as postnatal day (P) 8-10, and that these domains of co-active synapses increase their coverage of the dendritic arbor by P12-13. Interestingly, Leighton et al. demonstrate that synapses co-active with their neighbors are more likely to increase their activity across a single recording session, compared to synapses that are not co-active with their neighbors, suggesting local plasticity driven by coincident activity before eye-opening.

The current manuscript includes some replication of earlier results from the same research group (Winnubst et al., 2015), including the presence of clustered, co-active synapses in the visual cortex of mouse pups, and the finding that synapses co-active with their neighbors show an increase in transmission frequency during a recording session. The main novelty in the current study compared to Winnubst et al. (2015) is the inclusion of younger animals (P8-13 in the current study compared to P10-15 in Winnubst et al., 2015). The current manuscript is the first demonstration that active synapses are clustered on specific dendrite segments as early as P8-10 in the mouse visual cortex, and the first to show the progression in active synapse distribution along the dendrite during the 2nd postnatal week. These results from the visual cortex may help inform our understanding of sensory development more broadly.

Strengths:<br /> The authors ask a novel question about the emergence of synaptic spatial organization, and they use well-chosen techniques that directly address their questions despite the challenging nature of these techniques. To capture both structural and functional information from dendrites simultaneously, the authors performed a whole-cell voltage clamp to record synaptic currents arriving at the soma while imaging calcium influx at individual synaptic sites on dendrites. The simultaneous voltage clamp and calcium imaging allowed the authors to isolate individual synaptic inputs without their occlusion by widespread calcium influx from back-propagating action potentials. Achieving in vivo dendrite imaging in live mice that are as young as P8 is challenging, and the resulting data provides a unique view of synaptic activity along individual dendrites in the visual cortex at an early stage in development that is otherwise difficult to assess.

The authors provide convincing evidence that synapses are more likely to be co-active with their neighbors compared to synapses located farther away (Fig. 6F-H), and that synapses co-active with their neighbors increase their transmission frequency during a recording session (Figure 7C). These findings are particularly interesting given that the recordings occur before eye-opening, suggesting a relationship between co-activity and local synaptic plasticity even before the onset of detailed visual input. These results replicate previously published findings from P10-15 pups (Winnubst et al., 2015), increasing confidence in the reproducibility of the data.

The authors also provide novel data documenting for the first time spatially organized, co-active synapses in pups as young as P8. Comparing the younger (P8-10) and older (P12-13) pups, provides insight into how clusters of co-active synapses might emerge during development.

Weaknesses:<br /> This manuscript provides insufficient detail for assessing the rigor and reproducibility of the methods, particularly for age comparisons. The P8-10 vs P12-13 age comparisons are the primary novel finding in this manuscript, and it is, therefore, critical to avoid systematic age differences in the methods and analysis whenever possible. Specific concerns related to the age comparisons are listed below:

• Given that the same research group previously published P12-13 data (Winnubst et al., 2015), it is unclear whether both age groups in the current study were imaged/analyzed in parallel by the same researcher(s), or whether previous data was used for the P12-13 group.

• The authors mention that they used 2 different microscopes, and used a fairly wide range of imaging frame rates (5-15 Hz). It is unclear from the current manuscript whether the same imaging parameters were used across the two age groups. If data for the two experimental groups was collected separately, perhaps at different times, by a different person, or on a different microscope, there is a concern that some differences between the groups may not necessarily be due to age.

• It is unclear whether the image analysis was performed blind to age. Blinding to age during analysis is particularly important for this study, in which it was not possible to blind to age during imaging due to visible differences in size and developmental stage between younger and older pups.

• The relatively low N (where N is the number of dendrites or the number of mice) in this study is acceptable due to the challenging nature of the techniques used, but unintentional sampling bias is a concern. For example, if higher-order dendrites from the apical tuft were imaged at P12-13, while more segments of the apical trunk were imaged at P8-10, this could inadvertently create apparent age differences that were in fact due to dendrite location on the arbor or dendrite depth.

Additional general methodological concerns, which are not specifically related to the age comparisons, are listed below:

• The authors assert that clustered, co-active synapses emerge in the visual cortex before eye-opening, which is an important finding in that it suggests this phenomenon is driven by spontaneous activity rather than visual input. However, this finding hinges on the imaged cells being reliably located in the visual cortex, which is difficult to identify with certainty in animals that have not yet opened their eyes and therefore cannot undergo intrinsic signal imaging to demarcate the boundaries of the visual cortex. If the imaged cells were in, for example, nearby somatosensory cortex, then the observed spatial organization could be due to sensory input rather than spontaneous activity.

• It is unclear how the authors defined a synaptic transmission event in the GCaMP signal (e.g. whether there was a quantitative deltaF/F threshold).

• The authors' division of synapses into spine vs shaft is unconvincing due to the difficulty of identifying Z-projecting spines in images from 2-photon microscopy, where the Z resolution is insufficient to definitively identify Z-projecting spines, and the fact that spines in young animals may be thin and dim. The authors' examples of spine synapses (e.g. in Fig. 2A) are convincing, but some of the putative shaft synapses may in fact be on spines.

Reviewer #3 (Public Review):

Summary:

"Bridging the gap between presynaptic hair cell function and neural sound encoding" by Jaime Tobon and Moser uses patch-clamp electrophysiology in cochlear preparations to probe the pre- and post-synaptic specializations that give rise to the diverse activity of spiral ganglion afferent neurons (SGN). The experiments are quite an achievement! They use paired recordings from pre-synaptic cochlear inner hair cells (IHC) that allow precise control of voltage and therefore calcium influx, with post-synaptic recordings from type I SGN boutons directly opposed to the IHC for both presynaptic control of membrane voltage and post-synaptic measurement of synaptic function with great temporal resolution.

Strengths<br /> Any of these techniques by themselves are challenging, but the authors do them in pairs, at physiological temperatures, and in hearing animals, all of which combined make these experiments a real tour de force. The data is carefully analyzed and presented, and the results are convincing. In particular, the authors demonstrate that post-synaptic features that contribute to the spontaneous rate (SR) of predominantly monophasic post-synaptic currents (PSCs), shorter EPSC latency, and higher PSC rates are directly paired with pre-synaptic features such as a lower IHC voltage activation and tighter calcium channel coupling for release to give a higher probability of release and subsequent increase in synaptic depression. Importantly, IHCs paired with Low and High SR afferent fibers had the same total calcium currents, indicating that the same IHC can connect to both low and high SR fibers. These fibers also followed expected organizational patterns, with high SR fibers primarily contacting the pillar IHC face and low SR fibers primarily contacting the modiolar face. The authors also use in vivo-like stimulation paradigms to show different RRP and release dynamics that are similar to results from SGN in vivo recordings. Overall, this work systematically examines many features giving rise to specializations and diversity of SGN neurons.

Weaknesses / Comments / edits:<br /> 1) The careful analysis of calcium coupling and EPSC metrics is especially nice. Can the authors speculate as to why different synapses (likely in the same IHC) would have different calcium cooperativity?

2) On the bottom of page 6 it would be helpful to mention earlier how many pillar vs modiolar fibers were recorded from, otherwise the skewness of SRs (figure 2H could be thought to be due to predominantly recordings from modiolar fibers. As is, it reads a bit like a cliff-hanger.

3) The contrasts for some of the data could be used to point out that while significant differences occur between low and high SR fibers, some of these differences are no longer apparent when comparing modiolar vs pillar fibers (eg by contrasting Figure 2C and 2K). This can indicate that indeed there are differences between the fiber activity, but that the activity likely exists in a gradient across the hair cell faces. Pointing this out at the top of page 10 (end of the first paragraph) would be helpful, it would make the seemingly contradictory voltage-dependence data easier to understand on first read (voltage-dependence of release is significantly different between different SR fibers (figure 3) but is not significantly different between fibers on different HC faces (figure S3).

4) It should be acknowledged that although the use of post-hearing animals here (P14-23) ensures that SGN have begun to develop more mature activity patterns (Grant et al 2010), the features of the synapses and SGN activity may not be completely mature (Wu et al 2016 PMID: 27733610). Could this explain some of the 'challenges' (authors' section title) detailed on page 28, first full paragraph?

5) In the discussion on page 24, the authors compare their recorded SR of EPSCs to measure values in vivo which are higher. Could this indicate that in vivo, the resting membrane potential of IHCs is more depolarized than is currently used for in vitro cochlear experiments?

6) The results showing lower calcium cooperativity of high SR fibers are powerful, but do the authors have an explanation for why the calcium cooperativity of < 2 is different from that (m = 3-4) observed in other manuscripts?

Reviewer #3 (Public Review):

Summary:<br /> Sang et al. successfully demonstrate that a set of single sensory neurons in the pharynx of _Drosophila_ promotes avoidance of food with high salt concentrations, complementing previous findings on Ir7c neurons with an additional internal sensing mechanism. The experiments are well-conducted and presented, convincingly supporting their important findings and extending the understanding of internal sensing mechanisms. However, a few suggestions could enhance the clarity of the work.

Strengths:<br /> The authors convincingly demonstrate the avoidance phenotype using different behavioral assays, thus comprehensively analyzing different aspects of the behavior. The experiments are straightforward and well-contextualized within existing literature.

Weaknesses:<br /> Discussion<br /> While the authors effectively relate their findings to existing literature, expanding the discussion on the surprising role of Ir60b neurons in both sucrose and salt rejection would add depth. Additionally, considering Yang et al. 2021's (https://doi.org/10.1016/j.celrep.2021.109983) result that Ir60b neurons activate feeding-promoting IN1 neurons, the authors should discuss how this aligns with their own findings.

Lines 187ff: The discussion primarily focuses on taste sensillae outside the labellum, neglecting peg-type sensillae on the inner surface. Clarification on whether these pegs contribute to the described behaviors and if the Poxn mutants described also affect the pegs would strengthen the discussion.

In line 261 the authors state: "We attempted to induce salt activation in the I-type sensilla by ectopically expressing Ir60b, similar to what was observed with Ir56b 8; however, this did not generate a salt receptor (Figures S6A)"<br /> An obvious explanation would be that these neurons are missing the identified necessary co-receptors Ir76b and Ir25a. The authors should discuss here if the Gr33a neurons they target also express these co-receptors, if yes this would strengthen their conclusion that an additional receptor might be missing.

Methods<br /> The description of the Droso-X assay seems to be missing some details. Currently, it is not obvious how the two-choice is established. Only one capillary is mentioned, I assume there were two used? Also, the meaning of the variables used in the equation (DrosoX and DrosoXD) are not explained.

The description of the ex-vivo calcium imaging prep. is unclear in several points:<br /> 1. It is lacking information on how the stimulus was applied (was it manually washed in? If so how was it removed?).<br /> 2. The authors write: "A mild swallow deep well was prepared for sample fixation." I assume they might have wanted to describe a "shallow well"?<br /> 3. "...followed by excising a small portion of the labellum in the extended proboscis region to facilitate tastant access to pharyngeal organs." It is not clear to me how one would excise a small portion of the labellum, the labellum depicts the most distal part of the proboscis that carries the sensillae and pegs. Did the authors mean to say that they cut a part of the proboscis?

Reviewer #3 (Public Review):

In the current manuscript, Matsuo-Takasaki et al. have demonstrated that the addition of PKCβ and WNT signaling pathway inhibitors to the suspension cultures of iPSCs suppresses spontaneous differentiation. These conditions are suitable for large-scale expansion of iPSCs. The authors have shown that they can perform single-cell cloning, direct cryopreservation, and iPSC derivation from PBMCs in these conditions. Moreover, the authors have performed a thorough characterization of iPSCs cultured in these conditions, including an assessment of undifferentiated stem cell markers and genetic stability. The authors have elegantly shown that iPSCs cultured in these conditions can be differentiated into derivatives of three germ layers. By differentiating iPSCs into dopaminergic neural progenitors, cardiomyocytes, and hepatocytes they have shown that differentiation is comparable to adherent cultures. This new method of expanding iPSCs will benefit the clinical applications of iPSCs.

Recently, multiple protocols have been optimized for culturing human pluripotent stem cells in suspension conditions and their expansion. Additionally, a variety of commercially available media for suspension cultures are also accessible. However, the authors have not adequately justified why their conditions are superior to previously published protocols (indicated in Table 1) and commercially available media. They have not conducted direct comparisons. Additionally, the authors have not adequately addressed the observed variability among iPSC lines. While they claim in the Materials and Methods section to have tested multiple pluripotent stem cell lines, they do not clarify in the Results section which line they used for specific experiments and the rationale behind their choices. There is a lack of comparison among the different cell lines. It would also be beneficial to include testing with human embryonic stem cell lines. Additionally, there is a lack of information regarding the specific role of the two small molecules in these conditions. The authors have not attempted to elucidate the underlying mechanism other than RNA expression analysis.

For these reasons some aspects of the manuscript need to be extended:

1. It is crucial for authors to specify the culture media used for suspension cultures. In the Materials and Methods section, the authors mentioned that cells in suspension were cultured in either StemFit AK02N medium, 415 StemFit AK03N (Cat# AK03N, Ajinomoto, Co., Ltd., Tokyo, Japan), or StemScale PSC416 suspension medium (A4965001, Thermo Fisher Scientific, MA, USA). The authors should clarify in the text which medium was used for suspension cultures and whether they observed any differences among these media.

2. In the Materials and Methods section, the authors mentioned that they used multiple cell lines for this study. However, it is not clear in the text which cell lines were used for various experiments. Since there is considerable variation among iPSC lines, I suggest that the authors simultaneously compare 2 to 3 pluripotent stem cell lines for expansion, differentiation, etc.

3. Single-cell sorting can be confusing. Can iPSCs grown in suspensions be single-cell sorted? Additionally, what was the cloning efficiency? The cloning efficiency should be compared with adherent cultures.

4. The authors have not addressed the naïve pluripotent state in their suspension cultures, even though PKC inhibition has been shown to drive cells toward this state. I suggest the authors measure the expression of a few naïve pluripotent state markers and compare them with adherent cultures

Reviewer #3 (Public Review):

Summary: Irisin has previously been demonstrated to be a muscle-secreted factor that affects skeletal homeostasis. Through the use of different experimental approaches, such as genetic knockout models, recombinant Irisin treatment, or different cell lines, the role of Irisin on skeletal homeostasis has been revealed to be more complex than previously thought and this warrants further examination of its role. Therefore, the current study sought to rigorously examine the effects of global Irisin knockout (KO) in male and female mouse bone. Authors demonstrated that in calcium-demanding settings, such as lactation or low-calcium diet, female Irisin KO mice lose less bone compared to wild-type (WT) female mice. Interestingly male Irisin KO mice exhibited worse skeletal deterioration compared to WT male mice when fed a low-calcium diet. When examined for transcriptomic profiles of osteocyte-enriched cortical bone, authors found that Irisin KO altered the expression of osteocytic osteolysis genes as well as steroid and fatty acid metabolism genes in males but not in females. These data support the authors' conclusion that Irisin regulates skeletal homeostasis in sex-dependent manner.

Strengths: The major strength of the study is the rigorous examination of the effects of Irisin deletion in the settings of skeletal maturity and increased calcium demands in female and male mice. Since many of the common musculoskeletal disorders are dependent on sex, examining both sexes in the preclinical setting is crucial. Had the investigators only examined females or males in this study, the conclusions from each sex would have contradicted each other regarding the role of Irisin on bone. Also, the approaches are thorough and comprehensive that assess the functional (mechanical testing), morphological (microCT, BSEM, and histology), and cellular (RNA-seq) properties of bone.

Weaknesses: One of the weaknesses of this study is a lack of detailed mechanistic analysis of why Irisin has a sex-dependent role on skeletal homeostasis. This absence is particularly notable in the osteocyte transcriptomic results where such data could have been used to further probe potential candidate pathways between LC females vs. LC males.

Another weakness is authors did not present data that convincingly demonstrate that Irisin secretion is altered in the skeletal muscle between female vs. male WT mice in response to calcium restriction. The supplement skeletal muscle data only present functional and electrophysiolgical outcomes. Since Itgav or Itgb5 were not different in any of the experimental groups, it is assumed that the changes in the level of Irisin is responsible for the phenotypes observed in WT mice. Assessing Irisin expression will further strengthen the conclusion based on observing skeletal changes that occur in Irisin KO male and female mice.

Reviewer #3 (Public Review):

The work extends earlier studies on the Drosophila Id protein EMC to uncover a potential pathway that explains several tissue-scale developmental abnormalities in emc mutants. It also describes a non-apoptotic role for caspases in cell biology.

Strengths:<br /> The work adds to an emerging new set of functions for caspases beyond their canonical roles as cell death mediators. This novelty is a major strength as well as its reliance on genetic-based in vivo study. The study will be of interest to those who are curious about caspases in general.

Weaknesses:<br /> The manuscript relies on imaging experiments using genetic mosaic imaginal discs. It is for the most part a qualitative analysis, showing representative samples with a small number of mutant clones in each. Although the senior author has a long track record of using experiments like this to rigorously discover regulatory mechanisms in this system, it is straightforward in 2023 to use Fiji and other image analysis tools to measure fluorescence. Such measurements could be done for all replicate clones of a given genotype as well as genetic control sampling. These could be presented in plots that would not only provide quantitative and statistical measurements, but will be more reader-friendly to those who are not fly people.

Likewise, more details are needed to describe how clone areas were measured in Figure 1. Did they measure each clone and its twin spot, and then calculate the area ratio for each clone and its paired twin spot? This would be the correct way to analyze the data, yielding many independent measurements of the ratio. And doing so would obviate the need to log transform the data which is inexplicable unless they were averaging clones and twins within a disc and making replicates. More explanation is needed and if they indeed averaged, then they need to calculate the ratios pairwise for each clone and twin.

Die dänische Firma Ørsted investiert in die größteOoffshore-Windfarm der Welt. Hornsea 3 vor der britischen Ostküste soll ab 2027 2,9 Millionen Gigawatt liefern. https://www.liberation.fr/environnement/le-plus-grand-parc-eolien-offshore-du-monde-sortira-de-leau-au-large-de-langleterre-fin-2027-20231220_R5KQ3GIH6ZBBPAPWYCMGUCQBUA/

Reviewer #3 (Public Review):

Summary:

Allosteric regulations are complicated in multi-domain proteins and many large-scale mutational data cannot be explained by current theoretical models,, especially for those that are neither in the functional/allosteric sites nor on the allosteric pathways. This work provides a statistical thermodynamic model for a two-domain protein, in which one domain contains an effector binding site and the other domain contains a functional site. The authors build the model to explain the mutational experimental data of TetR, a transcriptional repress protein that contains a ligand and a DNA-binding domain. They incorporate three basic parameters, the energy change of the ligand and DNA binding domains before and after binding, and the coupling between the two domains to explain the free energy landscape of TetR's conformational and binding states. They go further to quantitatively explain the in vivo expression level of the TetR-regulated gene by fitting into the induction curves of TetR mutants. The effects of most of the mutants studied could be well explained by the model. This approach can be extended to understand the allosteric regulation of other two-domain proteins, especially to explain the effects of widespread mutants not on the allosteric pathways.

Strengths:

The effects of mutations that are neither in the functional or allosteric sites nor in the allosteric pathways are difficult to explain and quantify. This work develops a statistical thermodynamic model to explain these complicated effects. For simple two-domain proteins, the model is quite clean and theoretically solid. For the real TetR protein that forms a dimeric structure containing two chains with each of them composed of two domains, the model can explain many of the experimental observations. The model separates intra and inter-domain influences that provide a novel angle to analyse allosteric effects in multi-domain proteins.

Weaknesses:

As mentioned above, the TetR protein is not a simple two-main protein, but forms a dimeric structure in which the DNA binding domain in each chain forms contacts with the ligand-binding domain in the other chain. In addition, the two ligand-binding domains have strong interactions. Without considering these interactions, especially those mutants that are on these interfaces, the model may be oversimplified for TetR.

Reviewer #3 (Public Review):

Summary: This manuscript explores the development of a rodent voluntary oral THC consumption model. The authors use the model to demonstrate that similar effect levels of THC can be observed to what has previously been described for i.p. THC administration.

Strengths: Overall this is an interesting study with compelling data presented. There is a growing need within the field of cannabinoid research to explore more 'realistic' routes of cannabinoid administration, such as oral consumption or inhalation. The evidence presented here shows the utility of this oral administration model.

Weaknesses: The main weaknesses of the manuscript revolve around clarification of the Methods section. All of these weaknesses are described in the "Recommendations to authors" section. Revising the manuscript would account for many of these weaknesses.

Reviewer #3 (Public Review):

Summary:<br /> The paper investigates the effects of long-term linguistic experience on early auditory processing, a subject that has been relatively less studied compared to short-term influences. Using MEG, the study examines brain responses to auditory stimuli in speakers of Spanish and Basque, whose syntactic rules provide different degrees of exposure to durational patterns (long-short vs short-long). The findings suggest that both long-term language experience, as well as short-term transitional probabilities, can shape auditory predictive coding for non-linguistic sound sequences, evidenced by differences in mismatch negativity amplitudes localised to the left auditory cortex.

Strengths:<br /> The study integrates linguistics and auditory neuroscience in an interesting interdisciplinary way that may interest linguists as well as neuroscientists. The fact that long-term language experience affects early auditory predictive coding is important for understanding group and individual differences in domain-general auditory perception. It has importance for neurocognitive models of auditory perception (e.g. inclusion of long-term priors), and will be of interest to researchers in linguistics, auditory neuroscience, and the relationship between language and perception. The inclusion of a control condition based on pitch is also a strength.

Weaknesses:<br /> The main weaknesses are the strength of the effects and generalisability. The sample size is also relatively small by today's standards, with N=20 in each group. Furthermore, the crucial effects are all mostly in the .01>P<.05 range, such as the crucial interaction P=.03. It would be nice to see it replicated in the future, with more participants and other languages. It would also have been nice to see behavioural data that could be correlated with neural data to better understand the real-world consequences of the effect.

Reviewer #3 (Public Review):

Summary:<br /> Hauser et al. provide an exceptional study describing the role of resident mast cells in amphibian epidermis that produce anti-inflammatory cytokines that prevent Batrachochytrium dendrobatidis (Bd) infection from causing harmful inflammation, and also protect frogs from changes in skin microbiomes and loss of mucin in glands and loss of mucus integrity that otherwise cause changes to their skin microbiomes. Neutrophils, in contrast, were not protective against Bd infection. Beyond the beautiful cytology and transcriptional profiling, the authors utilized elegant cell enrichment experiments to enrich mast cells by recombinant stem cell factor, or to enrich neutrophils by recombinant colony-stimulating factor-3, and examined respective infection outcomes in Xenopus.

Strengths:<br /> Through the use of recombinant IL4, the authors were able to test and eliminate the hypothesis that mast cell production of IL4 was the mechanism of host protection from Bd infection. Instead, impacts on the mucus glands and interaction with the skin microbiome are implicated as the protective mechanism. These results will press disease ecologists to examine the relative importance of this immune defense among species, the influence of mast cells on the skin microbiome and mucosal function, and open the potential for modulating mucosal defense.

Weaknesses:<br /> A reduction of bacterial diversity upon infection, as described at the end of the results section, may not always be an "adverse effect," particularly given that anti-Bd function of the microbiome increased. Some authors (see Letourneau et al. 2022 ISME, or Woodhams et al. 2023 DCI) consider these short-term alterations as encoding ecological memory, such that continued exposure to a pathogen would encounter an enriched microbial defense. Regardless, mast cell-initiated protection of the mucus layer may negate the need for this microbial memory defense.

While the description of the mast cell location in the epidermal skin layer in amphibians is novel, it is not known how representative these results are across species ranging in chytridiomycosis susceptibility. No management applications are provided such as methods to increase this defense without the use of recombinant stem cell factor, and more discussion is needed on how the mast cell component (abundance, distribution in the skin) of the epidermis develops or is regulated.

Reviewer #3 (Public Review):

Summary:<br /> The authors have examined the 5-HT3 receptor using voltage clamp fluorometry, which enables them to detect structural changes at the same time as the state of receptor activation. These are ensemble measurements, but they enable a picture of the action of different agonists and antagonists to be built up.

Strengths:<br /> The combination of rigorously tested fluorescence reporters with oocyte electrophysiology is a solid development for this receptor class.

Weaknesses:<br /> The interpretation of the data is solid but relevant foundational work is ignored. Although the data represent a new way of examining the 5-HT3 receptor, nothing that is found is original in the context of the superfamily. Quantitative information is discussed but not presented.

Reviewer #3 (Public Review):

Summary:<br /> In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry, and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access to the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

Strengths:<br /> The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42 . In their clearly written discussion, the authors explain how PIP3, the cell membrane, and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

Weaknesses:<br /> There are a few weaknesses. While the resolution of the cryo-EM structure is modest, it is sufficient to identify the domain-domain interactions that are mechanistically important, since higher-resolution structures of various pREX-1 modules are available.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Yao et al. investigates the intracellular trafficking of Botulinum neurotoxin A (BoNT/A), a potent toxin used in clinical and cosmetic applications. Contrary to the prevailing understanding of BoNT/A translocation into the cytosol, the study suggests a retrograde migration from the synapse to the soma-localized Golgi in neurons. Using a genome-wide siRNA screen in genetically engineered neurons, the researchers identified over three hundred genes involved in this process. The study employs organelle-specific split-mNG complementation, revealing that BoNT/A traffics through the Golgi in a retromer-dependent manner before moving to the endoplasmic reticulum (ER). The Sec61 complex is implicated in the retro-translocation of BoNT/A from the ER to the cytosol. Overall, the research challenges the conventional model of BoNT/A translocation, uncovering a complex route from synapse to cytosol for efficient intoxication. The findings are based on a comprehensive approach, including the introduction of a fluorescent reporter for BoNT/A catalytic activity and genetic manipulations in neuronal cell lines. The conclusions highlight the importance of retrograde trafficking and the involvement of specific genes and cellular processes in BoNT/A intoxication.

Strengths:<br /> The major part of the experiments are convincing. They are well-controlled and the interpretation of their results is balanced and sensitive.

Weaknesses:<br /> To my opinion, the main weakness of the paper is in the interpretation of the data equating loss of tGFP signal (when using the Red SNAPR assay) with proteolytic cleavage by the toxin. Indeed, the first step for loss of tGFP signal by degradation of the cleaved part is the actual cleavage. However, this needs to be degraded (by the proteasome, I presume), a process that could in principle be affected (in speed or extent) by the toxin.

Reviewer #3 (Public Review):

In this manuscript, Gustison et al., describe the development of an automated whole-brain mapping pipeline, including the first 3D histological atlas of the prairie vole, and then use that pipeline to quantify Fos immunohistochemistry as a measure of neural activity during mating and pair bonding in male and female prairie voles. Prairie voles have become a useful animal model for examining the neural bases of social bonding due to their socially monogamous mating strategy. Prior studies have focused on identifying the role of a few neuromodulators (oxytocin, vasopressin, dopamine) acting in limited number of brain regions. The authors use this unbiased approach to determine which areas become activated during mating, cohabitation, and pair bonding in both sexes to identify 68 brain regions clustered in seven brain-wide neuronal circuits that are activated over the course of pair bonding. This is an important study because i) it generates a valuable tool and analysis pipeline for other investigators in the prairie vole research community and ii) it highlights potential involvement of many brain regions in regulating sexual behavior, social engagement and pair bonding that have not been previously investigated.

Strengths of the study include the unbiased assessment of neural activity using the automated whole brain activity mapped onto the 3D histological atlas. The design of the behavioral aspect of study is also a strength. Brains were collected at baseline and 2.5, 6 and 22 hrs after cohabitation with either a sibling or opposite sex partner. These times were strategically chosen to correspond to milestones in pair bond development. Behavior was also quantified during epochs over the 22 hr period providing useful information on the progression of behaviors (e.g. mating) during pair bonding and relating Fos activation to specific behaviors (e.g. sex vs bonding). The sibling co-housed group provided an important control, enabling identification of areas specifically activated by sex and bond formation. The analyses of the data were rigorous, resulting in convincing conclusions. While there was nothing particularly surprising in terms of the structures that were identified to be active during the mating and cohabitation, the statistical analysis revealed interesting relationships in terms of interactions of the various clusters, and also some level of synchrony in brain activation between partners. Furthermore, ejaculation was found to be the strongest predictor of Fos activation in both males and females. The sex differences identified in the study was subtle and less than the authors expected, which is interesting.

While the study provides a potentially useful tool and approach that may be general use to the prairie vole community and identifies in an anatomically precise manner areas that may be important for mating or pair bond formation, there are some weaknesses as well. The study is largely descriptive. It is impossible to determine whether the activated areas are simply involved in sex or in the pair bond process itself. In other words, the authors did not use the Fos data to inform functional testing of circuits in pair bonding or mating behaviors. However, that is likely beyond the scope of this paper in which the goal was more to describe the automated, unbiased approach. This weakness is offset by the value of the comprehensive and detailed analysis of the Fos activation data providing temporal and precise anatomical relationships between brain clusters and in relation to behavior. The manuscript concludes with some speculative interpretations of the data, but these speculations may be valuable for guiding future investigations.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors present a new automated image analysis pipeline named WormPsyQi which allows researchers to quantify various parameters of synapses in C. elegans. Using a collection of newly generated transgenic strains in which synaptic proteins are tagged with fluorescent proteins, the authors showed that WormPsyQi can reliably detect puncta of synaptic proteins, and measure several parameters, including puncta number, location, and size.

Strengths:

The image analysis of fluorescently labeled synaptic (or other types of) puncta pattern requires extensive experience such that one can tell which puncta likely represent bona fide synapse or background noise. The authors showed that WormPsyQi nicely reproduced the quantifications done manually for most of the marker strains they tested. Many researchers conducting such types of quantifications would receive significant benefits in saving their time by utilizing the pipeline developed by the authors. The collection of new markers would also help researchers examine synapse patterning in different neuron types which may have unique mechanisms in synapse assembly and specificity. The authors describe the limitations of the use of toolkits and potential solutions users can take.

Reviewer #3 (Public Review):

Summary:

This hybrid experimental/computational study by Hernandez-Hernandez sheds new light on sex-specific differences between male and female arterial myocytes from resistance arteries. The authors conduct careful experiments in isolated myocytes from male and female mice to obtain the data needed to parameterized sex-specific models of two important ionic currents (i.e., those mediated by CaV1.2 and KV2.1). Available experimental data suggest that KV1.5 channel currents from male and female myocytes are similar, but simulations conducted in the novel Hernandez-Hernandez sex-specific models provide a more nuanced view. This gives rise to the first of the authors' three key scientific claims: (1) In males, KV1.5 is the dominant current regulating membrane potential; whereas, in females, KV2.1 plays a primary role in voltage regulation. They further show that this (2) the latter distinction drives drive sex-specific differences in intracellular Ca2+ and cellular excitability. Finally, working with one-dimensional models comprising several copies of the male/female myocyte models linked by resistive junctions, they use simulations to (3) predict that sensitivity of arterial smooth muscle to Ca2+ channel-blocking drugs commonly used to treat hypertension is heightened in female compared to male cells.

In my opinion, the following strengths of the work are particularly notable:

• The Methodology is described in exquisite detail in straightforward language that will be easy to understand for most if not all peer groups working in computational physiology. The authors have deployed standard protocols (e.g., parameter fitting as described by Kernik et al., sensitivity analysis as described by Sobie et al.) and appropriate brief explanations of these techniques are provided. The manoeuvre used to represent stochastic effects on voltage dynamics is particularly clever and something I have not personally encountered before. Collectively, these strengthen the credibility of the model and greatly enrich the manuscript.<br /> • The Results section describes findings that robustly support the three key scientific claims outlined in my Summary. It is evident these experiments were carefully designed and carried out with care and intentionality. Several figures show experimental data side-by-side with outputs from the corresponding models. These are an excellent illustration of the power of the authors' novel sex-specific computational simulation platform.

Reviewer #3 (Public Review):

Summary:<br /> A mechanical model is used with input force patterns to generate output curvature patterns, corresponding to a number of different locomotion behaviors in C. elegans

Strengths:<br /> The use of a mechanical model to study a variety of locomotor sequences and the grounding in empirical data are strengths. The matching of speeds (though qualitative and shown only on agar) is a strength.

Weaknesses:<br /> What is the relation between input and output data? How does the input-output relation depend on the parameters of the model? What biological questions are addressed and can significant model predictions be made?

Reviewer #3 (Public Review):

The manuscript "Mechanical activation of TWIK-related potassium channel by nanoscopic movement and second messenger signaling" presents a new mechanism for the activation of TREK-1 channel. The mechanism suggests that TREK1 is activated by phosphatidic acids that are produced via a mechanosensitive motion of PLD2 to PIP2-enriched domains. Overall, I found the topic interesting but several typos and unclarities reduced the readability of the manuscript. Additionally, I have several major concerns on the interpretation of the results. Therefore, the proposed mechanism is not fully supported by the presented data. Lastly, the mechanism is based on several previous studies from the Hansen lab, however, the novelty of the current manuscript is not clearly stated. For example, in the 2nd result section, the authors stated, "fluid shear causes PLD2 to move from cholesterol dependent GM1 clusters to PIP2 clusters and this activated the enzyme". However, this is also presented as a new finding in section 3 "Mechanism of PLD2 activation by shear."<br /> In the revised manuscript, the authors addressed most of my concerns. I still have the following suggestions/confusions.<br /> 1. the reviewer would highly appreciate verification of the cholesterol assay, either by additional experiment or by citations of independent work.

2. The claim on "shear thinning" is still very confusing. First, asymmetric insertion of molecules to one monolayer of the membrane is a main mechanism for membrane bending and curvature formation. Second, why is "shear thinning" equivalent to entropy/order?

Reviewer #3 (Public Review):

Summary:<br /> The paper explores chemosensory behaviour in surface and cave morphs and F2 hybrids in the Mexican cavefish Astyanax mexicanus. The authors develop a new behavioural assay for the long-term imaging of individual fish in a parallel high-throughput setup. The authors first demonstrate that the different morphs show different basal exploratory swimming patterns and that these patterns are stable for individual fish. Next, the authors test the attraction of fish to various concentrations of alanine and other amino acids. They find that the cave morph is a lot more sensitive to chemicals and shows directional chemotaxis along a diffusion gradient of amino acids. For surface fish, although they can detect the chemicals, they do not show marked chemotaxis behaviour and have an overall lower sensitivity. These differences have been reported previously but the authors report longer-term observations on many individual fish of both morphs and their F2 hybrids. The data also indicate that the observed behavior is a quantitative genetic trait. The approach presented will allow the mapping of genes' contribution to these traits. The work will be of general interest to behavioural neuroscientists and those interested in olfactory behaviours and the individual variability in behavioural patterns.

Strengths:<br /> A particular strength of this paper is the development of a new and improved setup for the behavioural imaging of individual fish for extended periods and under chemosensory stimulation. The authors show that cavefish need up to 24 h of habituation to display a behavioural pattern that is consistent and unlikely to be due to the stressed state of the animals. The setup also uses relatively large tanks that allow the build-up of chemical gradients that are apparently present for at least 30 min.

The paper is well written, and the presentation of the data and the analyses are clear and to a high standard.

Weaknesses:<br /> One point that would benefit from some clarification or additional experiments is the diffusion of chemicals within the behavioural chamber. The behavioural data suggest that the chemical gradient is stable for up to 30 min, which is quite surprising. It would be great if the authors could quantify e.g. by the use of a dye the diffusion and stability of chemical gradients.

The paper starts with a statement that reflects a simplified input-output (sensory-motor) view of the organisation of nervous systems. "Their brains perceive the external world via their sensory systems, compute information and generate appropriate behavioral outputs." The authors' data also clearly show that this is a biased perspective. There is a lot of spontaneous organised activity even in fish that are not exposed to sensory stimulation. This sentence should be reworded, e.g. "The nervous system generates autonomous activity that is modified by sensory systems to adapt the behavioural pattern to the external world." or something along these lines.

Reviewer #3 (Public Review):

This research addresses a critical challenge in malaria research, specifically how to effectively access the highly polymorphic var gene family using short-read sequence data. The authors successfully tackled this issue by introducing an optimization of their original de novo assembler, which notably more than doubled the N50 metric and greatly improved the assembly of var genes.

The most intriguing aspect of this study lies in its methodologies, particularly the longitudinal analysis of assembled var transcripts within subjects. This approach allows for the construction of an unbiased var repertoire for each individual, free from the influence of a reference genome or other samples. These sample-specific var gene repertoires are then tracked over time in culture to evaluate the reliability of using cultured samples for inferences about in vivo expression patterns. The findings from this analysis are thought-provoking. While the authors conclude that culturing parasites can lead to unpredictable transcriptional changes, they also observe that the overall ranking of each var gene remains relatively robust over time. This resilience in the var gene ranking within individuals raises intriguing questions about the mechanisms behind var gene switching and adaptation during short-term culture.

In addition to the var gene-specific analysis, the study also delves into a comparison of ex vivo samples with generation 1 and generation 2 cultured parasites across the core genome. This analysis reveals substantial shifts in expression due to culture adaptation, shedding light on broader changes in the parasite transcriptome during short-term culture.

In summary, this research contributes to our understanding of var gene expression and potentially associations with disease. It emphasizes the importance of improved assembly techniques to access var genes and underscores the challenges of using short-term cultured parasites to infer in vivo characteristics. The longitudinal analysis approach offers a fresh perspective on var gene dynamics within individuals and highlights the need for further investigations into var gene switching and adaptation during culture.

Reviewer #3 (Public Review):

Peng et al. designed a computational framework for identifying pioneer factors using epigenomic data from five cell types. The identification of pioneer factors is important for our understanding of the epigenetic and transcriptional regulation of cells. A computational approach toward this goal can significantly reduce the burden of labor-intensive experimental validation.

The authors have addressed my previous comments.

The main issue identified in this re-review is based on the authors' additional experiments to investigate the reproducibility of the pioneer factors identified in the previous analysis that anchored on H1 ESCs.

The additional analysis that uses the other four cell types (HepG2, HeLa-S3, MCF-7, and K562) as anchors reveals the low reproducibility/concordance and high dependence on the selection of anchor cell type in the computational framework. In particular, now several stem cell related TFs (e.g. ESRRB, POU5F1) are ranked markedly higher when H1 ESC is not used as the anchor cell type as shown in Supplementary Figure 5.

Of note, the authors have now removed the shape labels that denote Yamanaka factors in Figure 2c (revised manuscript) that was presented in the main Figure 2a in the initial submission. The NFYs and ESRRB labels in Supplementary 4a are also removed and the boxplot comparing NFYs and ESRRB with other TF are also removed in this figure. Removing these results effectively hides the issues of the computational framework we identified in this revision. Please justify why this was done.

In summary, these new results reveal significant limitations of the proposed computational framework for identifying pioneer factors. The current identifications appear to be highly dependent on the choice of cell types.

Reviewer #3 (Public Review):

Summary:

This article demonstrates a Pax1-Col11a1-Mmp3 signaling axis associated with adolescent idiopathic scoliosis and finds that estrogen affects this signaling axis. In addition, the authors have identified a new COL11A1 mutation and verified its effect on the Pax1-Col11a1-Mmp3 axis.

Strengths:

1. Col11a1P1335L is verified in multicenter cohorts with high confidence.

2. The article identified a potential pathogenesis of AIS.

Weaknesses:

The SV40-immortalized cell line established from Col11a1fl/fl mouse rib cartilage was applied in the study, and overexpression system was used to confirm that P1335L variant in COL11A1 perturbs its regulation of MMP3. However, due to the absence of P1335L point mutant mice, it cannot be confirmed whether P1335L can actually cause AIS, and the pathogenicity of this mutation cannot be directly verified.

Reviewer #3 (Public Review):

Summary:<br /> This manuscript by Toth et al reveals a conserved phosphorylation site within the RIN4 (RPM1-interacting protein 4) R protein that is exclusive to two of the four nodulating clades, Fabales and Rosales. The authors present persuasive genetic and biochemical evidence that phosphorylation at the serine residue 143 of GmRIN4b, located within a 15-aa conserved motif with a core five amino acids 'GRDSP' region, by SymRK, is essential for optimal nodulation in soybean. While the experimental design and results are robust, the manuscript's discussion fails to clearly articulate the significance of these findings. Results described here are important to understand how the symbiosis signaling pathway prioritizes associations with beneficial rhizobia, while repressing immunity-related signals.

Strengths:<br /> The manuscript asks an important question in plant-microbe interaction studies with interesting findings.

Overall, the experiments are detailed, thorough, and very well-designed. The findings appear to be robust.

The authors provide results that are not overinterpreted and are instead measured and logical.

Weaknesses:<br /> No major weaknesses. However, a well-thought-out discussion integrating all the findings and interpreting them is lacking; in its current form, the discussion lacks 'boldness'. The primary question of the study - how plants differentiate between pathogens and symbionts - is not discussed in light of the findings. The concluding remark, "Taken together, our results indicate that successful development of the root nodule symbiosis requires cross-talk between NF-triggered symbiotic signaling and plant immune signaling mediated by RIN4," though accurate, fails to capture the novelty or significance of the findings, and left me wondering how this adds to what is already known. A clear conclusion, for eg, the phosphorylation of RIN4 isoforms by SYMRK at S143 modulates immune responses during symbiotic interactions with rhizobia, or similar, is needed.

Reviewer #3 (Public Review):

Summary:<br /> Regulated chloroplast breakdown allows plants to modulate these energy-producing organelles, for example during leaf aging, or during changing light conditions. This manuscript investigates how chloroplasts are broken down during light-limiting conditions.

The authors present very nice time-lapse imaging of multiple proteins as buds form on the surface of chloroplasts and pinch away, then associate with the vacuole. They use mutant analysis and autophagy markers to demonstrate that this process requires the ATG machinery, but not dynamin-related proteins that are required for chloroplast division. The manuscript concludes with a discussion of an internally-consistent model that summarizes the results.

Strengths:<br /> The main strength of the manuscript is the high-quality microscopy data. The authors use multiple markers and high-resolution time-lapse imaging to track chloroplast dynamics under light-limiting conditions.

Weaknesses:<br /> The main weakness of the manuscript is the lack of quantitative data. Quantification of multiple events is required to support the authors' claims, for example, claims about which parts of the plastid bud, about the dynamics of the events, about the colocalization between ATG8 and the plastid stroma buds, and the dynamics of this association. Without understanding how often these events occur and how frequently events follow the manner observed by the authors (in the 1 or 2 examples presented in each figure) it is difficult to appreciate the significance of these findings.

Reviewer #3 (Public Review):

The manuscript reports data collected in awake toddlers recording BOLD while watching videos. The authors analyse the BOLD time series using two different statistical approaches, both very complex but do not require any a priori determination of the movie features or contents to be associated with regressors. The two main messages are that 1) toddlers have occipital visual areas very similar to adults, given that an SRM model derived from adult BOLD is consistent with the infant brains as well; 2) the retinotopic organization and the spatial frequency selectivity of the occipital maps derived by applying correlation analysis are consistent with the maps obtained by standard and conventional mapping.

Clearly, the data are important, and the author has achieved important and original results. However, the manuscript is totally unclear and very difficult to follow; the figures are not informative; the reader needs to trust the authors because no data to verify the output of the statistical analysis are presented (localization maps with proper statistics) nor so any validation of the statistical analysis provided. Indeed what I think that manuscript means, or better what I understood, may be very far from what the authors want to present, given how obscure the methods and the result presentation are.

In the present form, this reviewer considers that the manuscript needs to be totally rewritten, the results presented each technique with appropriate validation or comparison that the reader can evaluate.

Reviewer #3 (Public Review):

Summary:

Mao and colleagues generated powerful reagents to genetically analyse chemical communication (CCT) in the brain, and in the process uncovered a function for the CNMa neuropeptide expressed in a subset of DN1p neurons that contributes to the temporal organization of locomotor activity, i.e., the timing of morning anticipation.

Strengths:

The strength of the manuscript relies on the generation/characterization of new tools for conditional targeting a well-defined set of CCT genes along with the design and testing of improved versions of Cas9 for efficient knockout. Such invaluable resources will be of interest to the whole community. The authors employed these tools and intersectional genetics to provide an alternative profiling of clock neurons, which is complementary to the ones already published. Furthermore, they uncovered a role for CNMamide, expressed in two DN1ps, in the timing of morning anticipation.

Weaknesses:

They targeted an extensive set of candidate genes putatively involved in communication (transporters, receptor subunits, neuropeptides, neurotransmitter synthesis, etc); they provide a list of efficient gRNAs to target even a longer list of candidate genes, however, it is not clear if all of those made it into transgenic lines that effectively mediate targeting all chemical transmission genes (as suggested by the authors).

Reviewer #3 (Public Review):

Though it is speculated that gene-environment interactions (GxE) contribute to disease heritability, they remain challenging to detect. Here, the authors use a massively parallel reporter assay in vascular endothelial cells treated with or without caffeine to explore context-specific gene regulation. They use a library of 200-bp candidate regions selected from a variety of genetic studies (eQTL, GWAS) and demonstrate allelic bias in activity across a large proportion, including variants with caffeine-specific allelic effects. The described assays represent a useful approach for examining GxE in complex traits, thus these results are of broad appeal. I have great enthusiasm for the experimental design, including the large library and sample size, testing the MPRA in an appropriate cell type with a relevant stimulus, and interesting functional analyses including transcription factor motif enrichment and comparison to GTEx data. My main critique is that the description of analyses and results lacks the clarity that would aid the reader in interpretation.

1. The abstract states that >43k variants are tested in the library while the methods section states that >43k constructs were tested. Because you tested allele pairs, my expectation is that you would have used ~86k constructs, and at various points, you mention denominators that are higher than 43k. Please address this discrepancy.<br /> 2. Previously, you reported allele-specific expression analysis across many conditions, including caffeine treatment. In that study, you observed high levels of differential expression induced by caffeine treatment (on the order of thousands of genes) with only a modest number of SNPs with allele-specific expression after caffeine treatment. In the current study, you report that only ~800 constructs are differentially active after caffeine treatment which you state as evidence that "caffeine overall increases the activity of the regulatory elements," but this is quite a small number given that you tested tens of thousands of constructs. Later you describe >8k constructs with conditional allele-specific expression. Do you mean that the former subset only displays caffeine effects without allele-specific expression? And taking both studies into account, what do you think accounts for the seeming discrepancies between the relative amount of conditional allele-specific expression measured by RNA-seq vs BiT-STARR-seq?<br /> 3. Your transcription factor motif enrichment analyses are interesting, and would benefit from a further grounding in the biology of the cells you're working with. To this end, what proportion of the transcription factor sets that you use for enrichment are expressed in your cell model? For those that are enriched, are they highly expressed, and does that expression vary with caffeine treatment? You provide some of this information for a specific example (rs228271), but a broader discussion is warranted.<br /> 4. I suggest elaborating on the choice of treatment conditions to provide valuable context. Acute responses to caffeine exposure may vary from chronic exposure. In this study, I think a single acute exposure is more than appropriate for reasons of feasibility and many of the regulatory pathways will be shared between acute and long-term; however, given that CAD is a chronic disease that develops over many years, it would be worthwhile to speculate on longer term effects of caffeine exposure in your model system.

Reviewer #3 (Public Review):

The authors inactivated the MurT-GatD of Mycobacterium bovis BCG using CRISPR interference and found that loss of these enzymes curbs growth but also alters the cell envelope and cell wall composition. As MurT-GatD are required for amidation of D-glutamate residues in the cell wall and amidation is required for cell wall crosslinking, depletion of MurT-GatD leads to envelope defects and increased sensitivity to cell wall-targeting antibiotics. Loss of D-glutamate amidation also leads in the accumulation of cell wall components that are detected by the cellular NOD1-innate immune surveillance system that is normally blind to amidated cell wall components. Such MurT-GatD-depleted BCG cells are more effective in protecting host cells towards infection by Mycobacterium tuberculosis (Mtb) in an in vitro monocyte model, but also in a murine lung infection model of Mtb.

The use of the cellular and animal models gave consistent findings for the recombinant BCG mutant strain in its protective effect against subsequent Mtb infections, importantly occurring in a concentration-dependent manner that correlates with the levels of CRISPR-mediated inhibition.

As no efficient vaccine exists against Mtb and the authors showed the potency of the mutant BCG over WT BCG to vaccinate mice against Mtb, this work identifies MurT-GatD-depleted BCG as a strong new and effective vaccine candidate against Mtb. It is possible that enhanced NOD1-signaling caused by loss of D-glutamate has a general self-adjuvanting effect on BCG bacteria and its conserved surface antigens towards Mtb. Alternatively, Mtb bacteria could alter their cell envelope structure during the course of an infection, rendering them more susceptible to immune responses already entrained by MurT-GatD-depleted BCG.

Reviewer #3 (Public Review):

This paper aims to understand the nature of collaborative hunting. It sets out by first defining simple conditions under which collaborative hunting emerges, which leads to the emergence of a toy environment. The environment itself is simple, K prey chasing a single predator with no occlusions. I find this a little strange, since it was my understanding that collaborative hunting emerges in part because the presence of occlusions allows for more complex strategies that require planning.

That being said, I do think the environment is sufficient for this paper, and I quite enjoyed using it to run some toy experiments. It reminds me of some of the simpler environments from Petting Zoo, a library for multi-agent learning in reinforcement learning.

Once a simple environment was established, the authors fit a reinforcement learning model to the environment. In this case, the model is Q-learning. The predator and prey are treated as separate agents in the environment, each with their own independent Q functions. Each agent gets full observability of the surroundings. As far as I understand, the predators do not share an action space, and so they can only collaborate implicitly by inferring the actions of the other predators. However, there is a version of these experiments wherein the reward function is shared, all agents receiving a 1 when the prey is caught. One limitation of the current work is that it does not consider reinforcement learning methods methods wherein a value function is shared. This is a current dominant strategy in multi-agent RL. See for example OpenAI Five and also Multi-Agent Actor-Critic for Mixed Cooperative-Competitive Environments. Missing these algorithms limits the scope of the work.

Having fit an RL model, the next order of business is to try and search for internal representations in the agent's model that correspond to collaboration. The author's use t-SNE embeddings of the agents last hidden layers in the policy network.

Analyzing these embeddings in Figure 3, we see that there are some representations that correspond to specific types of collaborative behavior, which indicates that the model is indeed learning to encode collaboration. I should note that this is not surprising from an RL perspective. Certainly, we are aware that Multi-Agent actor critic methods can exhibit cooperative behavior. See Emergent tool use from multi-agent interaction where agents jointly learn to push a table together. It is true that earlier work didn't specifically identify the units responsible for this behavior, and I think this work should be lauded for the novelty it brings to this discussion.

A large underlying point of this paper seems to be that we we need to consider these simple toy environments where we can easily train Q-learning, because it is impossible to analyze the behaviors that emerge from real animal behavior. See lines 187-189. This makes sense on the surface, because there are no policy weights in the case of real-world behavior. However, it is unfortunately misleading. It is entirely possible to take existing animal behavior, fit a linear model (or a deep net) to this behavior, and then do t-SNE on this fit model. This is referred to as behavioral cloning. What's more, offline RL makes it entirely possible to fit a Q-function to animal behaviors, in which case the exact same t-SNE analysis can be carried out without ever running Q-learning in the environment. From my perspective, the fact that RL is not needed to carry out the paper's main analysis is the biggest weakness of the paper.

Meanwhile, I do think the comparisons with human players was exceptionally interesting, and I'm glad it was included in this work.

Finally, I would like to speak to the reinforcement learning sections of this paper, as this is my personal area of expertise. I will note that the RL used in this paper is all valid and correct. The descriptions of Q-learning and its modifications are technically accurate. It's worth noting that the performance offered by the Q-learning methods in this paper is not particularly close to optimal. I mean this in two ways. First, cooperative RL methods are much more performant. Second, the Q-learning implementation considered by the author's is far below state of the art standards.

I will also note that, from the perspective of RL, there is no novelty in the paper. Indeed, many Deep Mind papers, including the original Q-learning paper, have similar t-SNE embeddings to try and understand the action space. And works such as Sentiment Neuron and Visualizing and Understanding Recurrent Networks, among many many others, have focused on the problem of understanding the correspondence between network weights and behaviors. Thus, the novelty must come from a biological perspective. Or perhaps from a perspective at the intersection of biology and RL. I do believe this is an area worth further studying.

Reviewer #3 (Public Review):

The authors elucidated the roles of cholecystokinin (CCK)-expressing excitatory neurons, which project from the rhinal cortex to the motor cortex, in motor skill learning. The authors found CCK knock-out mice exhibited learning defects in the pellet reaching task while the baseline success rate of the knock-out mice was similar to that of the wild-type mice. Application of a CCK B receptor (CCKBR) antagonist into the motor cortex lowered the success rate in the motor task. The authors found the population activity which was observed in the in vivo calcium imaging during motor learning was elevated after motor learning, but this increase disappeared in CCK knock-out mice and animals with CCKBR antagonist administration. Anterograde and retrograde viral tracing revealed that CCK-expressing excitatory neurons in the rhinal cortex projected to the motor cortex. Chemogenetic inhibition of the CCK-expressing neurons in the rhinal cortex lowered the ability for motor learning. The application of a CCKBR agonist increased the motor learning ability of CCK knock-out animals as well as long-term potentiation (LTP) observed in the slice of the motor cortex.

However, the manuscript contains several shortcomings:

First, the "Discussion" has several statements that are only supported weakly by the results, for example, ll. 429-431, ll. 432-433, and ll. 447-448. In addition, most of the sentences in this section are not divided into subsections. The paragraphs should be composed in multiple subsections with appropriate subheadings, even though the initial section summarizing the results can lack a subheading.

Second, it would be important that the authors showed which area(s) of the brain is affected by the CCKBR antagonist in the experiments described in ll. 166-206 and Fig. 2. The authors injected the drug into the motor cortex, but the chemical can spread to neighboring cortical areas (e.g. somatosensory cortex) or wider brain regions. If so, the blockade of the CCKBR in the brain areas other than the motor cortex could cause the defects of the motor task learning observed in these experiments. I think it is desirable that such a possibility should be excluded. Conversely, it is possible that the antagonist had an effect on a limited subarea of the motor cortex (e.g. only the primary motor cortex (M1)). In this case, the information about the field altered by the CCKBR blocker would be useful to interpret the results of the learning defects.

Third, the authors need to show bilateral data about their anterograde and retrograde tracking of CCK-expressing neurons in the rhinal cortex. In ll. 290-292, they described as follows: "Both anterograde and retrograde tracking results indicated that CCK-expressing neurons in the rhinal cortex projecting to the motor cortex were asymmetric, showing a preference for the ipsilateral hemisphere." However, they provided only unilateral data for the anterograde (Fig. 4B) and the retrograde (Fig. 4D) experiments.

Fourth, unilateral (contralateral to the dominant forelimb) experiments are needed in the chemogenetic inhibition of the CCK neurons. In ll. 301-338 and Fig. 5, the authors inhibited the CCK -expressing neurons in both hemispheres by injecting the virus into both sides. However, the CCKBR antagonist injection into the motor cortex contralateral to the dominant forelimb caused defects in motor learning ability, as described in ll. 166-206. The authors also observed that the population neuronal activity in the motor cortex contralateral to the dominant forelimb changed in accordance with the improvement of the motor skill in ll. 208-269. Therefore, it may be the case that inhibition of CCK neurons only in the side contralateral to the dominant forelimb - not bilaterally, as the authors did - could cause the lowered ability of motor learning. Such unilateral inhibition can be carried out by unilateral injection of the virus.

In relation to the point above, in the chemogenetic inhibition experiments, it would be important to show which neurons in which cortical area is inhibited. This could be done by examining the distributions of the mCherry-labeled somata in the rhinal cortex using histochemistry.

Fifth, it would be valuable to further examine differences in task performance across sessions and groups. The paragraph in ll. 138-153 needs a comparison of the "miss" rates of CCK-/- animals between Day 1 vs. Day 6 (related to ll. 429- 431). This paragraph also needs comparisons of the "no-grasp" and "drop" rates of CCK-/- animals between Day 1 vs. Day 6 (related to ll. 432- 433). The paragraph in ll. 175-190 needs comparisons of success rates between Day 1 and Day 5/6 within the antagonist group (related to ll. 447-448).

• for: 3 Deg C world - catastrophic

Reviewer #3 (Public Review):

The study by Ngodup and colleagues describes the contribution of sodium leak NALCN conductance on the effects of noradrenaline on cartwheel interneurons of the DCN. The manuscript is very well-written and the experiments are well-controlled. The scope of the study is of high biological relevance and recapitulates a primary finding of the Khaliq lab (Philippart et al., eLife, 2018) in ventral midbrain dopamine neurons, that Gi/o-coupled receptors inhibit NALCN current to reduce neuronal excitability. Together these studies provide unequivocable evidence for NALCN as a downstream target of these receptors.

In re-review of this study, the authors have addressed the concerns sufficiently. This is a very nice study.

Reviewer #3 (Public Review):

Summary:<br /> Much of the literature on attention has focused on static, non-contingent stimuli that can be easily controlled and replicated--a mismatch with the actual day-to-day deployment of attention. The same limitation is evident in the developmental literature, which is further hampered by infants' limited behavioral repertoires and the general difficulty in collecting robust and reliable data in the first year of life. The current study engages young infants as they play with age-appropriate toys, capturing visual attention, cardiac measures of arousal, and EEG-based metrics of cognitive processing. The authors find that the temporal relations between measures are different at age 5 months vs. age 10 months. In particular, at 5 months of age, cardiac arousal appears to precede attention, while at 10 months of age attention processes lead to shifts in neural markers of engagement, as captured in theta activity.

Strengths:<br /> The study brings to the forefront sophisticated analytical and methodological techniques to bring greater validity to the work typically done in the research lab. By using measures in the moment, they can more closely link biological measures to actual behaviors and cognitive stages. Often, we are forced to capture these measures in separate contexts and then infer in-the-moment relations. The data and techniques provide insights for future research work.

Weaknesses:

The sample is relatively modest, although this is somewhat balanced by the sheer number of data points generated by the moment-to-moment analyses. In addition, the study is cross-sectional, so the data cannot capture true change over time. Larger samples, followed over time, will provide a stronger test for the robustness and reliability of the preliminary data noted here. Finally, while the method certainly provides for a more active and interactive infant in testing, we are a few steps removed from the complexity of daily life and social interactions.

Reviewer #3 (Public Review):

Summary:<br /> The authors are looking for a spatially specific functional brain response to visualise non-invasively with 3T (clinical field strength) MRI. They propose a velocity-nulled weighting to remove the signal from draining veins in a submillimeter multiband acquisition.

Strengths:<br /> - This manuscript addresses a real need in the cognitive neuroscience community interested in imaging responses in cortical layers in-vivo in humans.<br /> - An additional benefit is the proposed implementation at 3T, a widely available field strength.

Weaknesses:<br /> - Although the VASO acquisition is discussed in the introduction section, the VN-sequence seems closer to diffusion-weighted functional MRI. The authors should make it more clear to the reader what the differences are, and how results are expected to differ. Generally, it is not so clear why the introduction is so focused on the VASO acquisition (which, curiously, lacks a reference to Lu et al 2013). There are many more alternatives to BOLD-weighted imaging for fMRI. CBF-weighted ASL and GRASE have been around for a while, ABC and double-SE have been proposed more recently.<br /> - The comparison in Figure 2 for different b-values shows % signal changes. However, as the baseline signal changes dramatically with added diffusion weighting, this is rather uninformative. A plot of t-values against cortical depth would be much more insightful.<br /> - Surprisingly, the %-signal change for a b-value of 0 is not significantly different from 0 in the gray matter. This raises some doubts about the task or ROI definition. A finger-tapping task should reliably engage the primary motor cortex, even at 3T, and even in a single participant.<br /> - The BOLD weighted images in Figure 3 show a very clear double-peak pattern. This contradicts the results in Figure 2 and is unexpected given the existing literature on BOLD responses as a function of cortical depth.<br /> - Given that data from Figures 2, 3, and 4 are derived from a single participant each, order and attention affects might have dramatically affected the observed patterns. Especially for Figure 4, neither BOLD nor VN profiles are really different from 0, and without statistical values or inter-subject averaging, these cannot be used to draw conclusions from.<br /> - In Figure 5, a phase regression is added to the data presented in Figure 4. However, for a phase regression to work, there has to be a (macrovascular) response to start with. As none of the responses in Figure 4 are significant for the single participant dataset, phase regression should probably not have been undertaken. In this case, the functional 'responses' appear to increase with phase regression, which is contra-intuitive and deserves an explanation.<br /> - Consistency of responses is indeed expected to increase by a removal of the more variable vascular component. However, the microvascular component is always expected to be smaller than the combination of microvascular+macrovascular responses. Note that the use of %signal changes may obscure this effect somewhat because of the modified baseline. Another expected feature of BOLD profiles containing both micro- and microvasculature is the draining towards the cortical surface. In the profiles shown in Figure 7, this is completely absent. In the group data, no significant responses to the task are shown anywhere in the cortical ribbon.<br /> - Although I'd like to applaud the authors for their ambition with the connectivity analysis, I feel that acquisitions that are so SNR starved as to fail to show a significant response to a motor task should not be used for brain wide directed connectivity analysis.

The claim of specificity is supported by the observation of the double-peak pattern in the motor cortex, previously shown in multiple non-BOLD studies. However, this same pattern is shown in some of the BOLD weighted data, which seems to suggest that the double-peak pattern is not solely due to the added velocity nulling gradients. In addition, the well-known draining towards the cortical surface is not replicated for the BOLD-weighted data in Figures 3, 4, or 7. This puts some doubt about the data actually having the SNR to draw conclusions about the observed patterns.

Reviewer #3 (Public Review):

Summary:<br /> This study studied the neural mechanisms underlying the shift of ocular dominance induced by "dichoptic-backward-movie" adaptation. The study is self-consistent.

Strengths:<br /> The experimental design is solid and progressive (relationship among three studies), and all of the raised research questions were well answered.

The logic behind the neural mechanisms is solid.

The findings regarding the cTMS (especially the position/site can be useful for future medical implications).

Weaknesses:<br /> Why does the "dichoptic-backward-movie" adaptation matter? This part is severely missing. This kind of adaptation is neither intuitive like the classical (Gbison) visual adaptation, nor practical as adaptation as a research paradigm as well as the fundamental neural mechanism. If this part is not clearly stated and discussed, this study is just self-consistent in terms of its own research question. There are tons of "cool" phenomena in which the neural mechanisms are apparent as "FEF controls vision-attention" but never tested using TMS & fMRI, but we all know that this kind of research is just of incremental implications.

Reviewer #3 (Public Review):

Summary:

Through a detailed methodology, the authors demonstrated that within 11 different primates, the shape of the brain matched a fractal of dimension 2.5. They enhanced the universality of this result by showing the concordance of their results with a previous study investigating 70 mammalian brains, and the discordance of their results with other folded objects that are not brains. They incidentally illustrated potential applications of this fractal property of the brain by observing a scale-dependent effect of aging on the human brain.

Strengths:

- New hierarchical way of expressing cortical shapes at different scales derived from the previous report through the implementation of a coarse-graining procedure.<br /> - Positioning of results in comparison to previous works reinforcing the validity of the observation.<br /> - Illustration of scale-dependence of effects of brain aging in the human.

Weaknesses:

- The impact of the contribution should be clarified compared to previous studies (implementation of new coarse graining procedure, dimensionality of primate brain vs previous studies, and brain aging observations).<br /> - The rather small sample sizes, counterbalanced by the strength of the effect demonstrated.<br /> - The use of either averaged or individual brains for the different sub-studies could be made clearer.<br /> - The model discussed hypothetically in the discussion is not very clear, and may not be state-of-the-art (axonal tension driving cortical folding? cf. https://doi.org/10.1115/1.4001683).

Reviewer #3 (Public Review):

Summary:<br /> The high heterogeneity nature of α-synuclein (α-syn) fibrils posed significant challenges in structural reconstruction of the ex vivo conformation. A deeper understanding of the factors influencing the formation of various α-syn polymorphs remains elusive. The manuscript by Frey et al. provides a comprehensive exploration of how pH variations (ranging from 5.8 to 7.4) affect the selection of α-syn polymorphs (specifically, Type1, 2, and 3) in vitro by using cryo-electron microscopy (cryo-EM) and helical reconstruction techniques. Crucially, the authors identify two novel polymorphs at pH 7.0 in PBS. These polymorphs bear resemblance to the structure of patient-derived juvenile-onset synucleinopathy (JOS) polymorph and diseased tissue amplified α-syn fibrils. The manuscript supports the notion that seeding is non-polymorph-specific in the context of secondary nucleation-dominated aggregation, underscoring the irreplaceable role of pH in polymorph formation. Nevertheless, certain areas within the manuscript would benefit from further refinement and elaboration to more robustly substantiate this hypothesis.

Strengths:<br /> This study systematically investigates the effects of environmental conditions and seeding on the structure of α-syn fibrils. It emphasizes the significant influence of environmental factors, especially pH, in determining the selection of α-syn polymorphs. The high-resolution structures obtained through cryo-EM enable a clear characterization of the composition and proportion of each polymorph in the sample. Collectively, this work provides strong support for the pronounced sensitivity of α-syn fibril structures to environmental conditions and systematically categorizes previously reported α-syn fibril structures. Furthermore, the identification of JOS-like polymorph also demonstrates the possibility of in vitro reconstruction of brain-derived α-syn fibril structures.

Weaknesses:<br /> 1. The authors reveal that both Type 1 monofilament fibril polymorph (reminiscent of JOS-like polymorph) and Type 5 polymorph (akin to tissue-amplified-like polymorph) can both form under the same condition. Additionally, this condition also fosters the formation of flat ribbon-like fibril across different batches. Notably, at pH 5.8, variations in experimental groups yield disparate abundance ratios between polymorph 3B and 3C, indicating a degree of instability in fibrillar formation. The variability would potentially pose challenges for replicability in subsequent research. In light of these situations, I propose the following recommendations:

(1) An explicit elucidation of the factors contributing to these divergent outcomes under similar experimental conditions is warranted. This should include an exploration of whether variations in purified protein batches are contributing factors to the observed heterogeneity.

(2) To enhance the robustness of the conclusions, additional replicates of the experiments under the same condition should be conducted, ideally a minimum of three times.

(3) Further investigation into whether different polymorphs formed under the same buffer condition could lead to distinct toxicological and pathology effects would be a valuable addition to the study.

2. The cross-seeding study presented in the manuscript demonstrates the pivotal role of pH conditions in dictating conformation. However, an intriguing aspect that emerges is the potential role of seed concentration in determining the resultant product structure. This raises a critical question: at what specific seed concentration does the determining factor for polymorph selection shift from pH condition to seed concentration? A methodological robust approach to address this should be conducted through a series of experiments across a range of seed concentrations. Such an approach could delineate a clear boundary at which seed concentration begins to predominantly dictate the conformation, as opposed to pH conditions. Incorporating this aspect into the study would not only clarify the interplay between seed concentration and pH conditions, but also add a fascinating dimension to the understanding of polymorph selection mechanisms.

Furthermore, the study prompts additional queries regarding the behavior of cross-seeding production under the same pH conditions when employing seeds of distinct conformation. Evidence from various studies, such as those involving E46K and G51D cross-seeding, suggests that seed structure plays a crucial role in dictating polymorph selection. A key question is whether these products consistently mirror the structure of their respective seeds.

3. In the Results section of "The buffer environment can dictate polymorph during seeded nucleation", the authors reference previous cell biological and biochemical assays to support the polymorph-specific seeding of MSA and PD patients under the same buffer conditions. This discussion is juxtaposed with recent research that compares the in vivo biological activities of hPFF, ampLB as well as LB, particularly in terms of seeding activity and pathology. Notably, this research suggests that ampLB, rather than hPFF, can accurately model the key aspects of Lewy Body Diseases (LBD) (refer to: https://doi.org/10.1038/s41467-023-42705-5). The critical issue here is the need to reconcile the phenomena observed in vitro with those in in-vivo or in-cell models. Given the low seed concentration reported in these studies, it is imperative for the authors to provide a more detailed explanation as to why the possible similar conformation could lead to divergent pathologies, including differences in cell-type preference and seeding capability.

4. In the Method section of "Image processing", the authors describe the helical reconstruction procedure, without mentioning much detail about the 3D reconstruction and refinement process. For the benefit of reproducibility and to facilitate a deeper understanding among readers, the authors should enrich this part to include more comprehensive information, akin to the level of detail found in similar studies (refer to: https://doi.org/10.1038/nature23002).

5. The abbreviation of amino acids should be unified. In the Results section "On the structural heterogeneity of Type 1 polymorphs", the amino acids are denoted using three-letter abbreviation. Conversely, in the same section under "On the structural heterogeneity of Type 2 and 3 structures", amino acids are abbreviated using the one-letter format. For clarity and consistency, it is essential that a standardized format for amino acid abbreviations be adopted throughout the manuscript.

Reviewer #3 (Public Review):

This manuscript focuses on defining the importance of UGGT1/2 in the process of protein degradation within the ER. The authors prepared cells lacking UGGT1, UGGT2, or both UGGT1/UGGT2 (DKO) HCT116 cells and then monitored the degradation of specific ERAD substrates. Initially, they focused on the ER stress sensor ATF6 and showed that loss of UGGT1 increased the degradation of this protein. This degradation was stabilized by deletion of ERAD-specific factors (e.g., SEL1L, EDEM) or treatment with mannose inhibitors such as kifunesine, indicating that this is mediated through a process involving increased mannose trimming of the ATF6 N-glycan. This increased degradation of ATF6 impaired the function of this ER stress sensor, as expected, reducing the activation of downstream reporters of ER stress-induced ATF6 activation. The authors extended this analysis to monitor the degradation of other well-established ERAD substrates including A1AT-NHK and CD3d, demonstrating similar increases in the degradation of destabilized, misfolding protein substrates in cells deficient in UGGT. Importantly, they did experiments to suggest that re-overexpression of wild-type, but not catalytically deficient, UGGT rescues the increased degradation observed in UGGT1 knockout cells. Further, they demonstrated the dependence of this sensitivity to UGGT depletion on N-glycans using ERAD substrates that lack any glycans. Ultimately, these results suggest a model whereby depletion of UGGT (especially UGGT1 which is the most expressed in these cells) increases degradation of ERAD substrates through a mechanism involving impaired re-glucosylation and subsequent re-entry into the calnexin/calreticulin folding pathway.

I must say that I was under the impression that the main conclusions of this paper (i.e., UGGT1 functions to slow the degradation of ERAD substrates by allowing re-entry into the lectin folding pathway) were well-established in the literature. However, I was not able to find papers explicitly demonstrating this point. Because of this, I do think that this manuscript is valuable, as it supports a previously assumed assertion of the role of UGGT in ER quality control. However, there are a number of issues in the manuscript that should be addressed.

Notably, the focus on well-established, trafficking-deficient ERAD substrates, while a traditional approach to studying these types of processes, limits our understanding of global ER quality control of proteins that are trafficked to downstream secretory environments where proteins can be degraded through multiple mechanisms. For example, in Figure 1-Figure Supplement 2, UGGT1/2 knockout does not seem to increase the degradation of secretion-competent proteins such as A1AT or EPO, instead appearing to stabilize these proteins against degradation. They do show reductions in secretion, but it isn't clear exactly how UGGT loss is impacting ER Quality Control of these more relevant types of ER-targeted secretory proteins.

Lastly, I don't understand the link between UGGT, ATF6 degradation, and ATF6 activation. I understand that the idea is that increased ATF6 degradation afforded by UGGT depletion will impair activation of this ER stress sensor, but if that is the case, how does UGGT2 depletion, which only minimally impacts ATF6 degradation (Fig. 1), impact activation to levels similar to the UGGT1 knockout (Fig 4)? This suggests UGGT1/2 may serve different functions beyond just regulating the degradation of this ER stress sensor. Also, the authors should quantify the impaired ATF6 processing shown in Fig 4B-D across multiple replicates.

Ultimately, I do think the data support a role for UGGT (especially UGGT1) in regulating the degradation of ERAD substrates, which provides experimental support for a role long-predicted in the field. However, there are a number of ways this manuscript could be strengthened to further support this role, some of which can be done with data they have in hand (e.g., the stats) or additional new experiments.

Reviewer #3 (Public Review):

In their paper entitled "Fear conditioning biases olfactory stem cell receptor fate" Liff et al. address the still enigmatic (and quite fascinating) phenomenon of intergenerationally inherited changes in the olfactory system in response to odor-dependent fear conditioning.

In the abstract / summary, the authors raise expectations that are not supported by the data. For example, it is claimed that "increases in F0 were due to biased stem cell receptor choice." While an active field of study that has seen remarkable progress in the past decade, olfactory receptor gene choice and its relevant timing in particular is still unresolved. Here, Liff et al., do not pinpoint at what stage during differentiation the "biased choice" is made.

Similarly, the concluding statement that the study provides "insight into the heritability of acquired phenotypes" is somewhat misleading. The experiments do not address the mechanisms underlying heritability.

The statement that "the percentage of newborn M71 cells is 4-5 times that of MOR23 may simply reflect differences in the birth rates of the two cell populations" should, if true, result in similar differences in the occurrence of mature OSNs with either receptor identity. According to Fig. 1H & J, however, this is not the case.

An important result is that Liff et al., in contrast to results from other studies, "do not observe the inheritance of odor-evoked aversion to the conditioned odor in the F1 generation." This discrepancy needs to be discussed.

The authors speculate that "the increase in neurons responsive to the conditioned odor could enhance the sensitivity to, or the discrimination of, the paired odor in F0 and F1. This would enable the F1 population to learn that odor predicts shock with fewer training cycles or less odorant when trained with the conditioned odor." This is a fascinating idea that, in fact, could have been readily tested by Liff and coworkers. If this hypothesis were found true, this would substantially enhance the impact of the study for the field.

Reviewer #3 (Public Review):

Summary:<br /> The authors food-deprived male and female mice and observed a much stronger reduction of leptin levels, energy consumption in the visual cortex, and visual coding performance in males than females. This indicates a sex-specific strategy for the regulation of the energy budget in the face of low food availability.

Strengths:<br /> This study extends a previous study demonstrating the effect of food deprivation on visual processing in males, by providing a set of clear experimental results, demonstrating the sex-specific difference. It also provides hypotheses about the strategy used by females to reduce energy budget based on the literature.

Weaknesses:<br /> The authors do not provide evidence that females are not impacted by visually guided behaviors contrary to what was shown in males in the previous study.