Reviewer #3 (Public Review):

Summary:

The manuscript by Kyusang Yoo et al. "Muscle-resident mesenchymal progenitors sense and repair peripheral nerve injury via the GDNF-BDNF axis" investigates the role and mechanisms of fibro-adipogenic progenitors (FAPs), that are muscle-resident mesenchymal progenitors, in the maturation and maintenance of the neuromuscular system. There is earlier evidence that absence of FAPs or its functional decline with age cause smaller regenerated myofibers. Role of FAPs on peripheral nerve regeneration is very poorly studied. This study has translational importance because traumatic injury to the peripheral nerve can cause lifelong paralysis of the injured limb.

This manuscript provides data indicating that GDNF-BDNF axis plays an important role in peripheral nerve regeneration and function.

Strengths:

Because the role of FAPs on peripheral nerve regeneration is very poorly studied this investigation is a major step towards understanding the mechanism on the role of FAPs. They use scRNA-seq, animal models, and cKO mice that is also important. This study has translational importance because traumatic injury to the peripheral nerve can cause lifelong paralysis of the injured limb.<br /> This is an interesting and original study focusing on the role of FAPs and indicating that GDNF-BDNF axis plays an important role in peripheral nerve regeneration and function.

Weaknesses:

In Fig. 1 and 2 authors provide data on scRNA seq and this is important information reporting the finding of RET and GFRa1 transcripts in the subpopulation of FAP cells. However, authors provide no data on the expression of RET and GFRa1 proteins in FAP cells.<br /> Another problem is the lack of information showing that GDNF secreted by Schwann cells can activate RET and its down-stream signaling in FAP cells.<br /> There is no direct experimental proof that GDNF activating GFRa1-RET signaling triggers BDNF upregulation In FAP cells.<br /> The data that GDNF signaling is inducing the synthesis and secretion of BDNF is also not conclusive.

Reviewer #3 (Public Review):

Summary:

The authors have tested for and demonstrated a physical (i.e., sensory nerves to the brain) connection between tumors and parts of the brain. This can explain why there is an increase in depressive disorders in HNSCC patients. While connections such as this have been suspected, this is a novel demonstration pointing to sensory neurons that is accompanied by a remarkable amount of complementary data.

Strengths:

There is substantial evidence provided for the hypotheses tested. The data are largely quite convincing.

Weaknesses:

The authors mention in their Discussion the need for additional experiments. Could they also include / comment on the potential impact on the anti-tumor immune system in their model?

Minor:

The authors mention the importance of inflammation contributing to pain in cancer but do not clearly highlight how this may play a role in their model. Can this be clarified?

The tumor model apparently requires isoflurane injection prior to tumor growth measurements. This is different from most other transplantable types of tumors used in the literature. Was this treatment also given to control (i.e., non-tumor) mice at the same time points? If not, can the authors comment on the impact of isoflurane (if any) in their model?

The authors emphasize in several places that this is a male mouse model. They mention this as a limitation in the Discussion. Was there an original reason why they only tested male mice?

Reviewer #3 (Public Review):

Summary:

This valuable study shows that shorter episodes (2 minutes duration) of energy depletion, as it occurs in ischemia, could lead to long-lasting dysregulation of synaptic transmission with presynaptic alterations of glutamate release at the CA3-CA1 synapses. A longer duration of chemical ischemia (5 minutes) permanently suppresses synaptic transmission. By using electrophysiological approaches, including field and patch clamp recordings, combined with imaging studies, the authors demonstrated that 2 minutes of chemical ischemia leads to a prolonged potentiation of synaptic activity with a long-lasting increase of glutamate release from presynaptic terminals. This was observed as an increase in iGluSnFR fluorescence, a sensor for glutamate expressed selectively on hippocampal astrocytes by viral injection. The increase in iGluSnFR fluorescence upon 2-minute chemical ischemia could not be ascribed to an altered glutamate uptake, which is unaffected by both 2-minute and 5-minute chemical ischemia. The presynaptic increase in glutamate release upon short episodes of chemical ischemia is confirmed by a reduced inhibitory effect of the competitive antagonist gamma-D-glutamylglycine on AMPA receptor-mediated postsynaptic responses. Fiber volley durations in field recording are prolonged in slices exposed to 2 min chemical ischemia. The authors interpret this data as an indication that the increase in glutamate release could be ascribed to a prolongation of the presynaptic action potential possibly due to inactivation of voltage-dependent K+ channels. However, more direct evidence is needed to support this hypothesis fully. This research highlights an important mechanism by which altered ionic homeostasis underlying metabolic failure can impact on neuronal activity. Moreover, it also showed a different vulnerability of mechanisms involved in glutamatergic transmission with a marked resilience of glutamate uptake to chemical ischemia.

Strengths:

(1) The authors use a variety of experimental techniques ranging from electrophysiology to imaging to study the contribution of several mechanisms underlying the effect of chemical ischemia on synaptic transmission.

(2) The experiments are appropriately designed and clearly described in the figures and in the text.

(3) The controls are appropriate.

Weaknesses:

- The data on fiber volley duration should be supported by more direct measurements to prove that chemical ischemia increases presynaptic Ca2+ influx due to a presynaptic broadening of action potentials. Given the influence that positioning of the stimulating and recording electrode can have on the fiber volley properties, I found this data insufficient to support the assumption of a relationship between increased iGluSnFR fluorescence, action potential broadening, and increased presynaptic Ca2+ levels.

- The results are obtained in an ex-vivo preparation, it would be interesting to assess if they could be replicated in vivo models of cerebral ischemia.

Impact:

This study provides a more comprehensive view of the long-term effects of energy depletion during short episodes of experimental ischemia leading to the notion that not only post-synaptic changes, as reported by others, but also presynaptic changes are responsible for long-lasting modification of synaptic transmission. Interestingly, the direction of synaptic changes is bidirectional and dependent on the duration of chemical ischemia, indicating that different mechanisms involved in synaptic transmission are differently affected by energy depletion.

Reviewer #3 (Public Review):

The manuscript by Tie et al. provides a quantitative assessment of intra-Golgi transport of diverse cargos. Quantitative approaches using fluorescence microscopy of RUSH synchronized cargos, namely GLIM and measurement of Golgi residence time, previously developed by the author's team (publications from 20216 to 2022), are being used here.

Most of the results have been already published by the same team in 2016, 2017, 2020 and 2021. In this manuscript, very few new data have been added. The authors have put together measurements of intra-Golgi transport kinetics and Golgi residence time of many cargos. The quantitative results are supported by a large number of Golgi mini-stacks/cells analyzed. They are discussed with regard to the intra-Golgi transport models being debated in the field, namely the cisternal maturation/progression model and the stable compartments model. However, over the past decades, the cisternal progression model has been mostly accepted thanks to many experimental data.

The authors show that different cargos have distinct intra-Golgi transport kinetics and that the Golgi residence time of glycosyltransferases is high. From this and the experiment using brefeldinA, the authors suggest that the rim progression model, adapted from the stable compartments model, fits with their experimental data.

Strengths:

The major strength of this manuscript is to put together many quantitative results that the authors previously obtained and to discuss them to give food for thought about the intra-Golgi transport mechanism.<br /> The analysis by fluorescence microscopy of intra-Golgi transport is tough and is a tour de force of the authors even if their approach show limitations, which are clearly stated. Their work is remarkable in regards to the numbers of Golgi markers and secretory cargos which have been analyzed.

Weaknesses:

As previously mentioned, most of the data provided here were already published and thus accessible for the community. Is there is a need to publish them again?<br /> The authors' discussion about the intra-Golgi transport model is rather simplistic. In the introduction, there is no mention of the most recent models, namely the rapid partitioning and the rim progression models. To my opinion, the tubular connections between cisternae and the diffusion/biochemical properties of cargos are not enough taken into account to interpret the results. Indeed, tubular connections and biochemical properties of the cargos may affect their transit through the Golgi and the kinetics with which they reach the TGN for Golgi exit.<br /> Nocodazole is being used to form Golgi mini-stacks, which are necessary to allow intra-Golgi measurement. The use of nocodazole might affect cellular homeostasis but this is clearly stated by the authors and is acceptable as we need to perturb the system to conduct this analysis. However, the manual selection of the Golgi mini-stack being analyzed raises a major concern. As far as I understood, the authors select the mini-stacks where the cargo and the Golgi reference markers are clearly detectable and separated, which might introduce a bias in the analysis.<br /> The terms 'Golgi residence time ' is being used but it corresponds to the residence time in the trans-cisterna only as the cargo has been accumulated in the trans-Golgi thanks to a 20{degree sign}C block. The kinetics of disappearance of the protein of interest is then monitored after 20{degree sign}C to 37{degree sign}C switch.<br /> Another concern also lies in the differences that would be introduced by different expression levels of the cargo on the kinetics of their intra-Golgi transport and of their packaging into post-Golgi carriers.

Reviewer #3 (Public Review):

Summary:

This study employs an optogenetics approach aimed at activating oncogene (KRASG12V) expression in a single somatic cell, with a focus on following the progression of activated cell to examine tumourigenesis probabilities under altered tissue environments. The research explores the role of stemness factors (VENTX/NANOG/OCT4) in facilitating oncogenic RAS (KRASG12V)-driven malignant transformations. Although the evidence provided are incomplete, the authors propose an important mechanism whereby reactivation of re-programming factors correlates with the increased likelihood of a mutant cell undergoing malignant transformation.

Strengths:

· Innovative Use of Optogenetics: The application of optogenetics for precise activation of KRAS in a single cell is valuable to the field of cancer biology, offering an opportunity to uncover insight into cellular responses to oncogenic mutations.<br /> · Important Observations: The findings concerning stemness factors' role in promoting oncogenic transformation are important, contributing data to the field of cancer biology.

Weaknesses:

Lack of Methodological Clarity: The manuscript lacks detailed descriptions of methodologies, making it difficult to fully evaluate the experimental design and reproducibility, rendering incomplete evidence to support the conclusion. Improving methodological transparency and data presentation will crucially strengthen the paper's contributions to understanding the complex processes of tumourigenesis.<br /> Sub-optimal Data Presentation and Quality:

The resolution of images throughout the manuscript are too low. Images presented in Figure 2 and Figure 4 are of very low resolution. It is very hard to distinguish individual cells and in which tissue they might reside.<br /> Lack of quantitative data and control condition data obtained from images of higher magnification limits the ability to robustly support the conclusions.

Here are some details:<br /> · Tissue specificity of the cells express KRASG12V oncogene: In this study, the ubiquitin promoter was used to drive oncogenic KRASG12V expression. Despite this, the authors claim to activate KRAS in a single brain cell based on their localized photo-activation strategy. However, upon reviewing the methods section, the description was provided that 'Localized uncaging was performed by illumination for 7 minutes on a Nikon Ti microscope equipped with a light source peaking at 405 nm, Figure 1. The size of the uncaging region was controlled by an iris that defines a circular illumination with a diameter of approximately 80 μm.' It is surprising that an epi-fluorescent microscope with an illumination diameter of around 80μm can induce activation in a single brain cell beneath skin tissue. Additionally, given that the half-life for mTFP maturation is around 60 minutes, it is likely that more cells from a variety of different lineages could be activated, but the fluorescence would not be visible until more than 1-hour post-illumination. Authors might want to provide more evidence to support their claim on the single cell KRAS activation.<br /> · Stability of cCYC: The manuscript does not provide information on the half-life and stability of cCYC. Understanding these properties is crucial for evaluating the system's reliability and the likelihood of leakiness, which could significantly influence the study's outcomes.<br /> · Metastatic Dissemination claim: Typically, metastatic cancer cells migrate to and proliferate within specific niches that are conducive to outgrowth, such as the caudal hematopoietic tissue (CHT) or liver. In figure 3 A, an image showing the presence of mTFP expressing cells in both the head and tail regions of the larva, with additional positive dots located at the fin fold. This is interpreted as "metastasis" by the authors. However, the absence of a supportive cellular compartment within the fin-fold tissue makes the presence of mTFP-positive metastatic cells there particularly puzzling. This distribution raises concerns about the spatial specificity of the optogenetic activation protocol.<br /> The unexpected locations of these signals suggest potential ectopic activation of the KRAS oncogene, which could be occurring alongside or instead of targeted activation. This issue is critical as it could affect the interpretation of whether the observed mTFP signal expansion over time is due to actual cell proliferation and infiltration, or merely a result of ectopic RAS transgene activation.<br /> · Image Resolution Concerns: The cells depicted in Figure 3C β, which appear to be near the surface of the yolk sac and not within the digestive system as suggested in the MS, underscore the necessity for higher-resolution imaging. Without clearer images, it is challenging to ascertain the exact locations and states of these cells, thus complicating the assessment of experimental results.<br /> · The cell transplantation experiment is lacking protocol details: The manuscript does not adequately describe the experimental protocols used for cell transplantation, particularly concerning the origin and selection of cells used for injection into individual larvae. This omission makes it difficult to evaluate the reliability and reproducibility of the results. Such as the source of transplanted cells:<br /> • If the cells are derived from hyperplastic growths in larvae where RAS and VX (presumably VENTX) were locally activated, the manuscript fails to mention any use of fluorescence-activated cell sorting (FACS) to enrich mTFP-positive cells. Such a method would be crucial for ensuring the specificity of the cells being studied and the validity of the results.<br /> • If the cells are obtained from whole larvae with induced RAS + VX expression, it is notable and somewhat surprising that the larvae survived up to six days post-induction (6dpi) before cells were harvested for transplantation. This survival rate and the subsequent ability to obtain single cell suspensions raise questions about the heterogeneity of the RAS + VX expressing cells that transplanted.<br /> · Unclear Experimental Conditions in Figure S3B: The images in Figure S3B lack crucial details about the experimental conditions. It is not specified whether the activation of KRAS was targeted to specific cells or involved whole-body exposure. This information is essential for interpreting the scope and implications of the results accurately.<br /> · Contrasting Data in Figure S3C compared to literature: The graph in Figure S3C indicates that KRAS or KRAS + DEX induction did not result in any form of hyperplastic growth. This observation starkly contrasts with previous literature where oncogenic KRAS expression in zebrafish led to significant hyper-proliferation and abnormal growth, as evidenced by studies such as those published in and Neoplasia (2018), DOI: 10.1016/j.neo.2018.10.002; Molecular Cancer (2015), DOI: 10.1186/s12943-015-0288-2; Disease Models & Mechanisms (2014) DOI: 10.1242/dmm.007831. The lack of expected hyperplasia raises questions about the experimental setup or the specific conditions under which KRAS was expressed. The authors should provide detailed descriptions of the conditions under which the experiments were conducted in Figure S3B and clarifying the reasons for the discrepancies observed in Figure S3C are crucial. The authors should discuss potential reasons for the deviation from previous reports.

Further comments:

Throughout the study, KRAS-activated cell expansion and metastasis are two key phenotypes discussed that Ventx is promoting. However, the authors did not perform any experiments to directly show that KRAS+ cells proliferate only in Ventx-activated conditions. The authors also did not show any morphological features or time-lapse videos demonstrating that KRAS+ cells are motile, even though zebrafish is an excellent model for in vivo live imaging. This seems to be a missed opportunity for providing convincing evidence to support the authors' conclusions.

There were minimal experimental details provided for the qPCR data presented in the supplementary figures S5 and S6, therefore, it is hard to evaluate result obtained.

Reviewer #3 (Public Review):

Summary:

In this manuscript Menon, Adhikari, and Mondal analyze explicit solvent molecular dynamics (MD) computer simulations of the intrinsically disordered protein (IDP) alpha-synuclein in the presence and absence of a small molecule ligand, Fasudil, previously demonstrated to bind alpha-synuclein by NMR spectroscopy without inducing folding into more ordered structures. In order to provide insight into the binding mechanism of Fasudil the authors analyze an unbiased 1500us MD simulation of alpha-synuclein in the presence of Fasudil previously reported by Robustelli et.al. (Journal of the American Chemical Society, 144(6), pp.2501-2510). The authors compare this simulation to a very different set of apo simulations: 23 separate1-4us simulations of alpha-synuclein seeded from different apo conformations taken from another previously reported by Robustelli et. al. (PNAS, 115 (21), E4758-E4766), for a total of ~62us.

To analyze the conformational space of alpha-synuclein - the authors employ a variational auto-encoder (VAE) to reduce the dimensionality of Ca-Ca pairwise distances to 2 dimensions, and use the latent space projection of the VAE to build Markov state Models. The authors utilize k-means clustering to cluster the sampled states of alpha-synuclein in each condition into 180 microstates on the VAE latent space. They then coarse grain these 180 microstates into a 3-macrostate model for apo alpha-synuclein and a 6-macrostate model for alpha-synuclein in the presence of fasudil using the PCCA+ course graining method. Few details are provided to explain the hyperparameters used for PCCA+ coarse graining and the rationale for selecting the final number of macrostates.

The authors analyze the properties of each of the alpha-synuclein macrostates from their final MSMs - examining intramolecular contacts, secondary structure propensities, and in the case of alpha-synuclein:Fasudil holo simulations - the contact probabilities between Fasudil and alpha-synuclein residues.

The authors utilize an additional variational autoencoder (a denoising convolutional VAE) to compare denoised contact maps of each macrostate, and project onto an additional latent space. The authors conclude that their apo and holo simulations are sampling distinct regions of the conformational space of alpha-synuclein projected on the denoising convolutional VAE latent space.

Finally, the authors calculate water entropy and protein conformational entropy for each microstate. To facilitate water entropy calculations - the author's take a single structure from each macrostate - and ran a 20ps simulation at a finer timestep (4 femtoseconds) using a previously published method (DoSPT), which computes thermodynamic properties of water from MD simulations using autocorrelation functions of water velocities. The authors report that water entropy calculated from these individual 20ps simulations is very similar.

For each macrostate the authors compute protein conformational entropy using a previously published Maximum Information Spanning tree approach based on torsion angle distributions - and observe that the estimated protein conformational entropy is substantially more negative for the macrostates of the holo ensemble.

The authors calculate mean first passage times from their Markov state models and report a strong correlation between the protein conformational entropy of each state and the mean first passage time from each state to the highest populated state.

As the authors observe the conformational entropy estimated from macrostates of the holo alpha-synuclein:Fasudil is greater than those estimated from macrostates of the apo holo alpha-synuclein macrostates - they suggest that the driving force of Fasudil binding is an increase in the conformational entropy of alpha-synuclein. No consideration/quantification of the enthalpy of alpha-synuclein Fasudil binding is presented.

Strengths:

The author's utilize MD simulations run with an appropriate force field for IDPs (a99SB-disp and a99SB-disp water (Robustelli et. al, PNAS, 115 (21), E4758-E4766) - which has previously been used to perform MD simulations of alpha-synuclein that have been validated with extensive NMR data.

The contact probability between Fasudil and each alpha-synuclein residue observed in the previously performed 1500us MD simulation of alpha-synuclein in the presence of Fasudil (Robustelli et. al., Journal of the American Chemical Society, 144(6), pp.2501-2510) was previously found to be in good agreement with experimental NMR chemical shift perturbations upon Fasudil binding - suggesting that this simulation is a reasonable choice for understanding IDP:small molecule interactions.

Weaknesses:

Major Weakness 1: Simulations of apo alpha-synuclein and holo simulations of alpha-synuclein and fasudil are not comparable.

The most robust way to determine how presence of Fasudil affects the conformational ensemble of alpha-synuclein conclusions is to run apo and holo simulations of the same length from the same starting structures using the same simulation parameters.

The 23 1-4 us independent simulations of apo alpha-synuclein and the long unbiased 1500us alpha-synuclein in the presence of fasudil are not directly comparable. The starting structures of simulations used to build a Markov state model to describe apo alpha-synuclein were taken from a previously reported 73us MD simulation of alpha-synuclein run with the a99SB-disp force field and water model) with 100mM NaCl, (Robustelli et. al, PNAS, 115 (21), E4758-E4766). As the holo simulation of alpha-synuclein and Fasudil was run in 50mM NaCl, snapshots from the original apo alpha-synuclein simulation were resolvated with 50mM NaCl - and new simulations were run.

No justification is offered for how starting structures were selected. We have no sense of the conformational variability of the starting structures selected and no sense of how these conformations compare to the alpha-synuclein conformations sampled in the holo simulation in terms of standard structural descriptors such as tertiary contacts, secondary structure, radius of gyration (Rg), solvent exposed surface area etc. (we only see a comparison of projections on an uninterpretable non-linear latent-space and average contact maps). Additionally, 1-4 us is a relatively short timescale for a simulation of a 140 residue IDP- and one is unlikely to see substantial evolution for many structural properties of interest (ie. secondary structure, radius of gyration, tertiary contacts) in simulations this short. Without any information about the conformational space sample in the 23 apo simulations (aside from a projection on an uninterpretable latent space)- we have no way to determine if we observe transitions between distinct states in these short simulations, and therefore if it is possible the construct a meaningful MSM from these simulations.

If the structures used for apo simulations are on average more compact or contain more tertiary contacts - then it is unsurprising that in short independent simulations they sample a smaller region of conformational space. Similarly, if the starting structures have similar dimensions - but we only observe extremely local sampling around starting structures in apo simulations in the short simulation times - it would also not be surprising that we sample a smaller amount of conformational space. By only presenting comparisons of conformational states on an uninformative VAE latent space - it is not possible for a reader to ask simple questions about how the conformational ensembles compare.

It is noted that the authors attempt to address questions about sampling by building an MSM of single contiguous 60us portion of the holo simulation of alpha-synuclein and Fasudil - noting that:

"the MSM built using lesser data (and same amount of data as in water) also indicated the presence of six states of alphaS in presence of fasudil, as was observed in the MSM of the full trajectory. Together, this exercise invalidates the sampling argument and suggests that the increase in the number of metastable macrostates of alphaS in fasudil solution relative to that in water is a direct outcome of the interaction of alphaS with the small molecule."

However, the authors present no data to support this assertion - and readers have no sense of how the conformational space sampled in this portion of the trajectory compares to the conformational space sampled in the independent apo simulations or the full holo simulation. As the analyzed 60us portion of the holo trajectory may have no overlap with conformational space sampled in the independent apo simulations - it is unclear if this control provides any information. There is no quantification of the conformational entropy of the 6 states obtained from this portion of the holo trajectory or the full conformational space sampled. No information is presented to determine if we observe similar states in the shorter portion of the holo trajectory. Furthermore - as the authors provide almost no justification for the criteria used to select of the final number of macrostates for any of the MSMs reported in this work- and the number of macrostates is effectively a free parameter in the PCCA+ method, arriving at an MSM with 6 macrostates does not convey any information about the conformational entropy of alpha-synuclein in the presence or absence of ligands. Indeed - the implied timescale plot for 60us holo MSM (Figure S2) - shows that at least 10 processes are resolved in the 120 microstate model - and there is no information to provided explaining/justifying how a final 6-macrostate model was determined. The authors also do not project the conformations sampled in this sub- trajectory onto the latent space of the final VAE.

One certainly expects that an MSM built with 1/20th of the simulation data should have substantial differences from an MSM built from the full trajectory - so failing additional information and hyperparameter justification - one wonders if the emergence of a 6-state model could be the direct result of hardcoded VAE and MSM construction hyperparameter choices.

Required Controls For Supporting the Conclusions of the Study: The authors should initiate apo and holo simulations from the same starting structures - using the same simulation software and parameters. This could be done by adding a Fasudil ligand to the apo structures - or by removing the Fasudil ligand from a subset of holo structures. This would enable them to make apples-to-apples comparisons about the effect of Fasudil on alpha-synuclein conformational space.

Failing to add direct apples-to-apples comparisons, which would be required to truly support the studies conclusions, the authors should at least compare the conformational space sampled in the independent apo simulations and holo simulations using standard interpretable IDP order parameters (ie. Rg, end-to-end distance, secondary structure order parameters) and/or principal components from PCA or tICA obtained from the holo simulation. The authors should quantify the number of transitions observed between conformational states in their apo simulations. The authors could also perform more appropriate holo controls, without additional calculations, by taking batches of a similar number of short 1-4us segments of simulations used to compute the apo MSMs and examining how the parameters/macrostates of the holo MSMs vary with the input with random selections.

Major Weakness 2: There is little justification of how the hyperparameters MSMs were selected. It is unclear if the results of the study depend on arbitrary hyperparameter selections such as the final number of macrostates in each model.

It is unclear what criteria were used to determine the appropriate number of microstates and macrostates for each MSM. Most importantly - as all analyses of water entropy and conformational entropy are restricted to the final macrostates - the criteria used to select the final number of macrostates with the PCCA+ are extremely important to the results of the conclusions of the study. From examining the ITS plots in Figure 3 - it seems both MSMs show the same number of resolved processes (at least 11) - suggesting that a 10-state model could be apropraite for both systems. If one were to simply select a large number of macrostates for the 20x longer holo simulation - do these states converge to the same conformational entropy as the states seen in the short apo simulations? Is there some MSM quality metric used to determine what number of macrostates is more appropriate?

Required Controls For Supporting the Conclusions of the Study: The authors should specify the criteria used to determine the appropriate number of microstates and macrostates for their MSMs and present controls that demonstrate that the conformational entropies calculated for their final states are not simply a function of the ratio of the number macrostates chosen to represent very disparate amounts of conformational sampling.

Major Weakness 3: The use of variational autoencoders (VAEs) obscures insights into the underlying conformational ensembles of apo and holo alpha-synuclein rather than providing new ones.

No rationale is offered for the selection of the VAE architecture or hyperparameters used to reduce the dimensionality of alpha-synuclein conformational space.

It is not clear the VAEs employed in this study are providing any new insight into the conformational ensembles and binding mechanisms of Fasudil to alpha-synuclein, or if the underlying latent space of the VAEs are more informative or kinetically meaningful than standard linear dimensionality reduction techniques like PCA and tICA. The initial VAE is used to reduce the dimensionality of alpha-synuclein conformational ensembles to 2 degrees of freedom - but it is unclear if this projection is structurally or kinetically meaningful. It is not clear why the authors choice to use a 2-dimeinsional projection instead of a higher number of dimensions to build their MSMs. Can they produce a more kinetically and structurally meaningful model using a higher dimensional VAE latent space?

Additionally - it is not clear what insights are provided by the Denoising Convolutional Variational Autoencoder. The authors appear to be noising-and-denoising the contact maps of each macrostate, and then projecting the denoised values onto a new latent space - and commenting that they are different. Does this provide additional insight that looking at the contact maps in Figures 4&5 does not? Is this more informative than examining the distribution of the Radii of gyration or the secondary structure propensities of each ensemble? It is not clear what insight this analysis adds to the manuscript.

Suggested controls to improve the study: The authors should project interpretable IDP structural descriptors (ie. secondary structure, radius of gyration, secondary structure content, # of intramolecular contacts, # of intermolecular contacts between alpha-synuclein and Fasudil ) onto this latent space to illustrate if any of these properties are meaningful separated by the VAE projection. The authors should compare these projections, and MSMs built from these projections, to projections and MSMs built from projections using standard linear dimensionality projection techniques like PCA and tICA.

Major Weakness 4: The MSMs produced in this study have large discrepancies with MSMs previously produced on the same dataset by the same authors that are not discussed.

Previously - two of the authors of this manuscript (Menon and Mondal) authored a preprint titled "Small molecule modulates α-synuclein conformation and its oligomerization via Entropy Expansion" (https://www.biorxiv.org/content/10.1101/2022.10.20.513005v1.full) that analyzed the same 1500us holo simulation of alpha-synuclein binding Fasudil. In this study - they utilized the variational approach to Markov processes (VAMP) to build an MSM using a 1D order parameter as input (the radius of gyration), first discretizing the conformational space into 300 microstates before similarly building a 6 macrostate model. From examining the contact maps and secondary structure propensities of the holo MSMs from the current study and the previous study- some of the macrostates appear similar, however there appear to be orders of magnitude differences in the timescales of conformational transitions between the two models. The timescales of conformational transitions in the previous MSM are on the order of 10s of microseconds, while the timescales of transitions in this manuscript are 100s-1000s microseconds. In the previous manuscript, a 3 state MSM is built from an apo α-synuclein obtained from a continuous 73ms unbiased MD simulation of alpha-synuclein run at a different salt concentration (100mM) and an additional 33 ms of shorter simulations. The apo MSM from the previous study similarly reports very fast timescales of transitions between apo states (on the order ~1ms) - while the MSM reported in the current study (Figure 9) are on the order of 10s-100s of microseconds).

These discrepancies raise further concerns that the properties of the MSMs built on these systems are extremely sensitive to the chosen projection methods and MSM modeling choices and hyperparameters, and that neither model may be an accurate description of the true underlying dynamics

Suggestions to improve the study: The authors should discuss the discrepancies with the MSMs reported in their previous studies.

Reviewer #3 (Public Review):

The manuscript by Chang, Quinodoz and Brangwynne describes the results of live cell imaging of fluorescently labeled Alu element genomic sites in combination with H2B-GFP marked chromatin in human cancer cells. The study includes dCas9 based genomic engineering for Suntag enhanced Alu element labeling. The motion of Alu elements and chromatin was analyzed in real time at 500 ms intervals over 1 min at high resolution. Advanced image analysis algorithms were developed.

The main objective of the study is to understand how motion of euchromatin or active chromatin relates to chromatin density. Alu elements, which are spread throughout the genome are used as a proxy for euchromatin or also A compartments. The study finds that Alu-rich chromatin is more mobile than Alu poor one and that actinomycin but not flavopyridol or alpha amanitin cause some decrease in the determined mobility. The authors emphasize the heterogeneity of motion, Alu clustering and chromatin density underscoring the complexity of the problem.

Although the topic is important and the imaging well performed, the study lacks depth and does not provide any truly new insights into our understanding of the link between genome activity and mobility nor diffusive behavior of the chromatin fiber in situ. Although the approach to record context dependent dynamics based on segmentation of pixels of varying intensity is elegant, the analysis of the trajectories requires further explanation and justification to be able to interpret the results. Important information on the biology of the engineered cell lines is lacking. Presented results are not discussed with respect to existing literature and knowledge.

Major concerns:<br /> - Are Alu elements a good proxy for A compartments? What consequences do massive dCas9 tags have on the genome and the engineered cells? How does the bulky dCas9-Suntag label impact mobility and transcription of Alu elements themselves? How many off target sites are potentially labeled?

(1) The authors should state the size of the dCas9-Suntag construct and perform FRAP analysis to compare the tag's behavior to the one of H2B-GFP<br /> (2) dCas9 locally unwinds DNA and is strongly bound to its target sequence impeding polymerase progression.<br /> (3) The authors need to check if DNA breaks are induced. An immunofluorescence using a gH2AX antibody is a minimum in all conditions tested. DNA breaks largely affect chromatin mobility which is a topic of major debate (see PMC5769766, PMID33061931).<br /> (4) The authors need to confirm that in dCas/sgAlu cells Alu elements are still transcribed similarly to wt cells (transcriptome or at least some qPCR).<br /> (5) Please compare H2B-GFP mobility of sgAlu tagged and untagged cells.<br /> (6) Figure 1D shows significant background in the Cut&run sgAlu line compared to H3K4me3 line. Are these off target sites? Was the H3K4me3 Cut&run performed in the engineered cell line? Did the authors test another guide RNA? Non-specific binding could also contribute to the observed heterogeneity in the measured dynamics.<br /> (7) Figure 3G shows that H2B MSND at tau=5s is high for high H2B density independently of Alu density questioning the value of using Alu sg tagging as a proxy for euchromatin.

- What are the physical principles of the measured motion? What is the rationale for the MSND analyses deployed in this study?<br /> (1) Please provide the equation used for MSND (seems to be different from the standard MSD one).<br /> (2) Figure 3: all MSD curves have a slope suggesting an alpha exponent significantly smaller than 0.5 reminiscent of subdiffusion (example panels A and E compare thick line to slope of the triangle bottom right). Please explain. Is it gaussian noise? Confinement? This was seen before for faster acquisition rates, but still requires explanation and interpretation.<br /> (3) What is the rationale for choosing the value at τ =5 s? Figure 3 panel E shows large variations in the MSND at all time points, curves do not start at the same lag time.<br /> (4) Figure S5 shows that for Alu elements, alpha is close to 0.5 at τ =<1 s but lower for larger tau, the relationship to intensity is inverse as well. Please explain.<br /> (5) It would be important to show the D values of your estimations. Plots for MSD curves in non log scale are important to be presented to show if there are different diffusion regimes (such as in Figure 4).<br /> (6) It is mentioned that the "Our measurements of total chromatin dynamics at lag time τ = 5 s are typically on the order of 10-2 μm2 (Figure 3 A, B), in agreement with past studies (Shaban et al., 2020; Zidovska et al., 2013)". This is inaccurate as both cited studies were performed at different time lags 0.2 sec. Change in time lag is supposed to show different diffusion behaviour. For consistency, the comparison should be done at the same time lag and the same number of analyzed video frames.<br /> (7) The study applies the MSND analysis for different time lags starting from 0.5 s to 11 s for videos of 60 s. Change in the number of data points affects the accuracy to calculate the diffusion coefficient. What is the impact of this uncertainty on the results and conclusions?

- Inhibition of polymerase 2 activity increases mobility as was shown before.<br /> (1) Figure 4: change in motion following alpha amanitin and Flavopiridol treatments recapitulate results from the Maeshima group (Nagashima 2019). Data shown for actinomycin treated cells appear extreme. A huge drop in H2B MSND (panel B and D). Please ensure that the cells are still alive after 4-6h exposure to ActD. ActD also affects cytoskeleton and replication, so different conclusion may be drawn if cells are still alive.<br /> (2) Treatment effects could also be enhanced should dCas9/ sgAlu induce massive DNA damage (see above). Check H2B-GFP motion in cells (both treated and not) not labeled with sgAlu.

- Positioning with respect to the literature:<br /> (1) The introduction, first paragraph is oversimplified, please review the literature citing work performed by many groups in the field using H2B-GFP, telomere or single site labeling in the past 10 years. Give details on the cell type used (mouse or human normal or cancer cells, amplified signals or single genes, same cell or cells at different stages of development, methodologies from whole genome to single particle tracking etc.).<br /> (2) The manuscript claims to introduce a novel mapping of the spatiotemporal dynamics of the A compartment in living cells. However, the authors did not discuss other previous approaches that were developed for the same purpose. The dynamic motion of active transcription chromatin domains/A compartment over the whole nucleus was investigated in different studies that used Mintbody labeling, please check PMCID: PMC7926250, PMCID: PMC8647360, PMID: 27534817, PMCID: PMC8491620<br /> (3) PIV applies a relatively large interrogation window size of micrometers to estimate the displacement vectors. Dynamic changes within the set window can include both A and B compartments, where the contribution of genomic processes to local chromatin motion, typically taking place at the nanometer scale, is missed. The Hi-D method ( PMCID: PMC7168861) introduced an Optical Flow approach to overcome this limitation of PIV (PMCID: PMC6061878 ). Could the authors test if Hi-D method to analyze the movies recorded in this study confirms their conclusions?

Heterogeneity of chromatin dynamics independent of chromatin density was shown by previous studies such as PMCID: PMC7775763 , and PMCID: PMC7168861 . Could the authors discuss their findings in the context of these studies?

Reviewer #3 (Public Review):

Summary:

The research by Qianqian Ju et al. found that the knockdown of TAK1 promoted ESCC migration and invasion, whereas overexpression of TAK1 resulted in the opposite outcome. These in vitro findings could be recapitulated in a xenograft metastasis mouse model.

Mechanistically, TAK1 phosphorylates PLCE1 S1060 in the cells, decreasing PLCE1 enzyme activity and repressing PIP2 hydrolysis. As a result, reducing DAG and inositol IP3, thereby suppressing signal transduction of PKC/GSK 3β/β Catenin. Consequently, cancer metastasis-related genes were impeded by TAK1.

Overall, this study offers some intriguing observations. Providing a potential druggable target for developing agents for dealing with ESCC.

The strengths of this research are:

(1) The research always uses different experimental approaches to address one question. The experiments are largely convincing and appear to be well executed.<br /> (2) The phenotypes were observed from different angles: at the mouse model, cellular level, and molecular level.<br /> (3) The molecular mechanism was down to a single amino acid modification on PLCE1.

The weaknesses part of this research are:

(1) Most of the phenotypes are only observed in the ECA-109 cell line. Whether TAK1-PLCE1 S1060 is a common pathway in other ESCC cells or just specific to the ECA-109 cell line is unclear. Using more cell lines to see whether this is a common mechanism of ESCC metastasis would greatly amplify the impact of this finding.<br /> (2) Most of the experiments were done in protein overexpression conditions, with the protein level increasing hundreds of folds in the cell, producing an artificial environment that would sometimes generate false positive results.<br /> (3) Whether TAK1 can directly phosphorylate PLCE1 S1060 needs more tests, especially the in vitro biochemical evidence.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors used a Drosophila model to show that exposure to repetitive mild TBI causes neurodegenerative conditions that emerge late in life and disproportionately affect females. In addition to well-known age-dependent impact, the authors identified Sex Peptide (SP) signaling as a key factor in female susceptibility to post-injury brain deficits.

Strengths:

The authors have presented a compelling set of results showing that female Sex Peptide signaling adversely affects late-life neurodegeneration after early-life exposure to repetitive mild head injury in Drosophila. They have (1) compared the phenotypes of adult male and female flies sustaining TBI at different ages, and the phenotypes of virgin females and mated females, (2) compared the phenotypes of eliminating SP signaling in mating females and introducing SP-signaling into virgin females, (3) compared transcriptomic changes of different groups in response to TBI. The results are generally consistent and robust.

Weaknesses:

The authors have made their claims largely based on assaying climbing index and vacuole formation as the only indicators of late-life neurodegeneration after TBI. However, these phenotypes are not really specific to TBI-related neurodegeneration, and the significance and mechanisms of especially vacuole formation are not clear. The authors should perform additional analyses on TBI-related neurodegeneration in flies, which have been shown before (Genetics. 2015 Oct; 201(2): 377-402). Furthermore, it is also really surprising to see so few DEGs even in wild-type males and mated females, and to see that none of the DEGs overlapped among groups or are even related to the SP-signaling. This raises questions about the validity of the RNA-seq analysis. It is critical to independently verify their RNA-sequencing results and to add some more molecular evidence to support their conclusion. Finally, it is unknown what the implication of female fly mating and its associated Sex Peptide signaling would be to mammalians or humans, and what are the mechanisms underlying the sexual dimorphism.

Reviewer #3 (Public Review):

Summary:

The manuscript uses high-content imaging and advanced image-analysis tools to monitor the infection of epithelial cells by Shigella. They perform some analysis on the state of the cells (through measurements of DNA and protein synthesis), and then they focus on differential recruitment of Sept7 to the bacteria. They link this recruitment with the activity of the bacterial T3SS, which is a very interesting discovery. Overall, I found numerous exciting elements in this manuscript, and I have a couple of reservations. Please see below for more details on my reservations. Nevertheless, I think that these issues can be addressed by the authors, and doing so will help to make it a convincing and interesting piece for the community working on intracellular pathogens. The authors should also carefully re-edit their manuscript to avoid overselling their data (see below for issues I see there). I would consider taking out the first figure and starting with Figure 3 (Figure 2 could be re-organized in the later parts)- that could help to make the flow of the manuscript better.

Strengths:

The high-content analysis including the innovative analytical workflows are very promising and could be used by a large number of scientists working on intracellular bacteria.

The finding that Septins (through SEPT7) are differentially regulated through actively secreting bacteria is very exciting and can steer novel research directions.

Weaknesses:

The manuscript makes a connection between two research lines (1: Shigella infection and DNA/protein synthesis, 2: regulation of septins around invading Shigella) that are not fully developed - this makes it sometimes difficult to understand the take-home messages of the authors.

It is not clear whether the analysis that was done on projected images actually reflects the phenotypes of the original 3D data. This issue needs to be carefully addressed.

Reviewer #3 (Public Review):

Summary:

The authors synthesized a compound which can inhibit ELF3 and MED23 interaction which leads to inhibition of HER2 expression in gastric cancer.

Strengths:

Enough evidence shows the potency of compound 10 in inhibiting ELF3 and MED23 interaction.

Weaknesses:

Compound 10 potency as PPI inhibitor has been shown in only one cell line NCI-N87.

Reviewer #3 (Public Review):

Genetic manipulation of Leishmania has some challenges, including some limitations in the DNA repair strategies that are present in the organism and the absence of RNA interference in many species. The senior author has contributed significantly to expanding the available routes towards Leishmania genetic manipulation by developing and adapting CRISPR-Cas9 tools to allow gene manipulation via DNA double-strand break repair and, more recently, base modification. This work seeks to improve on some limitations in the tools previously described for the latter approach of base modification leading to base change.

The work in the paper is meticulously described, with solid evidence for most of the improvements that are claimed: Figure1 clearly describes reduced impairment in the growth of parasites expressing sgRNAs via changes in promoters; Figures 2 and 3 compellingly document the usefulness of using AsCas12a for integration after transformation; and Figures 1 and 4 demonstrate the capacity of the combined modifications to efficiently edit a gene in three different Leishmania species. There is little doubt these new tools will be adopted by the Leishmania community, adding to the growing arsenal of approaches for genetic manipulation.

There are two weaknesses the authors may wish to address, one smaller and one larger.

(1) The main advance claimed here is in this section title: 'Integration of CBE sgRNA expression cassettes via AsCas12a ultra-introduced DSBs increase editing rates', with the evidence for this presented in Figure 4. It is hard work in the submission to discern what direct evidence there is for editing rates being improved relative to earlier, Cas9-based approaches. Did they directly compare the editing by the new and old approach? If not, can they more clearly explain how they are able to make this claim, either by adding text or a new figure? A side-by-side comparison would emphasise the advance of the new approach more clearly.

(2) The ultimate, stated goal of this work is (abstract) to 'enable a variety of loss-of-function screens', as the older approach had some limitations. This goal is not tested for the new tools that have been developed here; the experiment in Figure 5 merely shows that they can, not unexpectedly, make a gene mutant, which was already possible with available tools. Thus, to what extent is this paper describing a step forward? Why have the authors not run an experiment - even the same one that was described previously in Engstler and Beneke (2023) - to show that the new approach improves on previous tools in such a screen, either in scale or accuracy?

Reviewer #3 (Public Review):

Summary:

The goal of this study is to understand how, unlike other mammals, kangaroos are able to increase hopping speed without a concomitant increase in metabolic cost. They use a biomechancial analysis of kangaroo hopping data across a range of speeds to investigate how posture, effective mechanical advantage, and tendon stress vary with speed and mass. The main finding is that a change in posture leads to increasing effective mechanical advantage with speed, which ultimately increases tendon elastic energy storage and returns via greater tendon strain. Thus kangaroos may be able to conserve energy with increasing speed by flexing more, which increases tendon strain.

Strengths:

The approach and effort invested into collecting this valuable dataset of kangaroo locomotion is impressive. The dataset alone is a valuable contribution.

Weaknesses:

Despite these strengths, I have concerns regarding the strength of the results and the overall clarity of the paper and methods used (which likely influences how convincingly the main results come across).

(1) The paper seems to hinge on the finding that EMA decreases with increasing speed and that this contributes significantly to greater tendon strain estimated with increasing speed. It is very difficult to be convinced by this result for a number of reasons:<br /> • It appears that kangaroos hopped at their preferred speed. Thus the variability observed is across individuals not within. Is this large enough of a range (either within or across subjects) to make conclusions about the effect of speed, without results being susceptible to differences between subjects? In the literature cited, what was the range of speeds measured, and was it within or between subjects?<br /> • Assuming that there is a compelling relationship between EMA and velocity, how reasonable is it to extrapolate to the conclusion that this increases tendon strain and ultimately saves metabolic cost? They correlate EMA with tendon strain, but this would still not suggest a causal relationship (incidentally the p-value for the correlation is not reported). Tendon strain could be increasing with ground reaction force, independent of EMA. Even if there is a correlation between strain and EMA, is it not a mathematical necessity in their model that all else being equal, tendon stress will increase as ema decreases? I may be missing something, but nonetheless, it would be helpful for the authors to clarify the strength of the evidence supporting their conclusions.<br /> • The statistical approach is not well-described. It is not clear what the form of the statistical model used was and whether the analysis treated each trial individually or grouped trials by the kangaroo. There is also no mention of how many trials per kangaroo, or the range of speeds (or masses) tested. Related to this, there is no mention of how different speeds were obtained. It seems that kangaroos hopped at a self-selected pace, thus it appears that not much variation was observed. I appreciate the difficulty of conducting these experiments in a controlled manner, but this doesn't exempt the authors from providing the details of their approach.<br /> • Some figures (Figure 2 for example) present means for one of three speeds, yet the speeds are not reported (except in the legend) nor how these bins were determined, nor how many trials or kangaroos fit in each bin. A similar comment applies to the mass categories. It would be more convincing if the authors plotted the main metrics vs. speed to illustrate the significant trends they are reporting.

(2) The significance of the effects of mass is not clear. The introduction and abstract suggest that the paper is focused on the effect of speed, yet the effects of mass are reported throughout as well, without a clear understanding of the significance. This weakness is further exaggerated by the fact that the details of the subject masses are not reported.

(3) The paper needs to be significantly re-written to better incorporate the methods into the results section. Since the results come before the methods, some of the methods must necessarily be described such that the study can be understood at some level without turning to the dedicated methods section. As written, it is very difficult to understand the basis of the approach, analysis, and metrics without turning to the methods.

Reviewer #3 (Public Review):

Summary:

This is an interesting paper investigating fMRI changes during sensory (visual, tactile) stimulation and absence seizures in the GAERS model. The results are potentially important for the field and do suggest that sensory stimulation may not activate brain regions normally during absence seizures. However the findings are limited by substantial methodological issues that do not enable fMRI signals related to absence seizures to be fully disentangled from fMRI signals related to the sensory stimuli.

Strengths:

Investigating fMRI brain responses to sensory stimuli during absence seizures in an animal model is a novel approach with potential to yield important insights.

Use of an awake, habituated model is a valid and potentially powerful approach.

The major difficulty with interpreting the results of this study is that the duration of the visual and tactile stimuli were 6 seconds, which is very close to the mean seizure duration per Table 1. Therefore the HRF model looking at fMRI responses to visual or auditory stimuli occurring during seizures was simultaneously weighting both seizure activity and the sensory (visual or auditory) stimuli over the same time intervals on average. The resulting maps and time courses claiming to show fMRI changes from visual or auditory stimulation during seizures will therefore in reality contain some mix of both sensory stimulation-related signals and seizure-related signals. The main claim that the sensory stimuli do not elicit the same activations during seizures as they do in the interictal period may still be true. However the attempts to localize these differences in space or time will be contaminated by the seizure related signals.

In their repeated responses to this comment the authors have stated that some seizures had longer than average duration, and that they have attempted to model the effects of both seizures and sensory stimulation. However these factors do not mitigate the concern because the mean duration of seizures and sensory stimulation remain nearly identical, and the models used therefore will not be able to effectively separate signals related to seizures and related to sensory stimulation. Hemodynamic models can never in reality represent underlying signals in an orthogonal manner, and are only indirectly related to neural activity.

The only way to truly address the important weakness of this study would be to repeat the experiments using stimulus durations that do not match mean seizure duration, e.g. with much shorter duration stimuli.

The authors have clarified and improved the figure images and their description in the text based on previous specific comments. However, the main weakness in the results remains as summarized above.

Minor comments:

Aside from the concerns listed as weaknesses above which were not addressed, most of the more minor comments were addressed by the authors in the resubmissions. However, the comment made twice previously regarding Figure 6-figure supplement 1 was not addressed. It remains impossible to see any firing rate changes elicited by sensory stimuli during the ictal period in parts E and F of the figure vs. parts B and C (interictal), due to the very different scales used. The seizure signals should be removed or accounted for by the model so that any possible sensory stimulus-related signals could be seen, and/or displayed on the same scale as firing rates without seizures. The authors have simply restated their opinion that it is better to include the SWD dynamics in these figures parts, however this makes the figure wholly unconvincing. It is also concerning that part D (ictal), which is in fact shown on the same scale as part A (interictal), actually shows larger firing rates for both excitatory and inhibitory neurons in visual cortex for sensory stimulation during seizures. This contradicts the claims in the rest of the paper that neural activity and fMRI signals are smaller or are even decreased in visual cortex with sensory stimulation during seizures compared to the interictal period.

Reviewer #3 (Public Review):

Summary:

This manuscript reports an experiment that compared groups of rats acquisition and performance of a Pavlovian bi-conditional discrimination, in which the presence of one cue, A, signals that the presentation of one CS, X, will be followed by a reinforcer and a second CS, Y, will be nonreinforced. Periods of cue A alternated with periods of cue B, which signaled the opposite relationship, cue X is nonreinforced and cue Y is reinforced. This is a conditional discrimination problem in which the rats learned to approach the food cup in the presence of each CS conditional on the presence of the third background cue. The comparison groups consisted of the same conditional discrimination with the exception that each CS was paired with a different reinforcer. This makes the problem easier to solve as the background is now priming a differential outcome. A third group received simple discrimination training of X reinforced and Y nonreinforced in cues A and B, and the final group were trained with X and Y reinforced on half the trials (no discrimination). The results were clear that the latter two discrimination learning procedures resulted in rapid learning in comparison to the first. Rats required about 3 times as many 4-session blocks to acquire the bi-conditional discrimination than the other two discrimination groups. Within the biconditional discrimination group, female and male rats spent the same amount of time in the food cup during the rewarded CS, but females spent more time in the food cup during CS- than males. The authors interpret this as a deficit in discrimination performance in females on this task and use a measure that exaggerates the difference in CS+ and CS_ responding (a discrimination ratio) to support their point. When tested after acute restraint stress, the male rats spent less time in the food cup during the reinforced CS in comparison to the female rats, but did not lose discrimination performance entirely. The was also some evidence of more fos positive cells in the orbitofrontal cortex in females. Overall, I think the authors were successful in documenting performance on the biconditional discrimination task, showing that it is more difficult to perform than other discriminations is valuable and consistent with the proposal that accurate performance requires encoding of conditional information (which the authors refer to as "context"). There is evidence that female rats spend more time in the food cup during CS-, but this I hesitate to agree that this is an important sex difference. There is no cost to spending more time in the food cup during CS- and they spend much less time there than during CS+. Males and females also did not differ in their CS+ responding, suggesting similar levels of learning, A number of factors could contribute to more food cup time in CS-, such as smaller body size and more locomotor activity. The number of food cup entries during CS+ and CS- was not reported here. Nevertheless, I think the manuscript will make a useful contribution to the field and hopefully lead readers to follow up on these types of tasks. One area for development would be to test the associative properties of the cues controlling the conditional discrimination, can they be shown to have the properties of Pavlovian occasion setting stimuli? Such work would strengthen the justification/rationale for using the term "context" and "occasion setter" to refer to these stimuli in this task in the way the authors do in this paper.

Strengths:

Nicely designed and conducted experiment.<br /> Documents performance difference by sex.

Weaknesses:

Overstatement of sex differences.<br /> Inconsistent, confusing, and possibly misleading use of terms to describe/imply the underlying processes contributing to performance.

Reviewer #3 (Public Review):

Summary:

This work examined efference copy related to eye movements in healthy adults who have high autistic traits. Efference copies allow the brain to make predictions about sensory outcomes of self-generated actions, and thus serve important roles in motor planning and maintaining visual stability. Consequently, disrupted efference copies have been posited as a potential mechanism underlying motor and sensory symptoms in psychopathology such as Autism Spectrum Disorder (ASD), but so far very few studies have directly investigated this theory. Therefore, this study makes an important contribution as an attempt to fill in this knowledge gap. The authors conducted two eye-tracking experiments examining the accuracy of motor planning and visual perception following a saccade, and found that participants with high autistic traits exhibited worse task performance (i.e., less accurate second saccade and biased perception of object displacement), consistent with their hypothesis of less impact of efference copies on motor and visual updating. Moreover, the motor and visual biases are positively correlated, indicative of a common underlying mechanism. These findings are promising and can have important implications for clinical intervention, if they can be replicated in a clinical sample.

Strengths:

The authors utilized well-established and rigorously designed experiments and sound analytic methods. This enables easy translations between similar work in non-human primates and humans and readily points to potential candidates for underlying neural circuits that could be further examined in follow-up studies (e.g., superior colliculus, frontal eye fields, mediodorsal thalamus). The finding of no association between initial saccade accuracy and level of autistic trait in both experiments also serves as an important control analysis and increases one's confidence in the conclusion that the observed differences in task performance were indeed due to disrupted efference copies, not confounding factors such as basic visual/motor deficits or issues with working memory. The strong correlation between the observed motor and visual biases further strengthens the claim that the findings from both experiments may be explained by the same underlying mechanism - disrupted efference copies. Lastly, the authors also presented a thoughtful and detailed mechanistic theory of how efference copy impairment may lead to ASD symptomatology, which can serve as a nice framework for more research into the role of efference copies in ASD.

Weaknesses:

Although the paper has a lot of strengths, the main weakness of the paper is that a direct link with sensory/motor symptoms cannot be established. As the authors have discussed, the most likely symptoms resulting from disrupted efference copies would be sensory overload and motor inflexibility. The measure used to quantify the level of autistic traits, Autistic Quotient (AQ), does not capture any sensory or motor characteristics of the Autism spectrum. Therefore, it is unknown whether those scored high on AQ in this study experienced high, or even any, sensory or motor difficulties. In other words, more evidence is needed to demonstrate a direct link between disrupted efference copies and sensory/motor symptoms in ASD.

Reviewer #3 (Public Review):

Summary:

In this study, Manley and Vaziri designed and built a Fourier light-field microscope (fLFM) inspired by previous implementations but improved and exclusively from commercially available components so others can more easily reproduce the design. They combined this with the design of novel algorithms to efficiently extract whole-brain activity from larval zebrafish brains.

This new microscope was applied to the question of the origin of behavioral variability. In an assay in which larval zebrafish are exposed to visual dots of various sizes, the fish respond by turning left or right or not responding at all. Neural activity was decomposed into an activity that encodes the stimulus reliably across trials, a 'noise' mode that varies across trials, and a mode that predicts tail movements. A series of analyses showed that trial-to-trial variability was largely orthogonal to activity patterns that encoded the stimulus and that these noise modes were related to the larvae's behavior.

To identify the origins of behavioral variability, classifiers were fit to the neural data to predict whether the larvae turned left or right or did not respond. A set of neurons that were highly distributed across the brain could be used to classify and predict behavior. These neurons could also predict spontaneous behavior that was not induced by stimuli above chance levels. The work concludes with findings on the distributed nature of single-trial decision-making and behavioral variability.

Strengths:

The design of the new fLFM microscope is a significant advance in light-field and computational microscopy, and the open-source design and software are promising to bring this technology into the hands of many neuroscientists.

The study addresses a series of important questions in systems neuroscience related to sensory coding, trial-to-trial variability in sensory responses, and trial-to-trial variability in behavior. The study combines microscopy, behavior, dynamics, and analysis and produces a well-integrated analysis of brain dynamics for visual processing and behavior. The analyses are generally thoughtful and of high quality. This study also produces many follow-up questions and opportunities, such as using the methods to look at individual brain regions more carefully, applying multiple stimuli, investigating finer tail movements and how these are encoded in the brain, and the connectivity that gives rise to the observed activity. Answering questions about variability in neural activity in the entire brain and its relationship to behavior is important to neuroscience and this study has done that to an interesting and rigorous degree.

Points of improvement and weaknesses:

The results on noise modes may be a bit less surprising than they are portrayed. The orthogonality between neural activity patterns encoding the sensory stimulus and the noise modes should be interpreted within the confounds of orthogonality in high-dimensional spaces. In higher dimensional spaces, it becomes more likely that two random vectors are almost orthogonal. Since the neural activity measurements performed in this study are quite high dimensional, a more explicit discussion is warranted about the small chance that the modes are not almost orthogonal.

The conclusion that sparsely distributed sets of neurons produce behavioral variability needs more investigation because the way the results are shown could lead to some misinterpretations. The prediction of behavior from classifiers applied to neural activity is interesting, but the results are insufficiently presented for two reasons.

(1) The neurons that contribute to the classifiers (Figures 4H and J) form a sufficient set of neurons that predict behavior, but this does not mean that neurons outside of that set cannot be used to predict behavior. Lasso regularization was used to create the classifiers and this induces sparsity. This means that if many neurons predict behavior but they do so similarly, the classifier may select only a few of them. This is not a problem in itself but it means that the distributions of neurons across the brain (Figures 4H and J) may appear sparser and more distributed than the full set of neurons that contribute to producing the behavior. This ought to be discussed better to avoid misinterpretation of the brain distribution results, and an alternative analysis that avoids the confound could help clarify.

(2) The distribution of neurons is shown in an overly coarse manner in only a flattened brain seen from the top, and the brain is divided into four coarse regions (telencephalon, tectum, cerebellum, hindbrain). This makes it difficult to assess where the neurons are and whether those four coarse divisions are representative or whether the neurons are in other non-labeled deeper regions. For these two reasons, some of the statements about the distribution of neurons across the brain would benefit from a more thorough investigation.

Reviewer #3 (Public Review):

Summary:

Taking advantage of the existence in fish of two genes coding for estrogen synthase, the enzyme aromatase, one mostly expressed in the brain (Cyp19a1b) and the other mostly found in the gonads (Cyp19a1a), this study investigates the role of neuro-estrogens in the control of sexual and aggressive behavior in teleost fish. The constitutive deletion of Cyp19a1b reduced brain estrogen content by 87% in males and about 50% in females. It led to reduced sexual and aggressive behavior in males and reduced sexual behavior in females. These effects are reversed by adult treatment with estradiol thus indicating that they are activational in nature. The deletion of Cyp19a1b is associated with a reduced expression of the genes coding for the two androgen receptors, ara, and arb, in brain regions involved in the regulation of social behavior. The analysis of the gene expression and behavior of mutants of estrogen receptors indicates that these effects are likely mediated by the activation of the esr1 and esr2a isoforms. These results provide valuable insight into the role of neuro-estrogens in social behavior in the most abundant vertebrate taxa. While estrogens are involved in the organization of the brain and behavior of some birds and rodents, neuro-estrogens appear to play an activational role in fish through a facilitatory action of androgen signaling.

Strengths:

- Evaluation of the role of brain "specific" Cyp19a1 in male teleost fish, which as a taxa are more abundant and yet proportionally less studied than the most common birds and rodents. Therefore, evaluating the generalizability of results from higher vertebrates is important. This approach also offers great potential to study the role of brain estrogen production in females, an understudied question in all taxa.

- Results obtained from multiple mutant lines converge to show that estrogen signaling drives aspects of male sexual behavior.

- The comparative discussion of the age-dependent abundance of brain aromatase in fish vs mammals and its role in organization vs activation is important beyond the study of the targeted species.

Weaknesses:

- The new transgenic lines are under-characterized. There is no evaluation of the mRNA and protein products of Cyp19a1b and ESR2a.

- The stereotypic sequence of sexual behavior is poorly described, in particular, the part played by the two sexual partners, such that the conclusions are not easily understandable, notably with regards to the distinction between motivation and performance. The behavior of females is only assessed from the perspective of the male, which raises questions about the interpretation of the reduced behavior of the males.<br /> At no point do the authors seem to consider that a reduced behavior of one sex could result from a reduced sensory perception from this sex or a reduced attractivity or sensory communication from the other sex.

- Aspects of the methods are not detailed enough to allow proper evaluation of their quality or replication of the data.

- It seems very dangerous to use the response to a mutant abnormal behavior (ESR2-KO females) as a test, given that it is not clear what is the cause of the disrupted behavior.

- Most experiments are weakly powered (low sample size) and analyzed by multiple T-tests while 2 way ANOVA could have been used in several instances. No mention of T or F values, or degrees of freedom.

- The variability of the mRNA content for the same target gene between experiments (genotype comparison vs E2 treatment comparison) raises questions about the reproducibility of the data (apparent disappearance of genotype effect).

- The discussion confuses the effects of estrogens on sexual differentiation (developmental programming = permanent) and activation (= reversible activation of brain circuits in adulthood) of the brain and behavior. Whether sex differences in the circuits underlying social behaviors exist is not clear.

Conclusions :

Overall, the claims regarding the activational role of neuro-estrogens on male sexual behavior are supported by converging evidence from multiple mutant lines. The role of neuroestrogens on gene expression in the brain is mostly solid too. The data for females are comparatively weaker. Conclusions regarding sexual differentiation should be considered carefully.

Reviewer #3 (Public Review):

This study explores the use of an adenine base editing strategy to knock down PTBP1 in astrocytes and neurons of a Parkinson's disease mouse model, as a potential AAV-BE therapy. The results indicate that editing Ptbp1 in neurons, but not astrocytes, leads to the formation of tyrosine hydroxylase (TH)+ cells, rescuing some motor symptoms.

Several aspects of the manuscript stand out positively. Firstly, the clarity of the presentation. The authors communicate their ideas and findings in a clear and understandable manner, making it easier for readers to follow.

The Materials and methods section is well-elaborated, providing sufficient detail for reproducibility.

The logical flow of the manuscript makes sense, with each section building upon the previous one coherently.

The ABE strategy employed by the authors appears sound, and the manuscript presents a coherent and well-supported argument.

Positively, some of the data in this study effectively counteracts previous work in line with more recent publications, demonstrating the authors' ability to contribute to the ongoing conversation in the field.

However, while the in vitro data yields promising results, it may have been overly optimistic to assume that the efficiencies observed in dividing cells will directly translate to in vivo conditions. This consideration is important given the added complexities of vector optimization, different cell types targeted in vitro versus in vivo, as well as unknown intrinsic limitations of the base editing technology.

In addition, certain aspects of the manuscript would benefit from a more in-depth and comprehensive discussion rather than being only briefly touched upon. Such a discussion would enhance the relevance of the obtained results and provide the foundation for improvement when using similar approaches.

Reviewer #3 (Public Review):

Summary:

Zhang et al. explored strategies for aligning electrophysiological recordings from high-density laminar electrode arrays (Neuropixels) with the pattern of lamination across cortical depth in macaque primary visual cortex (V1), with the goal of improving the spatial resolution of layer identification based on electrophysiological signals alone. The authors compare the current commonly used standard in the field - current source density (CSD) analysis - with a new set of measures largely derived from action potential (AP) frequency band signals. Individual AP band measures provide distinct cues about different landmarks or potential laminar boundaries, and together they are used to subdivide the spatial extent of array recordings into discrete layers, including the very thin layer 4A, a level of resolution unavailable when relying on CSD analysis alone for laminar identification. The authors compare the widths of the resulting subdivisions with previously reported anatomical measurements as evidence that layers have been accurately identified. This is a bit circular, given that they also use these anatomical measurements as guidelines limiting the boundary assignments; however, the strategy is overall sensible and the electrophysiological signatures used to identify layers are generally convincing. Furthermore, by varying the pattern of visual stimulation to target chromatically sensitive inputs known to be partially segregated by layer in V1, they show localized response patterns that lend confidence to their identification of particular sublayers.

The authors compellingly demonstrate the insufficiency of CSD analysis for precisely identifying fine laminar structure, and in some cases its limited accuracy at identifying coarse structure. CSD analysis produced inconsistent results across array penetrations and across visual stimulus conditions and was not improved in spatial resolution by sampling at high density with Neuropixels probes. Instead, in order to generate a typical, informative pattern of current sources and sinks across layers, the LFP signals from the Neuropixels arrays required spatial smoothing or subsampling to approximately match the coarser (50-100 µm) spacing of other laminar arrays. Even with smoothing, the resulting CSDs in some cases predicted laminar boundaries that were inconsistent with boundaries estimated using other measures and/or unlikely given the typical sizes of individual layers in macaque V1. This point alone provides an important insight for others seeking to link their own laminar array recordings to cortical layers.

They next offer a set of measures based on analysis of AP band signals. These measures include analyses of the density, average signal spread, and spike waveforms of single- and multi-units identified through spike sorting, as well as analyses of AP band power spectra and local coherence profiles across recording depth. The power spectrum measures in particular yield compact peaks at particular depths, albeit with some variation across penetrations, whereas the waveform measures most convincingly identified the layer 6-white matter transition. In general, some of the new measures yield inconsistent patterns across penetrations, and some of the authors' explanations of these analyses draw intriguing but rather speculative connections to properties of anatomy and/or responsivity. However, taken as a group, the set of AP band analyses appear sufficient to determine the layer 6-white matter transition with precision and to delineate intermediate transition points likely to correspond to actual layer boundaries.

Strengths:

The authors convincingly demonstrate the potential to resolve putative laminar boundaries using only electrophysiological recordings from Neuropixels arrays. This is particularly useful given that histological information is often unavailable for chronic recordings. They make a clear case that CSD analysis is insufficient to resolve the lamination pattern with the desired precision and offer a thoughtful set of alternative analyses, along with an order in which to consider multiple cues in order to facilitate others' adoption of the strategy. The widths of the resulting layers bear a sensible resemblance to the expected widths identified by prior anatomical measurements, and at least in some cases there are satisfying signatures of chromatic visual sensitivity and latency differences across layers that are predicted by the known connectivity of the corresponding layers. Thus, the proposed analytical toolkit appears to work well for macaque V1 and has strong potential to generalize to use in other cortical regions, though area-targeted selection of stimuli may be required.

Weaknesses:

The waveform measures, and in particular the unit density distribution, are likely to be sensitive to the criteria used for spike sorting, which differ widely among experimenters/groups, and this may limit the usefulness of this particular measure for others in the community. The analysis of detected unit density yields fluctuations across cortical depth which the authors attribute to variations in neural density across layers; however, these patterns seemed particularly variable across penetrations and did not consistently yield peaks at depths that should have high neuronal density, such as layer 2. Therefore, this measure has limited interpretability.

More generally, although the sizes of identified layers comport with typical sizes identified anatomically, a more powerful confirmation would be a direct per-penetration comparison with histologically identified boundaries. Ultimately, the absence of this type of independent confirmation limits the strength of their claim that veridical laminar boundaries can be identified from electrophysiological signals alone.

Reviewer #3 (Public Review):

Summary:

Yu et al harness the capabilities of mesoscopic 2P imaging to record simultaneously from populations of neurons in several visual cortical areas and measure their correlated variability. They first divide neurons into 65 classes depending on their tuning to moving gratings. They found the pairs of neurons of the same tuning class show higher noise correlations (NCs) both within and across cortical areas. Based on these observations and a model they conclude that visual information is broadcast across areas through multiple, discrete channels with little mixing across them.

NCs can reflect indirect or direct connectivity, or shared afferents between pairs of neurons, potentially providing insight on network organization. While NCs have been comprehensively studied in neuron pairs of the same area, the structure of these correlations across areas is much less known. Thus, the manuscripts present novel insights into the correlation structure of visual responses across multiple areas.

Strengths:

The study uses state-of-the art mesoscopic two-photon imaging.

The measurements of shared variability across multiple areas are novel.

The results are mostly well presented and many thorough controls for some metrics are included.

Weaknesses:

I have concerns that the observed large intra-class/group NCs might not reflect connectivity but shared behaviorally driven multiplicative gain modulations of sensory-evoked responses. In this case, the NC structure might not be due to the presence of discrete, multiple channels broadcasting visual information as concluded. I also find that the claim of multiple discrete broadcasting channels needs more support before discarding the alternative hypothesis that a continuum of tuning similarity explains the large NCs observed in groups of neurons.

Specifically:

Major concerns:

(1) Multiplicative gain modulation underlying correlated noise between similarly tuned neurons

(1a) The conclusion that visual information is broadcasted in discrete channels across visual areas relies on interpreting NC as reflecting, direct or indirect connectivity between pairs, or common inputs. However, a large fraction of the activity in the mouse visual system is known to reflect spontaneous and instructed movements, including locomotion and face movements, among others. Running activity and face movements are some of the largest contributors to visual cortex activity and exert a multiplicative gain on sensory-evoked responses (Niell et al, Stringer et al, among others). Thus, trial-by-fluctuations of behavioral state would result in gain modulations that, due to their multiplicative nature, would result in more shared variability in cotuned neurons, as multiplication affects neurons that are responding to the stimulus over those that are not responding ( see Lin et al, Neuron 2015 for a similar point).

As behavioral modulations are not considered, this confound affects most of the conclusions of the manuscript, as it would result in larger NCs the more similar the tuning of the neurons is, independently of any connectivity feature. It seems that this alternative hypothesis can explain most of the results without the need for discrete broadcasting channels or any particular network architecture and should be addressed to support its main claims.

(1b) In Figure 5 the observations are interpreted as evidence for NCs reflecting features of the network architecture, as NCs measured using gratings predicted NC to naturalistic videos. However, it seems from Figure 5 A that signal correlations (SCs) from gratings had non-zero correlations with SCs during naturalistic videos (is this the case?). Thus, neurons that are cotuned to gratings might also tend to be coactivated during the presentation of videos. In this case, they are also expected to be susceptible to shared behaviorally driven fluctuations, independently of any circuit architecture as explained before. This alternative interpretation should be addressed before concluding that these measurements reflect connectivity features.

(2) Discrete vs continuous communication channels

(2a) One of the author's main claims is that the mouse cortical network consists of discrete communication channels. This discreteness is based on an unbiased clustering approach to the tuning of neurons, followed by a manual grouping into six categories in relation to the stimulus space. I believe there are several problems with this claim. First, this clustering approach is inherently trying to group neurons and discretise neural populations. To make the claim that there are 'discrete communication channels' the null hypothesis should be a continuous model. An explicit test in favor of a discrete model is lacking, i.e. are the results better explained using discrete groups vs. when considering only tuning similarity? Second, the fact that 65 classes are recovered (out of 72 conditions) and that manual clustering is necessary to arrive at the six categories is far from convincing that we need to think about categorically different subsets of neurons. That we should think of discrete communication channels is especially surprising in this context as the relevant stimulus parameter axes seem inherently continuous: spatial and temporal frequency. It is hard to motivate the biological need for a discretely organized cortical network to process these continuous input spaces.

(2b) Consequently, I feel the support for discrete vs continuous selective communication is rather inconclusive. It seems that following the author's claims, it would be important to establish if neurons belong to the same groups, rather than tuning similarity is a defining feature for showing large NCs.

Finally, as stated in point 1, the larger NCs observed within groups than across groups might be due to the multiplicative gain of state modulations, due to the larger tuning similarity of the neurons within a class or group.

Reviewer #3 (Public Review):

Summary:

Cells and tissues are viscoelastic materials. However, metabolic processes that underly survival, growth and migration render the cell as an active matter at non-equilibrium. These two facts contribute to the difficulty of probing mechanical properties especially with sub-cellular resolution. However, the concept that the mechanical phenotype can be indicative of normal physiology necessitates approaches of defining the cellular phenotype. Here, Muenker et al evokes a powerful argument for mapping intracellular mechanics using optical tweezer- active microrheology. They present a suite of parameters towards a definition of a mechanical fingerprint. This is a compelling idea. There are some concerns as detailed below

Strengths:

These are technically challenging experiments and the authors provide systematic approaches to probe a system at non-equilibrium.

Weaknesses:

The importance of the mechanical fingerprint is diluted due to some missing controls needed for biological relevance.

Reviewer #3 (Public Review):

In this manuscript by Kroll and colleagues, the authors describe combining behavioral pharmacology with sleep profiling to predict disease and potential treatment pathways at play in AD. AD is used here as a case study, but the approaches detailed can be used for other genetic screens related to normal or pathological states for which sleep/arousal is relevant. The data are for the most part convincing, although generally the phenotypes are relatively small and there are no major new mechanistic insights. Nonetheless, the approaches are certainly of broad interest and the data are comprehensive and detailed.

A notable weakness is the introduction, which overly generalizes numerous concepts and fails to provide the necessary background to set the stage for the data.

Major points

(1) The authors should spend more time explaining what they see as the meaning of the large number of behavioral parameters assayed and specifically what they tell readers about the biology of the animal. Many are hard to understand--e.g. a "slope" parameter.

(2) Because in the end the authors did not screen that many lines, it would increase confidence in the phenotypes to provide more validation of KO specificity. Some suggestions include:<br /> a. The authors cite a psen1 and psen2 germline mutant lines. Can these be tested in the FramebyFrame R analysis? Do they phenocopy F0 KO larvae?<br /> b. psen2KO is one of the larger centerpieces of the paper. The authors should present more compelling evidence that animals are truly functionally null. Without this, how do we interpret their phenotypes?<br /> c. Related to the above, for cd2AP and sorl1 KO, some of the effect sizes seem to be driven by one clutch and not the other. In other words, great clutch-to-clutch variability. Should the authors increase the number of clutches assayed?

(3) The authors make the point that most of the AD risk genes are expressed in fish during development. Is there public data to comment on whether the genes of interest are expressed in mature/old fish as well? Just because the genes are expressed early does not at all mean that early-life dysfunction is related to future AD (though this could be the case, of course). Genes with exclusive developmental expression would be strong candidates for such an early-life role, however. I presume the case is made because sleep studies are mainly done in juvenile fish, but I think it is really a pretty minor point and such a strong claim does not even need to be made.

(4) A common quandary with defining sleep behaviorally is how to rectify sleep and activity changes that influence one another. With psen2 KOs, the authors describe reduced activity and increased sleep during the day. But how do we know if the reduced activity drives increased behavioral quiescence that is incorrectly defined as sleep? In instances where sleep is increased but activity during periods during wake are normal or elevated, this is not an issue. But here, the animals might very well be unhealthy, and less active, so naturally they stop moving more for prolonged periods, but the main conclusion is not sleep per se. This is an area where more experiments should be added if the authors do not wish to change/temper the conclusions they draw. Are psen2 KOs responsive to startling stimuli like controls when awake? Do they respond normally when quiescent? Great care must be taken in all models using inactivity as a proxy for sleep, and it can harm the field when there is no acknowledgment that overall health/activity changes could be a confound. Particularly worrisome is the betamethasone data in Figure 6, where activity and sleep are once again coordinately modified by the drug.

(5) The conclusions for the serotonin section are overstated. Behavioural pharmacology purports to predict a signaling pathway disrupted with sorl1 KO. But is it not just possible that the drug acts in parallel to the true disrupted pathway in these fish? There is no direct evidence for serotonin dysfunction - that conclusion is based on response to the drug. Moreover, it is just 1 drug - is the same phenotype present with another SSRI? Likewise, language should be toned down in the discussion, as this hypothesis is not "confirmed" by the results (consider "supported"). The lack of measured serotonin differences further raises concern that this is not the true pathway. This is another major point that deserves further experimental evidence, because without it, the entire approach (behavioral pharm screen) seems more shaky as a way to identify mechanisms. There are any number of testable hypotheses to pursue such as a) Using transient transgenesis to visualize 5HT neuron morphology (is development perturbed: cell number, neurite morphology, synapse formation); b) Using transgenic Ca reporters to assay 5HT neuron activity.

Reviewer #3 (Public Review):

This study from the Flores group aims at understanding neuronal circuit changes during adolescence which is an ill-defined, transitional period involving dramatic changes in behavior and anatomy. They focus on DA innervation of the prefrontal cortex, and their interaction with the guidance cue Netrin-1. They propose DA axons in the PFC increase in the postnatal period, and their density is reduced in a Netrin 1 knockdown, suggesting that Netrin abets the development of this mesocortical pathway. In such mice impulsivity gauged by a go-no-go task is reduced. They then provide some evidence that Unc5c is developmentally regulated in DA axons. Finally they use an interesting hamster model, to study the effect of light hours on mesocortical innervation, and make some interesting observations about the timing of innervation and Unc5c expression, and the fact that females housed in winter day length conditions display an accelerated innervation of the prefrontal cortex.

Comments on the revision. Several points were addressed; some remain to be addressed.

4. It's not clear to me that TH doesn't stain noradrenergic axons in the PFC. See Islam and Blaess, 2021, and references therein.

6. The Netrin knockdown data provided is from a previous study/samples.

8. While the authors make the argument that the behavior is linked to DA, they still haven't formally tested it, in my opinion.

13. Fig 3, UNc 5c levels are not yet quantified. Furthermore, I agree with the previous reviewer that Unc5C knockdown would corroborate key aspects of the model.

New - Developmental trajectory of prefrontal TH-positive axons from early adolescence to adulthood is similar in male and female rats, (Willing Juraska et al., 2017). This needs discussion.

Editors note:<br /> should you choose to revise your manuscript, please include degrees of freedom in your statistical reporting.

Reviewer #3 (Public Review):

Summary:

The authors investigate the extent to which the responses of different layers of a vision model (VGG-11) can be linked to the cascade of responses (namely, type-I, type-II, and N400) in the human brain when reading words. To achieve maximal consistency, they add noisy-activations to VGG and finetune it on a character recognition task. In this setup, they observe various similarities between the behavior of VGG and the brain when when presented with various transformations of the words (added noise, font modification, etc).

Strengths:

- The paper is well-written and well-presented.

- The topic studied is interesting.

- The fact that the response of the CNN on unseen experimental contrasts such as adding noise correlated with previous results on the brain is compelling.

Weaknesses:

- The paper is rather qualitative in nature. In particular, the authors show that some resemblance exists between the behavior of some layers and some parts of the brain, but it is hard to quantitively understand how strong the resemblances are in each layer, and the exact impact of experimental settings such as the frequency balancing (which seems to only have a very moderate effect according to Figure 5).

- The experiments only consider a rather outdated vision model (VGG).

Reviewer #3 (Public Review):

Summary:

Lin et al., performed a scRNA-seq-based study of tea roots, as an example, to elucidate the biosynthesis and regulatory processes for theanine, a root-specific secondary metabolite, and established the first map of tea roots comprised of 8 cell clusters. Their findings contribute to deepening our understanding of the regulation of the synthesis of important flavor substances in tea plant roots. They have presented some innovative ideas.

It is notable that the authors - based on single-cell analysis results - proposed that TFs and target genes are not necessarily always highly expressed in the same cells. Many of the important TFs they previously identified, along with their target genes (CsTSI or CsAlaDC), were not found in the same cell cluster. Therefore, they proposed a model in which the theanine biosynthesis pathway occurs via multicellular compartmentation and does not require high co-expression levels of transcription factors and their target genes within the same cell cluster. Since it is not known whether the theanine content is absolutely high in the cell cluster 1 containing a high CsAlaDC expression level (due to the lack of cell cluster theanine content determination, which may be a current technical challenge), it is difficult to determine whether this non-coexpressing cell cluster 1 is a precise regulatory mechanism for inhibiting theanine content in plants. In fact, there are actually a small number of cells where TFs and CsAlaDC are simultaneously highly expressed, but the quantity is insufficient to form a separate cluster. However, these few cells may be sufficient to meet the current demands for theanine synthesis. This possibility may better align with some previous experiments and validation results in this study. Moreover, I feel that under normal conditions, plants may not mobilize a large number of cells to synthesize a particular substance. Perhaps, cell cluster 1 is actually a type of cell that inhibits the synthesis of theanine, aiming to prevent excessive theanine production? I do not oppose the model proposed by the author, but I feel there is a possibility as I mentioned. If it seems reasonable, the author may consider adding it to an appropriate position in the discussion.

Reviewer #3 (Public Review):

The aim of the study was to map, a) whether different tissues exhibit different metabolic profiles (this is known already), what differences are found between female and male mice and how the profiles changes with age. In particular, the study recorded the activity of respirasomes, i.e. the concerted activity of mitochondrial respiratory complex chains consisting of CI+CIII2+CIV, CII+CIII2+CIV or CIV alone.

The strength is certainly the atlas of oxidative metabolism in the whole mouse body, the inclusion of the two different sexes and the comparison between young and old mice. The measurement was performed on frozen tissue, which is possible as already shown (Acin-Perez et al, EMBO J, 2020).

Weakness:

The assay reveals the maximum capacity of enzyme activity, which is an artificial situation and may differ from in vivo respiration, as the authors themselves discuss. The material used was a very crude preparation of cells containing mitochondria and other cytosolic compounds and organelles. Thus, the conditions are not well defined and the respiratory chain activity was certainly uncoupled from ATP synthesis. Preparation of more pure mitochondria and testing for coupling would allow evaluation of additional parameters: P/O ratios, feedback mechanism, basal respiration, and ATP-coupled respiration, which reflect in vivo conditions much better. The discussion is rather descriptive and cautious and could lead to some speculations about what could cause the differences in respiration and also what consequences these could have, or what certain changes imply.

Nevertheless, this study is an important step towards this kind of analysis.

Reviewer #3 (Public Review):

Summary:

This study by Jiang et al. aims to establish the streptozotocin (STZ)-induced type 1 diabetes mellitus (T1DM) mouse model in vivo and the STZ-induced pancreatic β cell MIN6 cell model in vitro to explore the protective effects of Eugenol (EUG) on T1DM. The authors tried to elucidate the potential mechanism by which EUG inhibits the NRF2-mediated anti-oxidative stress pathway. Overall, this study is well executed with solid data, offering an intriguing report from animal studies for a potential new treatment strategy for T1DM.

Strengths:

The in vivo efficacy study is comprehensive and solid. Given that STZ-induced T1DM is a devastating and harsh model, the in vivo efficacy of this compound is really impressive.

Weaknesses:

The Mechanism is linked with the anti-oxidant property of the compound, which is common for many natural compounds, such as flavonoids and polyphenol. However, rarely, this kind of compound has been successfully developed into therapeutics in clinical usage. Indeed, if that is the case, Vitamin C or Vitamin E could be used here as the positive control.

Reviewer #3 (Public Review):

Summary:

In this study, the authors utilize biophysical modeling to investigate differences in free energies and nucleosomal configuration probability density of CpG islands and nonmethylated regions in the genome. Toward this goal, they develop and apply the cgNA+ coarse-grained model, an extension of their prior molecular modeling framework.

Strengths:

The study utilizes biophysical modeling to gain mechanistic insight into nucleosomal occupancy differences in CpG and nonmethylated regions in the genome.

Weaknesses:

Although the overall study is interesting, the manuscripts need more clarity in places. Moreover, the rationale and conclusion for some of the analyses are not well described.

Reviewer #3 (Public Review):

Summary:

The manuscript from Zhang et al. utilizes a multi-omics approach to analyze lung adenocarcinoma cases in female never smokers from the Xuanwei area (XWLC cohort) compared with cases associated with smoking or other endogenous factors to identify mutational signatures and proteome changes in lung cancers associated with air pollution. Mutational signature analysis revealed a mutation hotspot, EGFR-G719X, potentially associated with BaP exposure, in 20% of the XWLC cohort. This correlated with predicted MAPK pathway activations and worse outcomes relative to other EGFR mutations. Multi-omics clustering, including RNA-seq, proteomics, and phosphoproteomics identified 4 clusters with the XWLC cohort, with additional feature analysis pathway activation, genetic differences, and radiomic features to investigate clinical diagnostic and therapeutic strategy potential for each subgroup. The study, which nicely combines multi-modal omics, presents potentially important findings, that could inform clinicians with enhanced diagnosis and therapeutic strategies for more personalized or targeted treatments in lung adenocarcinoma associated with air pollution. The authors successfully identify four distinct clusters with the XWLC cohort, with distinct diagnostic characteristics and potential targets. However, many validating experiments must be performed, and data supporting BaP exposure linkage to XWLC subtypes is suggestive but incomplete to conclusively support this claim. Thus, while the manuscript presents important findings with the potential for significant clinical impact, the data presented are incomplete in supporting some of the claims and would benefit from validation experiments.

Strengths:

Integration of omics data from multimodalities is a tremendous strength of the manuscript, allowing for cross-modal comparison/validation of results, functional pathway analysis, and a wealth of data to identify clinically relevant case clusters at the transcriptomic, translational, and post-translational levels. The inclusion of phosphoproteomics is an additional strength, as many pathways are functional and therefore biologically relevant actions center around activation of proteins and effectors via kinase and phosphatase activity without necessarily altering the expression of the genes or proteins.

Clustering analysis provides clinically relevant information with strong therapeutic potential both from a diagnostic and treatment perspective. This is bolstered by the individual microbiota, radiographic, wound healing, outcomes, and other functional analyses to further characterize these distinct subtypes.

Visually the figures are well-designed and presented and for the most part easy to follow. Summary figures/histograms of proteogenomic data, and specifically highlighted genes/proteins are well presented.

Molecular dynamics simulations and 3D binding analysis are nice additions.

While I don't necessarily agree with the authors' interpretation of the microbiota data, the experiment and results are very interesting, and clustering information can be gleaned from this data.

Weaknesses:

Statistical methods for assessing significance may not always be appropriate.

Necessary validating experiments are lacking for some of the major conclusions of the paper.

Many of the conclusions are based on correlative or suggestive results, and the data is not always substantive to support them.

Experimental design is not always appropriate, sometimes lacking necessary controls or large disparity in sample sizes.

Conclusions are sometimes overstated without validating measures, such as in BaP exposure association with the identified hotspot, kinase activation analysis, or the EMT function.

Reviewer #3 (Public Review):

The manuscript by Goyal et al reports substrate-bound and substrate-free structures of a tripartite ATP-independent periplasmic (TRAP) transporter from a previously uncharacterized homolog, F. nucleatum. This is one of the most mechanistically fascinating transporter families, by means of its QM domain (the domain reported in his manuscript) operating as a monomeric 'elevator', and its P domain functioning as a substrate-binding 'operator' that is required to deliver the substrate to the QM domain; together, this is termed an 'elevator with an operator' mechanism. Remarkably, previous structures had not demonstrated the substrate Neu5Ac bound. In addition, they confirm the previously reported Na+ binding sites and report a new metal binding site in the transporter, which seems to be mechanistically relevant. Finally, they mutate the substrate binding site and use proteoliposomal uptake assays to show the mechanistic relevance of the proposed substrate binding residues.

The structures are of good quality, the functional data is robust, the text is well-written, and the authors are appropriately careful with their interpretations. Determination of a substrate-bound structure is an important achievement and fills an important gap in the 'elevator with an operator' mechanism. Nevertheless, I have concerns with the data presentation, which in its current state does not intuitively demonstrate the discussed findings. Furthermore, the structural analysis appears limited, and even slight improvements in data processing and resulting resolution would greatly improve the authors' claims. I have several suggestions to hopefully improve the clarity and quality of the manuscript.

DOI: 10.1007/978-3-031-44907-9_3

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Reviewer #3 (Public Review):

Wu et al. present cryo-EM structures of the potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and presumably sodium-bound states at 3.2 Å, 2.5 Å, 2.8 Å, and 2.9 Å. The work builds on a large body of structural work on Kv1.2 and related voltage-gated potassium channels. The manuscript presents a plethora of structural work, and the authors are commended on the breadth of the studies. The structural studies are well-executed. Although the findings are mostly confirmatory, they do add to the body of work on this and related channels. Notably, the authors present structures of DTx-bound Kv1.2 and of Kv1.2 in a low concentration of potassium (which may contain sodium ions bound within the selectivity filter). These two structures add considerable new information. The DTx structure has been markedly improved in the revised version and the authors arrive at well-founded conclusions regarding its mechanism of block. Regarding the Na+ structure, the authors claim that the structure with sodium has "zero" potassium - I caution them to make this claim. It is likely that some K+ persists in their sample and that some of the density in the "zero potassium" structure may be due to K+ rather than Na+. This can be clarified by revisions to the text and discussion. I do not think that any additional experiments are needed. Overall, the manuscript is well-written, a nice addition to the field, and a crowning achievement for the Sigworth lab.

Most of this reviewer's initial comments have been addressed in the revised manuscript. Some comments remain that could be addressed by revisions of the text.

Specific comments on the revised version:<br /> Quotations indicate text in the manuscript.<br /> (1) "While the VSD helices in Kv1.2s and the inactivated Kv1.2s-W17'F superimpose very well at the top (including the S4-S5 interface described above), there is a general twist of the helix bundle that yields an overall rotation of about 3o at the bottom of the VSD."

Comment: This seemed a bit confusing. I assume the authors aligned the complete structures - the differences they indicate seem to be slight VSD repositioning relative to the pore rather than differences between the VSD conformations themselves. The authors may wish to clarify. As they point out in the subsequent paragraph, the VSDs are known to be loosely associated with the pore.

(2) Comment: The modeling of DTx into the density is a major improvement in the revision. Figure 3 displays some interactions between the toxin and Kv1.2 - additional side views of the toxin and the channel might allow the reader to appreciate the interactions more fully. The overall fit of the toxin structure into the density is somewhat difficult to assess from the figure. (The authors might consider using ChimeraX to display density and model in this figure.)

(3) "We obtained the structure of Kv1.2s in a zero K+ solution, with all potassium replaced with sodium, and were surprised to find that it is little changed from the K+ bound structure, with an essentially identical selectivity filter conformation (Figure 4B and Figure 4-figure supplement 1)."

Comment: It should be noted in the manuscript that K+ and Na+ ions cannot be distinguished by the cryo-EM studies - the densities are indistinguishable. The authors are inferring that the observed density corresponds to Na+ because the protein was exchanged from K+ into Na+ on a gel filtration (SEC) column. It is likely that a small amount of K+ remains in the protein sample following SEC. I caution the authors to claim that there is zero K+ in solution without measuring the K+ content of the protein sample. Additionally, it should be considered that K+ may be present in the blotting paper used for cryo-EM grid preparation (our laboratory has noted, for example, a substantial amount of Ca2+ in blotting paper). The affinity of Kv1.2 for K+ has not been determined, to my knowledge - the authors note in the Discussion that the Shaker channel has "tight" binding for K+. It seems possible that some portion of the density in the selectivity filter could be due to residual K+. This caveat should be clearly stated in the main text and discussion. More extensive exchange into Na+, such as performing the entire protein purification in NaCl, or by dialysis (as performed for obtaining the structure of KcsA in low K+ by Y. Zhou et al. & Mackinnon 2001), would provide more convincing removal of K+, but I suspect that the Kv1.2 protein would not have sufficient biochemical stability without K+ to endure this treatment. One might argue that reduced biochemical stability in NaCl could be an indication that there was a meaningful amount of K+ in the final sample used for cryo-EM (or in the particles that were selected to yield the final high-resolution structure).

(4) Referring to the structure obtained in NaCl: "The ion occupancy is also similar, and we presume that Kv1.2 is a conducting channel in sodium solution."

Comment: Stating that "Kv1.2 is a conducting channel in sodium solution" and implying that conduction of Na+ is achieved by an analogous distribution of ion binding sites as observed for K+ are strong statements to make - and not justified by the experiments provided. Electrophysiology would be required to demonstrate that the channel conducts sodium in the absence of K+. More complete ionic exchange, better control of the ionic conditions (Na+ vs K+), and affinity measurements for K+ would be needed to determine the distribution of Na+ in the filter (as mentioned above). At minimum, the authors should revise and clarify what the intended meaning of the statement "we presume that Kv1.2 is a conducting channel in sodium solution". As mentioned above, it seems possible/likely that a portion of the density in the filter may be due to K+.

Reviewer #3 (Public Review):

In this manuscript, the authors explored the interaction between the pattern recognition receptor MDA5 and 5'ppp-RNA in the Miiuy croaker. They found that MDA5 can serve as a substitute for RIG-I in detecting 5'ppp-RNA of Siniperca cheilinus rhabdovirus (SCRV) when RIG-I is absent in Miiuy croaker. Furthermore, they observed MDA5's recognition of 5'ppp-RNA in chickens (Gallus gallus), a species lacking RIG-I. Additionally, the authors documented that MDA5's functionality can be compromised by m6A-mediated methylation and degradation of MDA5 mRNA, orchestrated by the METTL3/14-YTHDF2/3 regulatory network in Miiuy croaker during SCRV infection. This impairment compromises the innate antiviral immunity of fish, facilitating SCRV's immune evasion. These findings offer valuable insights into the adaptation and functional diversity of innate antiviral mechanisms in vertebrates.

Reviewer #3 (Public Review):

Summary:

Dong et al. described a deep learning-based framework of antimicrobial (AMP) generator and regressor to design and rank de novo antimicrobial peptides (AMPs). For generated AMPs, they predicted their minimum inhibitory concentration (MIC) using a model that combines the Morgan fingerprint, contact map, and ESM language model. For their selected AMPs based on predicted MIC, they also use a combination of antiviral peptide (AVP) prediction models to select AMPs with potential antiviral activity. They experimentally validated 3 candidates for antimicrobial activity against S. aureus, A. baumannii, E. coli, and P. aeruginosa, and their toxicity on mouse blood and three human cell lines. The authors select their most promising AMP (P076) for in vivo experiments in A. baumannii-infected mice. They finally test the antiviral activity of their 3 AMPs against viruses.

Strengths:

-The development of de novo antimicrobial peptides (AMPs) with the novelty of being bifunctional (antimicrobial and antiviral activity).

-Novel, combined approach to AMP activity prediction from their amino acid sequence.

Weaknesses:

-I missed justification on why training AMPs without information of their antiviral activity would generate AMPs that could also have antiviral activity with such high frequency (32 out of 104).

-The justification for AMP predictor advantages over previous tools lacks rationale, comparison with previous tools (e.g., with the very successful AMP prediction approach described by Ma et al. 10.1038/s41587-022-01226-0), and proper referencing.

-Experimental validation of three de novo AMPs is a very low number compared to recent similar studies.

-I have concerns regarding the in vivo experiments including i) the short period of reported survival compared to recent studies (0.1038/s41587-022-01226-0, 10.1016/j.chom.2023.07.001, 0.1038/s41551-022-00991-2) and ii) although in Figure 2 f and g statistics have been provided, log scale y-axis would provide a better comparative representation of different conditions.

-I had difficulty reading the story because of the use of acronyms without referring to their full name for the first time, and incomplete annotation in figures and captions.

Reviewer #3 (Public Review):

The manuscript Kroon et al. described two algorithms, which when combined achieve high throughput automation of "martinizing" protein structures with selected protonation states and post-translational modifications. After the revisions provided by the authors, I recommend minor revision.

The authors have addressed most of my concerns provided previously. Specifically, showcasing the capability of coarse-graining other types of molecules (Figure 7) is a useful addition, especially for the booming field of therapeutic macrocycles.

My only additional concern is that to justify Martinize2 and Vermouth as a "high-throughput" method, the speed of these tools needs to be addressed in some form in the manuscript as a guideline to users.

Reviewer #3 (Public Review):

The authors address the process of community evolution under collective-level selection for a prescribed community composition. They mostly consider communities composed of two types that reproduce at different rates, and that can mutate one into the other. Due to such differences in 'fitness' and to the absence of density dependence, within-collective selection is expected to always favour the fastest grower, but the collective-level selection can oppose this tendency, to a certain extent at least. By approximating the stochastic within-generation dynamics and solving it analytically, the authors show that not only high frequencies of fast growers can be reproducibly achieved, aligned with their fitness advantage. Small target frequencies can also be maintained, provided that the initial proportion of fast growers is sufficiently small. In this regime, similar to the 'stochastic corrector' model, variation upon which selection acts is maintained by a combination of demographic stochasticity and of sampling at reproduction. These two regions of achievable target compositions are separated by a gap, encompassing intermediate frequencies that are only achievable when the bottleneck size is small enough or the number of communities is (disproportionately) larger.

A similar conclusion, that stochastic fluctuations can maintain the system over evolutionary time far from the prevalence of the faster-growing type, is then confirmed by analyzing a three-species community, suggesting that the qualitative conclusions of this study are generalizable to more complex communities.

I expect that these results will be of broad interest to the community of researchers who strive to improve community-level selection, but are often limited to numerical explorations, with prohibitive costs for a full characterization of the parameter space of such embedded populations. The realization that not all target collective functions can be as easily achieved and that they should be adapted to the initial conditions and the selection protocol is also a sobering message for designing concrete applications.

A major strength of this work is that the qualitative behaviour of the system is captured by an analytically solvable approximation so that the extent of the 'forbidden region' can be directly and generically related to the parameters of the selection protocol.

I however found the description of the results too succinct and I think that more could be done to unpack the mathematical results in a way that is understandable to a broader audience. Moreover, the phenomenon the authors characterize is of purely ecological nature. Here, mutations of the growth rate are, in my understanding, neither necessary (non-trivial equilibria can be maintained also when \mu =0) nor sufficient (community-level selection is necessary to keep the system far from the absorbing state) for the phenomenon described. Calling this dynamics community evolution reflects a widespread ambiguity, and is not ascribable just to this work. I find that here the authors have the opportunity to make their message clearer by focusing on the case where the 'mutation' rate \mu vanishes (Equations 39 & 40 of the SI) - which is more easily interpretable, at least in some limits - while they may leave the more general equations 3 & 4 in the SI. Combined with an analysis of the deterministic equations, that capture the possibility of maintaining high frequencies of fast growers, the authors could elucidate the dynamics that are induced by the presence of a second level of selection, and speculate on what would be the result of real open-ended evolution (not encompassed by the simple 'switch mutations' generally considered in evolutionary game theory), for instance discussing the invasibility (or not) of mutant types with slightly different growth rates.

The single most important model hypothesis that I would have liked to be discussed further is that the two types do not interact. Species interactions are not only essential to achieve inheritance of composition in the course of evolution but are generally expected to play a key role even on ecological time scales. I hope the authors plan to look at this in future work.

Reviewer #3 (Public Review):

Summary:

This study reports on a novel NAD+ and Zn2+-independent protein lysine deacetylase (KDAC) in Aeromonas hydrophila, termed AhCobQ (AHA_1389). This protein is annotated as a CobQ/CobB/MinD/ParA family protein and does not show similarity with known NAD+-dependent or Zn2+-dependent KDACs. The authors show that AhCobQ has NAD+ and Zn2+-independent deacetylase activity with acetylated BSA by western blot and MS analyses. They also provide evidence that the 195-245 aa region of AhCobQ is responsible for the deacetylase activity, which is conserved in some marine prokaryotes and has no similarity with eukaryotic proteins. They identified target proteins of AhCobQ deacetylase by proteomic analysis and verified the deacetylase activity using site-specific acetyllysine-incorporated target proteins. Finally, they show that AhCobQ activates isocitrate dehydrogenase by deacetylation at K388.

Strengths:

The finding of a new type of KDAC has a valuable impact on the field of protein acetylation. The characters (NAD+ and Zn2+-independent deacetylase activity in an unknown domain) shown in this study are very unexpected.

Weaknesses:

(1) As the characters of AhCobQ are very unexpected, to convince readers, MSMS data would be needed to exactly detect deacetylation at the target site in deacetylase activity assays. The authors show the MSMS data in assays with acetylated BSA, but other assays only rely on western blot.

(2) They prepared site-specific Kac proteins and used them in deacetylase activity assays. The incorporation of acetyllysine at the target site needs to be confirmed by MSMS and shown as supplementary data.

(3) The authors imply that the 195-245 aa region of AhCobQ may represent a new domain responsible for deacetylase activity. The feature of the region would be of interest but is not sufficiently described in Figure 5. The amino acid sequence alignments with representative proteins with conserved residues would be informative. It would be also informative if the modeled structure predicted by AlphaFold is shown and the structural similarity with known deacetylases is discussed.

Reviewer #3 (Public Review):

Although this work represents a massive screening effort to find new drugs targeting P. vivax hypnozoites, the authors should balance their statement that they identified targetable epigenetic pathways in hypnozoites.

• They should emphasize the potential role of the host cell in the presentation of the results and the discussion, as it is known that other pathogens modify the epigenome of the host cell (i.e. toxoplasma, HIV) to prevent cell division. Also, hydrazinophtalazines target multiple pathways (notably modulation of calcium flux) and have been shown to inhibit DNA-methyl transferase 1 which is lacking in Plasmodium.

• In a drug repurposing approach, the parasite target might also be different than the human target.

• The authors state that host-cell apoptotic pathways are downregulated in P. vivax infected cells (p. 5 line 162). Maybe the HDAC inhibitors and DNA-methyltransferase inhibitors are reactivating these pathways, leading to parasite death, rather than targeting parasites directly.

It would make the interpretation of the results easier if the authors used EC50 in µM rather than pEC50 in tables and main text. It is easy to calculate when it is a single-digit number but more complicated with multiple digits.

Authors mention hypnozoite-specific effects but in most cases, compounds are as potent on hypnozoite and schizonts. They should rather use "liver stage specific" to refer to increased activity against hypnozoites and schizonts compared to the host cell. The same comment applies to line 351 when referring to MMV019721. Following the same idea, it is a bit far-fetched to call MMV019721 "specific" when the highest concentration tested for cytotoxicity is less than twice the EC50 obtained against hypnozoites and schizonts.

Page 5 lines 187-189, the authors state "...hydrazinophtalazines were inactive when tested against P. berghei liver schizonts and P. falciparum asexual blood stages, suggesting that hypnozoite quiescence may be biologically distinct from developing schizonts". The data provided in Figure 1B show that these hydrazinophtalazines are as potent in P. vivax schizonts than in P. vivax hypnozoites, so the distinct activity seems to be Plasmodium species specific and/or host-cell specific (primary human hepatocytes rather than cell lines for P. berghei) rather than hypnozoite vs schizont specific.

Why choose to focus on cadralazine if abandoned due to side effects? Also, why test the pharmacokinetics in monkeys? As it was a marketed drug, were no data available in humans?

In the counterscreen mentioned on page 6, the authors should mention that the activity of poziotinib in P. berghei and P. cynomolgi is equivalent to cell toxicity, so likely not due to parasite specificity.

To improve the clarity and flow of the manuscript, could the authors make a recapitulative table/figure for all the data obtained for poziotinib and hydrazinophtalazines in the different assays (8-days vs 12-days) and laboratory settings rather than separate tables in main and supplementary figures. Maybe also reorder the results section notably moving the 12-day assay before the DNA methylation part.

The isobologram plot shows an additive effect rather than a synergistic effect between cadralazine and 5-azacytidine, please modify the paragraph title accordingly. Please put the same axis scale for both fractional EC50 in the isobologram graph (Figure 2A).

Concerning the immunofluorescence detection of 5mC and 5hmC, the authors should be careful with their conclusions. The Hoechst signal of the parasites is indistinguishable because of the high signal given by the hepatocyte nuclei. The signal obtained with the anti-5hmC in hepatocyte nuclei is higher than with the anti-5mC, thus if a low signal is obtained in hypnozoites and schizonts, it might be difficult to dissociate from the background. In blood stages (Figure S18), the best to obtain a good signal is to lyse the red blood cell using saponin, before fixation and HCl treatment.

To conclude that 5mC marks are the predominate DNA methylation mark in both P. falciparum and P. vivax, authors should also mention that they compare different stages of the life cycle, that might have different methylation levels.

Also, the authors conclude that "[...] 5mC is present at low level in P. vivax and P. cynomolgi sporozoites and could control liver stage development and hypnozoite quiescence". Based on the data shown here, nothing, except presence the of 5mC marks, supports that DNA methylation could be implicated in liver stage development or hypnozoite quiescence.

How many DNA-methyltransferase inhibitors were present in the epigenetic library? Out of those, none were identified as hits, maybe the hydrazinophtalazines effect is not linked to DNMT inhibition but another target pathway of these molecules like calcium transport?

The authors state (line 344): "These results corroborate our hypothesis that epigenetic pathways regulate hypnozoites". This conclusion should be changed to "[...] that epigenetic pathways are involved in P. vivax liver stage survival" because:<br /> • The epigenetic inhibitors described here are as active on hypnozoite than liver schizonts.<br /> • Again, we cannot rule out that the host cell plays a role in this effect and that the compound may not act directly on the parasite.

The same comment applies to the quote in lines 394 to 396. There is no proof in the results presented here that DNA methylation plays any role in the effect of hydrazinophtalazines in the anti-plasmodial activity obtained in the assay.

Reviewer #3 (Public Review):

Distant metastasis is the major cause of death in patients with breast cancer. In this manuscript, Liu et al. show that RGS10 deficiency elicits distant metastasis via epithelial-mesenchymal transition in breast cancer. As a prognostic indicator of breast cancer, RGS10 regulates the progress of breast cancer and affects tumor phenotypes such as epithelial-mesenchymal transformation, invasion, and migration. The conclusions of this paper are mostly well supported by data.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in the Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces the proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific to Flamingo as it cannot be recapitulated with other components of the PCP pathway, and does not rely on the interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo does not just suppress the competitive advantage of winner clones, but even turns them into putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long-term avenue for therapeutic purposes as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantification, and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provided some hints on the putative mechanism (specifically by comparing its localisation in winner and loser cells).

Also, on a more interpretative note, the absence of the impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

Strengths:

- A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition.

- One of the rare genetic conditions that affects very specifically winner cells without any impact on losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective in the long term)

Weaknesses:

- The mechanistic understanding obviously remains quite limited at this stage especially since the signaling does not go through the PCP pathway.

Reviewer #3 (Public Review):

Summary:

The current manuscript investigates the effect of 2-oxoglutarate and the Glk1 protein as modulators of the enzymatic reactivity of glutamine synthetase. To do this, the authors rely on mass photometry, specific activity measurements, and single-particle cryo-EM data.

From the results obtained, the authors convey that glutamine synthetase from Methanosarcina mazei exists in a non-active monomeric/dimeric form under low concentrations of 2-oxoglutarate, and its oligomerization into a dodecameric complex is triggered by higher concentration of 2-oxoglutarate, also resulting in the enhancement of the enzyme activity.

Strengths:

Glutamine synthetase is a crucial enzyme in all domains of life. The dodecameric fold of GS is recurrent amongst prokaryotic and archaea organisms, while the enzyme activity can be regulated in distinct ways. This is a very interesting work combining protein biochemistry with structural biology.

The role of 2-OG is here highlighted as a crucial effector for enzyme oligomerization and full reactivity.

Weaknesses:

Various opportunities to enhance the current state-of-the-art were missed. In particular, omissions of the ligand-bound state of GnK1 leave unexplained the lack of its interaction with GS (in contradiction with previous results from the authors). A finer dissection of the effect and role of 2-oxoglurate are missing and important questions remain unanswered (e.g. are dimers relevant during early stages of the interaction or why previous GS dodecameric structures do not show 2-oxoglutarate).

Reviewer #3 (Public Review):

Summary:<br /> In human patients with Huntington's disease (HD), caused by a CAG repeat expansion mutation, the number of uninterrupted CAG repeats at the genomic level influences age-at-onset of clinical signs independent of the number of polyglutamine repeats at the protein level. In most patients, the CAG repeat terminates with a CAA-CAG doublet. However, CAG repeat variants exist that either do not have that doublet or have two doublets. These variants consequently differ in their number of uninterrupted CAG repeats, while the number of glutamine repeats is the same as both CAA and CAG codes for glutamine. The authors first confirm that a shorter uninterrupted CAG repeat number in human HD patients is associated with developing the first clinical signs of HD later. They predict that introducing a further CAA-CAG doublet will result in years of delay of clinical onset. Based on this observation, the authors tested the hypothesis that turning CAG to CAA within a CAG repeat sequence using base editing techniques will benefit HD biology. They show that, indeed, in HD cell models (HEK293 cells expressing 16/17 CAG repeats; a single human stem cell line carrying a CAG repeat expansion in the fully penetrant range with 42 CAG repeats), their base editing strategies do induce the desired CAG-CAA conversion. The efficiency of conversion differed depending on the strategy used. In stem cells, delivery posed a problem, so to test allele specificity, the authors then used a HEK 293 cell line with 51 CAG repeats on the expanded allele. Conversion occurred in both alleles with huntingtin protein and mRNA levels; transcriptomics data was unchanged. In knock-in mice carrying 110 CAG repeats, however, base editing did not work as well for different, mainly technical, reasons.

Strengths:<br /> The authors use state-of-the-art methods and carefully and thoroughly designed experiments. The data support the conclusions drawn. This work is a very valuable translation from the insight gained from large GWAS studies into HD pathogenesis. It rightly emphasises the potential this has as a causal treatment in HD, while the authors also acknowledge important limitations.

Weaknesses:<br /> They could dedicate a little more to discussing several of the mentioned challenges. The reader will better understand where base editing is in HD currently and what needs to be done before it can be considered a treatment option. For instance,

-It is important to clarify what can be gained by examining again the relationship between uninterrupted CAG repeat length and age-at-onset. Could the authors clarify why they do this and what it adds to their already published GWAS findings? What is the n of datasets?<br /> -What do they think an ideal conversion rate would be, and how that could be achieved?<br /> -Is there a dose-effect relationship for base editing, and would it be realistic to achieve the ideal conversion rate in target cells, given the difficulties described by the authors in differentiated neurons from stem cells?<br /> - The liver is a good tool for in-vivo experiments examining repeat instability in mouse models. However, the authors could comment on why they did not examine the brain.<br /> - Is there a limit to judging the effects of base editing on somatic instability with longer repeats, given the difficulties in measuring long CAG repeat expansions?<br /> - Given the methodological challenges for assessing HTT fragments, are there other ways to measure the downstream effects of base editing rather than extrapolate what it will likely be?<br /> - Sequencing errors could mask low-level, but biologically still relevant, off-target effects (such as gRNA-dependent and gRNA-independent DNA, Off-targets, RNA off-targets, bystander editing). How likely is that?<br /> - How worried are the authors about immune responses following base editing? How could this be assessed?

Reviewer #3 (Public Review):

Summary:

In the present study, the authors aimed to achieve a better understanding of the mechanisms underlying the attentional blink, that is, a deficit in processing the second of two target stimuli when they appear in rapid succession. Specifically, they used a concurrent detection and identification task in- and outside of the attentional blink and decoupled effects of perceptual sensitivity and response bias using a novel signal detection model. They conclude that the attentional blink selectively impairs perceptual sensitivity but not response bias, and link established EEG markers of the attentional blink to deficits in stimulus detection (N2p, P3) and discrimination (fronto-parietal high-beta coherence), respectively. Taken together, their study suggests distinct mechanisms mediating detection and discrimination deficits in the attentional blink.

Strengths:

Major strengths of the present study include its innovative approach to investigating the mechanisms underlying the attentional blink, an elegant, carefully calibrated experimental paradigm, a novel signal detection model, and multifaceted data analyses using state-of-the-art model comparisons and robust statistical tests. The study appears to have been carefully conducted and the overall conclusions seem warranted given the results. In my opinion, the manuscript is a valuable contribution to the current literature on the attentional blink. Moreover, the novel paradigm and signal detection model are likely to stimulate future research.

Weaknesses:

Weaknesses of the present manuscript mainly concern the negligence of some relevant literature, unclear hypotheses, potentially data-driven analyses, relatively low statistical power, potential flaws in the EEG methods, and the absence of a discussion of limitations. In the following, I will list some major and minor concerns in detail.

Major points

Hypotheses:<br /> I appreciate the multifaceted, in-depth analysis of the given dataset including its high amount of different statistical tests. However, neither the Introduction nor the Methods contain specific statistical hypotheses. Moreover, many of the tests (e.g., correlations) rely on selected results of previous tests. It is unclear how many of the tests were planned a priori, how many more were performed, and how exactly corrections for multiple tests were implemented. Thus, I find it difficult to assess the robustness of the results.

Power:<br /> Some important null findings may result from the rather small sample sizes of N = 24 for behavioral and N = 18 for ERP analyses. For example, the correlation between detection and discrimination d' deficits across participants (r=0.39, p=0.059) (p. 12, l. 263) and the attentional blink effect on the P1 component (p=0.050, no test statistic) (p. 14, 301) could each have been significant with one more participant. In my opinion, such results should not be interpreted as evidence for the absence of effects.

Neural basis of the attentional blink:<br /> The introduction (e.g., p. 4, l. 56-76) and discussion (e.g., p. 19, 427-447) do not incorporate the insights from the highly relevant recent review by Zivony & Lamy (2022), which is only cited once (p. 19, l. 428). Moreover, the sections do not mention some relevant ERP studies of the attentional blink (e.g., Batterink et al., 2012; Craston et al., 2009; Dell'Acqua et al., 2015; Dellert et al., 2022; Eiserbeck et al., 2022; Meijs et al., 2018).

Detection versus discrimination:<br /> Concerning the neural basis of detection versus discrimination (e.g., p. 6, l. 98-110; p. 18, l. 399-412), relevant existing literature (e.g., Broadbent & Broadbent, 1987; Hillis & Brainard, 2007; Koivisto et al., 2017; Straube & Fahle, 2011; Wiens et al., 2023) is not included.

Pooling of lags and lag 1 sparing:<br /> I wonder why the authors chose to include 5 different lags when they later pooled early (100, 300 ms) and late (700, 900 ms) lags, and whether this pooling is justified. This is important because T2 at lag 1 (100 ms) is typically "spared" (high accuracy) while T2 at lag 3 (300 ms) shows the maximum AB (for reviews, see, e.g., Dux & Marois, 2009; Martens & Wyble, 2010). Interestingly, this sparing was not observed here (p. 43, Figure 2). Nevertheless, considering the literature and the research questions at hand, it is questionable whether lag 1 and 3 should be pooled.

Discrimination in the attentional blink<br /> Concerning the claims that previous attentional blink studies conflated detection and discrimination (p. 6, l. 111-114; p. 18, l. 416), there is a recent ERP study (Dellert et al., 2022) in which participants did not perform a discrimination task for the T2 stimuli. Moreover, since the relevance of all stimuli except T1 was uncertain in this study, irrelevant distractors could not be filtered out (cf. p. 19, l. 437). Under these conditions, the attentional blink was still associated with reduced negativities in the N2 range (cf. p. 19, l. 427-437) but not with a reduced P3 (cf. p. 19, l 439-447).

General EEG methods:<br /> While most of the description of the EEG preprocessing and analysis (p. 31/32) is appropriate, it also lacks some important information (see, e.g., Keil et al., 2014). For example, it does not include the length of the segments, the type and proportion of artifacts rejected, the number of trials used for averaging in each condition, specific hypotheses, and the test statistics (in addition to p-values).

EEG filters:<br /> P. 31, l. 728: "The data were (...) bandpass filtered between 0.5 to 18 Hz (...). Next, a bandstop filter from 9-11 Hz was applied to remove the 10 Hz oscillations evoked by the RSVP presentation." These filter settings do not follow common recommendations and could potentially induce filter distortions (e.g., Luck, 2014; Zhang et al., 2024). For example, the 0.5 high-pass filter could distort the slow P3 wave. Mostly, I am concerned about the bandstop filter. Since the authors commendably corrected for RSVP-evoked responses by subtracting T2-absent from T2-present ERPs (p. 31, l. 746), I wonder why the additional filter was necessary, and whether it might have removed relevant peaks in the ERPs of interest.

Coherence analysis:<br /> P. 33, l. 786: "For subsequent, partial correlation analyses of coherence with behavioral metrics and neural distances (...), we focused on a 300 ms time period (0-300 ms following T2 onset) and high-beta frequency band (20-30 Hz) identified by the cluster-based permutation test (Fig. 5A-C)." I wonder whether there were any a priori criteria for the definition and selection of such successive analyses. Given the many factors (frequency bands, hemispheres) in the analyses and the particular shape of the cluster (p. 49, Fig 5C), this focus seems largely data-driven. It remains unclear how many such tests were performed and whether the results (e.g., the resulting weak correlation of r = 0.22 in one frequency band and one hemisphere in one part of a complexly shaped cluster; p. 15, l. 327) can be considered robust.

References<br /> Batterink, L., Karns, C. M., & Neville, H. (2012). Dissociable mechanisms supporting awareness: The P300 and gamma in a linguistic attentional blink task. Cerebral Cortex, 22(12), 2733-2744. https://doi.org/10.1093/cercor/bhr346<br /> Broadbent, D. E., & Broadbent, M. H. P. (1987). From detection to identification: Response to multiple targets in rapid serial visual presentation. Perception & Psychophysics, 42(2), 105-113. https://doi.org/10.3758/BF03210498<br /> Craston, P., Wyble, B., Chennu, S., & Bowman, H. (2009). The attentional blink reveals serial working memory encoding: Evidence from virtual and human event-related potentials. Journal of Cognitive Neuroscience, 21(3), 550-566. https://doi.org/10.1162/jocn.2009.21036<br /> Dell'Acqua, R., Dux, P. E., Wyble, B., Doro, M., Sessa, P., Meconi, F., & Jolicœur, P. (2015). The attentional blink impairs detection and delays encoding of visual information: Evidence from human electrophysiology. Journal of Cognitive Neuroscience, 27(4), 720-735. https://doi.org/10.1162/jocn_a_00752<br /> Dellert, T., Krebs, S., Bruchmann, M., Schindler, S., Peters, A., & Straube, T. (2022). Neural correlates of consciousness in an attentional blink paradigm with uncertain target relevance. NeuroImage, 264C, 119679. https://doi.org/10.1016/j.neuroimage.2022.119679<br /> Dux, P. E., & Marois, R. (2009). The attentional blink: A review of data and theory. Attention, Perception, & Psychophysics, 71(8), 1683-1700. https://doi.org/10.3758/APP.71.8.1683<br /> Hillis, J. M., & Brainard, D. H. (2007). Distinct mechanisms mediate visual detection and identification. Current Biology, 17(19), 1714-1719. https://doi.org/10.1016/j.cub.2007.09.012<br /> Keil, A., Debener, S., Gratton, G., Junghöfer, M., Kappenman, E. S., Luck, S. J., Luu, P., Miller, G. A., & Yee, C. M. (2014). Committee report: Publication guidelines and recommendations for studies using electroencephalography and magnetoencephalography. Psychophysiology, 51(1), 1-21. https://doi.org/10.1111/psyp.12147<br /> Koivisto, M., Grassini, S., Salminen-Vaparanta, N., & Revonsuo, A. (2017). Different electrophysiological correlates of visual awareness for detection and identification. Journal of Cognitive Neuroscience, 29(9), 1621-1631. https://doi.org/10.1162/jocn_a_01149<br /> Luck, S. J. (2014). An introduction to the event-related potential technique. MIT Press.<br /> Martens, S., & Wyble, B. (2010). The attentional blink: Past, present, and future of a blind spot in perceptual awareness. Neuroscience & Biobehavioral Reviews, 34(6), 947-957. https://doi.org/10.1016/j.neubiorev.2009.12.005<br /> Meijs, E. L., Slagter, H. A., de Lange, F. P., & Gaal, S. van. (2018). Dynamic interactions between top-down expectations and conscious awareness. Journal of Neuroscience, 38(9), 2318-2327. https://doi.org/10.1523/JNEUROSCI.1952-17.2017<br /> Straube, S., & Fahle, M. (2011). Visual detection and identification are not the same: Evidence from psychophysics and fMRI. Brain and Cognition, 75(1), 29-38. https://doi.org/10.1016/j.bandc.2010.10.004<br /> Wiens, S., Andersson, A., & Gravenfors, J. (2023). Neural electrophysiological correlates of detection and identification awareness. Cognitive, Affective, & Behavioral Neuroscience. https://doi.org/10.3758/s13415-023-01120-5<br /> Zhang, G., Garrett, D. R., & Luck, S. J. (2024). Optimal filters for ERP research II: Recommended settings for seven common ERP components. Psychophysiology, n/a(n/a), e14530. https://doi.org/10.1111/psyp.14530

Reviewer #3 (Public Review):

Summary:

The authors aimed to improve single-nucleus RNA sequencing (snRNA-seq) to address current limitations and challenges with nuclei and RNA isolation quality. They successfully developed a protocol that enhances RNA preservation and yields high-quality snRNA-seq data from multiple tissues, including a challenging model of adipose tissue. They then applied this method to eWAT and iWAT from mice fed either a normal or high-fat diet, exploring depot-specific cellular dynamics and gene expression changes during obesity. Their analysis included subclustering of SVF cells and revealed that obesity promotes a transition in APCs from an early to a committed state and induces a pro-inflammatory phenotype in immune cells, particularly in eWAT. In addition to SVF cells, they discovered six adipocyte subpopulations characterized by a gradient of unique gene expression signatures. Interestingly, a novel subpopulation, termed Ad6, comprised stressed and dying adipocytes with reduced transcriptional activity, primarily found in eWAT of mice on a high-fat diet. Overall, the methodology is sound, the writing is clear, and the conclusions drawn are supported by the data presented. Further research based on these findings could pave the way for potential novel interventions in obesity and metabolic disorders, or for similar studies in other tissues or conditions.

Strengths:

• The authors developed a robust snRNA-seq technique that preserves the integrity of the nucleus and RNA across various tissue types, overcoming the challenges of existing methods.

• They identified adipocyte subpopulations that follow adaptive or pathological trajectories during obesity.

• The study reveals depot-specific differences in adipose tissues, which could have implications for targeted therapies.

Weaknesses:

• The adipose tissues were collected after 10 weeks of high-fat diet treatment, lacking the intermediate time points for identifying early markers or cell populations during the transition from healthy to pathological adipose tissue.

• The expansion of the Ad6 subpopulation in obese iWAT and gWAT is interesting. The author claims that Ad6 exhibited a substantial increase in eWAT and a moderate rise in iWAT (Figure 4C). However, this adipocyte subpopulation remains the most altered in iWAT upon obesity. Could the authors elaborate on why there is a scarcity of adipocytes with ROS reporter and B2M in obese iWAT?

• While the study provides extensive data on mouse models, the potential translation of these findings to human obesity remains uncertain.

Reviewer #3 (Public Review):

Summary:

This study by Beath et al. investigated the mechanisms by which sperm DNA is excluded from the meiotic spindle after fertilization. Time-lapse imaging revealed that sperm DNA is surrounded by paternal mitochondria and maternal ER that is permeable to proteins. By increasing cytoplasmic streaming using kinesin-13 or katanin RNAi, the authors demonstrated that limiting cytoplasmic streaming in the embryo is an important step that prevents the capture of sperm DNA by the oocyte meiotic spindle. Further experiments showed that the Ataxin-2 protein is required to hold paternal mitochondria together and close to the sperm DNA. Finally, double depletion of kinesin-13 and Ataxin-2 suggested an increased risk of meiotic spindle capture of sperm DNA.

Overall, this is an interesting finding that could provide a new understanding of how meiotic spindle capture of sperm DNA and its accidental expulsion into the polar body is prevented. However, some conceptual gaps need to be addressed and further experiments and improved data analyses would strengthen the paper.

• It would be helpful if the authors could discuss in good detail how they think maternal ER surrounds the sperm DNA and why is it not disrupted following Ataxin disruption.

• Since important phenotypes revealed in RNAi experiments (e.g. kinesin-13 and ataxin-2 double depletion) are not very robust, the authors should consider toning down their conclusions and revising some of their section headings. I appreciate that they are upfront about some limitations, but they do nonetheless make strong concluding sentences.

• The discussion section could be improved further to present the authors' findings in the larger context of current knowledge in the field.

• The authors previously demonstrated that F-actin prevents meiotic spindle capture of sperm DNA in this system. However, the current manuscript does not discuss how the katanin, kinesin-13 and Ataxin-2 mechanisms could work together with previously established functions of F-actin in this process.

• How can the authors exclude off-target effects in their RNAi depletion experiments? Can kinesin-13, katanin, and Ataxin phenotypes be rescued for instance?

• How are the authors able to determine if the paternal genome was actually captured by the spindle? Does lack of movement definitively suggest capture without using a spindle marker?

Reviewer #3 (Public Review):

This study describes the first structure of Gram-positive bacterial AdhE spirosomes that are in a native extended conformation. All the previous structures of AdhE spirosomes obtained come from Gram-negative bacterial species with native compact spirosomes (E. coli, V. cholerae). In E. coli, AdhE spirosomes can be found in two different conformational states, compact and extended, depending on the substrates and cofactors they are bound to.

The high-resolution cryoEM structure of the extended C. thermocellum AdhE spirosomes produced in E. coli in an apo state (without any substrate or cofactors) is compared to the E. coli extended and compact AdhE spirosomes structures previously published. The authors have modeled (in Swiss-Model) the structure of compact C. thermocellum AdhE spirosomes, using E. coli compact AdhE spirosome conformation as a template, and performed molecular dynamics simulations. They have identified a channel in which the toxic reaction intermediate aldehyde could transit from the aldehyde dehydrogenase active site to the alcohol dehydrogenase active site, in an analogous manner to E. coli spirosomes. These findings are in line with the hypothesis that the extended spirosomes could correspond to the active form of the enzyme.

In this work, the authors speculate that the C. thermocellum AdhE spirosomes could switch from the native extended conformation to a compact conformation, in a way that is inverse of E. coli spirosomes. Although attractive, this hypothesis is not supported by the literature. Amazingly, in some Gram-positive bacterial species (S. pneumoniae, S. sanguinis or C. difficile...), AdhE spirosomes are natively extended and have never been observed in a compact conformation. On the opposite, E. coli (and other Gram-negative bacteria) native AdhE spirosomes are compact and are able to switch to an extended conformation in the presence of the cofactors (NAD+, coA, and iron). The data presented as they are now are not convincing to confirm the existence of C. thermocellum AdhE spirosomes in a compact conformation.

Reviewer #3 (Public Review):

Summary:

The pathomechanism underlying Sjögren's syndrome (SS) remains elusive. The authors have studied if altered calcium signaling might be a factor in SS development in a commonly used mouse model. They provide a thorough and straightforward characterization of the salivary gland fluid secretion, cytoplasmic calcium signaling, mitochondrial morphology, and respiration. A special strength of the study is the spectacular in vivo imaging, very few if any groups could have succeeded with the studies. The authors show that the cytoplasmic calcium signaling is upregulated in the SS model and the Ca2+ regulated Cl- channels are normally localized and function, but still fluid secretion is suppressed. They also find altered localization of the IP3R and speculate about lesser exposure of Cl- channels to high local [Ca2+]. In addition, they describe changes in mitochondrial morphology and function that might also contribute to the attenuated secretory response. Although the exact contribution of calcium and mitochondria to secretory dysfunction remains to be determined, the results seem to be useful for a range of scientists.

Specific points to consider:

(1) Are all the effects of DMXAA mediated through STING? DMXAA has been reported to inhibit NAD(P)H quinone oxidoreductase (NQO1) PMID: 10423172, which might be relevant both for the calcium and mitochondrial phenotypes. I would recommend that the authors either test the dependency of the DMXAA effects on STING or avoid attributing all effects of DMXAA to STING.

(2) "mitochondrial membrane potential (ΔΨm), the driving force of ATP production" the driving force is the electrochemical H+ gradient.

(3) ΔΨm is assessed as decreased in the DMXAA model without a change in TMRE steady state. Higher post-uncoupler fluorescence caused a lesser uncoupler-sensitive pool. This is not a very common observation. Was the autofluorescence of the DMXAA-treated cells higher in the red channel?

(4) The EM study indicated ER structure disruption. Are there any clues to the contribution of this to the augmented agonist/electrical stimulation-induced calcium signaling and decreased fluid secretion?

Reviewer #3 (Public Review):

Summary:

In this paper, Curran et al investigate the role of Ntn, Slit1 and Slit 2 in axon patterning of DRG neurons. The paper uses mouse genetics to perturb each guidance molecule and its corresponding receptor. Cre-based approaches and immunostaining of DRG neurons are used to assess the phenotypes. Overall, the study uses the strength of mouse genetics and imaging to reveal new genetic modifiers of DRG axons. The conclusions of the experiments match the presented results. The paper is an important contribution to the field, as evidence that dorsal funiculus formation is impacted by Ntn and Slit signaling. The paper clearly demonstrates molecules that impact the patterning of the dorsal funiculus formation, which can provide a foundation for future studies on the specific steps in that patterning that require the studied molecules.

Strengths:

The manuscript uses the advantage of mouse genetics to investigate axon patterning of DRG neurons. The work does a great job of assessing individual phenotypes in single and double mutants. This reveals an intriguing cooperative and independent function of Ntn, Slit1 and Slit2 in DRG axon patterning. The sophisticated triple mutant analysis is lauded and provides important insight.

Weaknesses:

Overall, the manuscript is sound in technique and analysis. While not a weakness, the paper provides the foundation for future studies that investigate the specific molecular mechanisms of each step in the patterning of the dorsal funiculus.

Reviewer #3 (Public Review):

The manuscript from Amen et al reports the isolation and characterization of human antibodies that recognize proteins expressed at different sexual stages of Plasmodium falciparum. The isolation approach was antigen agnostic and based on the sorting, activation, and screening of memory B cells from a donor whose serum displays high transmission-reducing activity. From this effort, 14 antibodies were produced and further characterized. The antibodies displayed a range of transmission-reducing activities and recognized different Pf sexual stage proteins. However, none of these antibodies had higher TRA than previously described antibodies.

The authors then performed further characterization of antibody B1E11K, which was unique in that it recognized multiple proteins expressed during sexual and asexual stages. Using protein microarrays, B1E11K was shown to recognize glutamate-rich repeats, following an EE-XX-EE pattern. An impressive set of biophysical experiments was performed to extensively characterize the interactions of B1E11K with various repeat motifs and lengths. Ultimately, the authors succeeded in determining a 2.6 A resolution crystal structure of B1E11K bound to a 16AA repeat-containing peptide. Excitingly, the structure revealed that two Fabs bound simultaneously to the peptide and made homotypic antibody-antibody contacts. This had only previously been observed with antibodies directed against CSP repeats.

Overall I found the manuscript to be very well written, although there are some sections that are heavy on field-specific jargon and abbreviations that make reading unnecessarily difficult. For instance, 'SIFA' is never defined. Strengths of the manuscript include the target-agnostic screening approach and the thorough characterization of antibodies. The demonstration that B1E11K is cross-reactive to multiple proteins containing glutamate-rich repeats, and that the antibody recognizes the repeats via homotypic interactions, similar to what has been observed for CSP repeat-directed antibodies, should be of interest to many in the field.

DOI: 10.1186/s13578-023-00970-3

Resource: (Abcam Cat# ab89780, RRID:AB_2042411)

Curator: @abever99

SciCrunch record: RRID:AB_2042411

What is this?

DOI: 10.1186/s13578-023-00970-3

Resource: (Cell Signaling Technology Cat# 78896, RRID:AB_2799920)

Curator: @abever99

SciCrunch record: RRID:AB_2799920

What is this?

Reviewer #3 (Public Review):

Na-Phatthalung et al observed that transcripts of the zinc transporter Slc30a1 was upregulated in Salmonella-infected murine macrophages and in human primary macrophages therefore they sought to determine if, and how, Slc30a1 could contribute to the control of bacterial pathogens. Using a reporter mouse the authors show that Slc30a1 expression increases in a subset of peritoneal and splenic macrophages of Salmonella-infected animals. Specific deletion of Slc30a1 in LysM+ cells resulted in a significantly higher susceptibility of mice to Salmonella infection which, counter to the authors conclusions, is not explained by the small differences in the bacterial burden observed in vivo and in vitro. Although loss of Slc30a1 resulted in reduced iNOS levels in activated macrophages, the study lacks experiments that mechanistically link loss of NO-mediated bactericidal activity to Salmonella survival in Slc30a1 deficient cells. The additional deletion of Mt1, another zinc binding protein, resulted in even lower nitrite levels of activated macrophages but only modest effects on Salmonella survival. By combining genetic approaches with molecular techniques that measure variables in macrophage activation and the labile zinc pool, Na-Phattalung et al successfully demonstrate that Slc30a1 and metallothionein 1 regulate zinc homeostasis in order to modulate effective immune responses to Salmonella infection. The authors have done a lot of work and the information that Slc30a1 expression in macrophages contributes to control of Salmonella infection in mice is a new finding that will be of interest to the field. Whether the mechanism by which SLC30A1 controls bacterial replication and/or lethality of infection involves nitric oxide production by macrophages remains to be shown.

Reviewer #3 (Public Review):

The results of the deep mutagenesis screen represent a wealth of information on the expression and function of SERT that everyone studying this protein will appreciate. However, as the authors explain, the screen identified mutations that increased APP+ transport but inhibited transport of the cognate substrate, 5-HT. Because of the methods used, 5-HT could not be used as a substrate, somewhat limiting the usefulness of the screen.

However, the authors have taken advantage of this limitation to address the mechanistic features of SERT that discriminate between 5-HT and APP+. From the position of mutations that augment APP+ transport, they have identified the aqueous pathway created in inward facing SERT conformations as a region of importance. Based on the MD simulations, transition to inward facing conformations is facilitated by 5-HT but less so by APP+. The authors conclude, quite reasonably, that mutations interfering with the stability of inward-closed SERT states could overcome the reduced ability of APP+ to open the pathway.

Another reasonable conclusion based on the mutant screen, is that mutations detrimental to surface expression were found in packed hydrophobic regions of the protein, but similar mutations in the permeation pathways were less likely to decrease expression. The authors postulate that this provides an evolutionary advantage by maintaining the structural fold while allowing modification of ion and substrate binding and coupling sites, a reasonable but speculative conclusion.

Not all gain-of-function mutations have to be specific to APP+. The authors point out that Ala173Gly converts SERT to the residue found in NET and DAT at this position. It would have been interesting to know how this mutation and others affect 5-HT transport. Indeed, the lack of any 5-HT transport measurements with the mutants is a glaring weakness of the manuscript.

Reviewer #3 (Public Review):

The manuscript by Rubin and Agrawal et al presents a very nice imaging analysis of clonal cell organization in the fetal and late juvenile mouse growth cartilages. The authors have performed a thorough quantification of the orientations of clusters and of clones of cells with respect to the growth axis. They conclude that growth cartilage is not as strictly 'columnar' as has been commonly described, especially at the fetal stage. There is value to having such quantifications in the literature as a reminder that interpretations of phenotypes need to be rooted in the cell biology of the stage at hand, as emphasized by the authors. However, although the approach is comprehensive, aspects of the quantification methods are not described adequately to determine if they are correct for the questions. There are also some inequivalent comparisons to prior literature and an oversight of important published observations showing that some of these conclusions have been known for decades, though not as thoroughly quantitative. There have long been observations that some growth cartilages do not have proliferative columns oriented in the axis of growth and that not all columns of a growth cartilage are perfectly organized; these facts do not negate the observations that columnar organization does exist, as re-confirmed here, and that it correlates with and contributes to rapid growth rates. Each of these points is further elaborated below.

Reviewer #3 (Public Review):

It has been reported that the sympathetic nervous system (SNS) mediates bone metabolism and nociceptive functions. However, the exact localization and organization of the central SNS circuitry innervating bone and the brain sites have not been mapped and efferent SNS outflow to bone has not yet been characterized yet. Authors used pseudorabies (PRV) viral transneuronal tracing approach to identify central SNS outflow sites that innervate bone. The authors found that the central SNS outflow to bone originates from brain nuclei, sub-nuclei and regions of six brain divisions (midbrain and pons, hypothalamus, hindbrain medulla, forebrain, cerebral cortex, and thalamus). The authors provided compelling evidence for a brain-bone SNS neuroaxis that may regulate bone metabolism and nociceptive functions, which provided a greater understanding of the neural regulation of bone metabolism and would stimulate further research into bone pain and the neural regulation of bone metabolism. Authors may discuss and summarize their results in detail for a better understanding of their findings and enhancing the manuscript's utility for readers.

Reviewer #3 (Public Review):

The manuscript from Barekatain et al. is investigating heterogeneity within the population of insulin vesicles from an insulinoma cell line (INS-1E) in response to glucose stimulation. Prevailing dogma in the beta-cell field suggests that there are distinct pools of mature insulin granules, such as ready-releasable and a reserve pool, which contribute to distinct phases of insulin release in response to glucose stimulation. Whether these pools (and others) are distinct in protein/lipid composition or other aspects is not known, but has been suggested. In this manuscript, the authors use density gradient sedimentation to enrich for insulin vesicles, noting the existence of a number of co-purifying contaminants (ER and mitochondrial markers). Following immunolabeling with synaptotagmin V and fluorescent-conjugated secondary antibodies, insulin vesicles were applied to a microfluidic device and separated by dielectrophoretic and electrokinetic forces following an applied voltage. The equilibrium between these opposing forces was used to physically separate insulin granules. Here some differences were observed in the insulin (Syt V positive) granule populations, when isolated from cells that were either non-stimulated or stimulated with glucose, which has been suggested previously by other studies as noted by the authors; however in the current manuscript, the inclusion of a number of control experiments may provide a better context for what the data reveal about these changes.

The major strength of the paper is in the use of the novel, highly sophisticated methodology to examine physical attributes of insulin granules and thus begin to provide some insight into the existence of distinct insulin granule populations within a beta-cell -these include insulin granules that are maturing, membrane-docked (i.e. readily releasable), in reserve, newly-synthesized, aged, etc. Whether physical differences exist between these various granule pools is not known. In this capacity, the technical abilities of the current manuscript may begin to offer some insight into whether these perceived distinctions are physical.

The major weakness of the manuscript is that the study falls short in terms of linking the biology to the sophisticated changes observed and primarily focuses on differences in response to glucose. Without knowing what the various populations of granules are, it is challenging to understand what the changes in response to glucose mean.

Specific concerns are as follows:

(1) There is confusion on what the DC-iDEP separation between stimulated and stimulated cells reveals. Do these changes reflect maturation state of granules, nascent vs. old granules? Ready-releasable vs. reserve pool? The comments in the text seem to offer all possibilities.

(2) It is unclear what we can infer regarding the physical changes of granules between the stimulated states of the cells. Without an understanding of the magnitude of the effect, it is unclear how biologically significant these changes are. For example, what degree of lipid or protein remodeling would be necessary to give a similar change?

(3) The reliance on a single vesicle marker, Syt V, is concerning given that granule remodeling is the focus.

(4) Additional confirmation that the isolated vesicles are in fact insulin granules would be helpful. As noted, granules were gradient enriched, but did carry contaminants. Note that the microscopy image provided does not provide any real validation for this marker.

Further confirmation that the immune-isolated vesicles are in fact insulin granules should be included. EM with immunogold labeling post-SytV enrichment would be a potential methodology to confirm.

(5) It would be useful to understand if the observed effects are specific to the INS-1E cell line or are a more universal effect of glucose on beta-cells.

Reviewer #3 (Public Review):

Summary:

This study considers how to model distinct host cell states that correspond to different stages of a viral infection: from naïve and susceptible cells to infected cells and a minority of important interferon-secreting cells that are the first line of defense against viral spread. The study first considers the distinct host cell states by analyzing previously published single-cell RNAseq data. Then an agent-based model on a square lattice is used to probe the dependence of the system on various parameters. Finally, a simplified version of the model is explored, and shown to have some similarity with the more complex model, yet lacks the dependence on the interferon range. By exploring these models one gains an intuitive understanding of the system, and the model may be used to generate hypotheses that could be tested experimentally, telling us "when to be surprised" if the biological system deviates from the model predictions.

Strengths:

- Clear presentation of the experimental findings and a clear logical progression from these experimental findings to the modeling.<br /> - The modeling results are easy to understand, revealing interesting behavior and percolation-like features.<br /> - The scaling results presented span several decades and are therefore compelling.<br /> - The results presented suggest several interesting directions for theoretical follow-up work, as well as possible experiments to probe the system (e.g. by stimulating or blocking IFN secretion).

Weaknesses:

- The fixed time-step of the agent-based modeling may introduce biases. I would consider simulating the system with Gillespie dynamics where the reaction rates depend on the ambient system parameters.<br /> - Single-cell RNAseq data requires careful handling or it may generate false leads. The strength of the RNAseq evidence presented is not clear.

Two places where the manuscript could be extended:

- Since the "range" of IFN is an important parameter, it makes sense to consider other lattice geometries other than the square lattice, which is somewhat pathological. Perhaps a hexagonal lattice would generalize better.<br /> - Tissues are typically three-dimensional, not two-dimensional. (Epithelium is an exception). It would be interesting to see how the modeling translates to the three-dimensional case. Percolations transitions are known to be very sensitive to the dimensionality of the system.

Justification of claims and conclusions:

The claims and conclusions are well justified.

Reviewer #3 (Public Review):

In this manuscript, Pierre Ferrer and colleagues explore the exciting possibility that, in the male germ line, the composition and function of deeply conserved chromatin remodeling complexes is fine-tuned by the addition of testis-specific actin-related proteins (ARPs). In this regard, the Authors aim to extend previously reported non-canonical (transcriptional) roles of ARPs in somatic cells to the unique developmental context of the germ line. The manuscript is focused on the potential regulatory role in post-meiotic transcription of two ARPs: ACTL7A and ACTL7B (particularly the latter). The canonical function of both testis-specific ARPs in spermatogenesis is well established, as they have been previously shown to be required for the extensive cellular morphogenesis program driving post-meiotic development (spermiogenesis). Disentangling the actual functions of ACTL7A and ACTL7B as transcriptional regulators from their canonical role in the profound morphological reshaping of post-meiotic cells (a process that also deeply impacts nuclear architecture and regulation) represents a key challenge in terms of interpreting the reported findings (see below).

The authors begin by documenting, via fluorescence microscopy, the intranuclear localization of ACTL7B. This ARP is convincingly shown to accumulate in the nucleus of spermatocytes and spermatids. Using a series of elegant reporter-based experiments in a somatic cell line, the authors map the driver of this nuclear accumulation to a potential NLS sequence in the ACTL7B actin-like body domain. Ferrer and colleagues then performed a testicular RNA-seq analysis in ACTL7B KO mice to define the putative role of ACTL7B in male germ cell transcription. They report substantial changes to the testicular transcriptome - particularly the upregulation of several classes of genes - in ACTL7B KO mice. However, wild-type testes were used as controls for this experiment, thus introducing a clear confounding effect to the analysis (ACTL7B KO testes have extensive post-meiotic defects due to the canonical role of ACTL7B in spermatid development). Then, the authors employ cutting-edge AI-driven approaches to predict that both ACTL7A and ACTL7B are likely to bind to four key chromatin remodeling complexes. Although these predictions are based on a robust methodology, they would certainly benefit from experimental validation. Finally, the authors associate the loss of ACTL7B with decreased lysine acetylation and lower levels of the HDAC1 and HDAC3 chromatin remodelers in the nucleus of developing spermatids.

Globally, these data may provide important insight into the unique processes male germ cells employ to sustain their extraordinarily complex transcriptional program. Furthermore, the concept that (comparably younger) testis-specific proteins can be incorporated into ancient chromatin remodeling complexes to modulate their function in the germ line is timely and exciting.

It is my opinion that the manuscript would benefit from additional experimental validation to better support the authors' conclusions. In particular, I believe that addressing two critical points would substantially strengthen the message of the manuscript:

(1) The proposed role of ACTL7B in post-meiotic transcriptional regulation temporally overlaps with the protein's previously reported canonical functions in spermiogenesis (PMID: 36617158 and 37800308). Indeed, the canonical functions of ACTL7B have been shown to have a profound effect at the level of spermatid morphology and to impact nuclear organization. This potentially renders the observed transcriptional deregulation in ACTL7B KO testes an indirect consequence of spermatid morphology defects. I acknowledge that it is experimentally difficult to disentangle the proposed intranuclear roles of ACTL7B from the protein's well-documented cytoplasmic function. Perhaps the generation of a NLS-scrambled ACTL7B variant could offer some insight. In light of the substantial investment this approach would represent, I would suggest, as an alternative, that instead of using wild-type testes as controls for the transcriptome and chromatin localization assays, the authors consider the possibility of using testicular tissue from a mutant with similarly abnormal spermiogenesis but due to transcription-independent defects. This would, in my opinion, offer a more suitable baseline to compare ACTL7B KO testes with.

(2) The manuscript would greatly benefit if experimental validation of the AI-driven predictions were to be provided (in terms of the binding capacity of ACTL7A and ACTL7B to key chromatin remodeling complexes). More so it seems that the authors have the technical expertise / available mass spectrometry data required for this purpose (lines 664-665). Still on this topic, given the predicted interactions of ACTL7A and ACTL7B with the SRCAP, EP400, SMARCA2 and SMARCA4 complexes (Figure 7), it is rather counter-intuitive that the Authors chose for their immunofluorescence assays, in ACTL7B KO testes, to determine the chromatin localization of HDAC1 and HDAC3, rather than that of any of above four complexes.

DOI: 10.1186/s41232-024-00336-3

Resource: (MMRRC Cat# 011015-UCD,RRID:MMRRC_011015-UCD)

Curator: @bandrow

SciCrunch record: RRID:MMRRC_011015-UCD

What is this?

Reviewer #3 (Public Review):

Summary:

In Okholm et al., the authors evaluate the functional impact of circHIPK3 in bladder cancer cells. By knocking it down and performing an RNA-seq analysis, the authors found thousand deregulated genes which look unaffected by miRNAs sponging function and that are, instead, enriched for a 11-mer motif. Further investigations showed that the 11-mer motif is shared with the circHIPK3 and able to bind the IGF2BP2 protein. The authors validated the binding of IGF2BP2 and demonstrated that IGF2BP2 KD antagonizes the effect of circHIPK3 KD and leads to the upregulation of genes containing the 11-mer. Among the genes affected by circHIPK3 KD and IGF2BP2 KD, resulting in downregulation and upregulation respectively, the authors found STAT3 gene which also consistently leads to the concomitant upregulation of one of its targets TP53. The authors propose a mechanism of competition between circHIPK3 and IGF2BP2 triggered by IGF2BP2 nucleation, potentially via phase separation.

Strengths:

The number of circRNAs continues to drastically grow however the field lacks detailed molecular investigations. The presented work critically addresses some of the major pitfalls in the field of circRNAs and there has been a careful analysis of aspects frequently poorly investigated. The time-point KD followed by RNA-seq, investigation of miRNAs-sponge function of circHIPK3, identification of 11-mer motif, identification and validation of IGF2BP2, and the analysis of copy number ratio between circHIPK3 and IGF2BP2 in assessing the potential ceRNA mode of action have been extensively explored and, comprehensively convincing.

Weaknesses:

The authors addressed the majority of the weak points raised initially. However the role played by the circHIPK3 in cancer remains elusive and not elucidated in full in this study.

Overall, the presented study surely adds some further knowledge in describing circHIPK3 function, its capability to regulate some downstream genes, and its interaction and competition for IGF2BP2. However, whereas the experimental part sounds technically logical, it remains unclear the overall goal of this study and the achieved final conclusions.

This study is a promising step forward in the comprehension of the functional role of circHIPK3. These data could possibly help to better understand the circHIPK3 role in cancer

Reviewer #3 (Public Review):

In the present study, the authors examined how dPAG neurons respond to predatory threats and how dPAG and BLA communicate threat signals. The authors employed single-unit recording and optogenetics tools to address these issues in an 'approach food-avoid predator' paradigm. They characterized dPAG and BLA neurons responsive to a looming robot predator and found that dPAG opto-stimulation elicited fleeing and increased BLA activity. Importantly, they found that dPAG stimulation produces activity changes in subpopulations of BLA neurons related to predator detection, thus supporting the idea that dPAG conveys innate fear signals to the amygdala. In addition, injections of anterograde and retrograde tracers into the dPAG and BLA, respectively, along with the examination of c-FOS activity in midline thalamic relay stations, suggest that the paraventricular nucleus of the thalamus (PVT) may serve as a mediator of dPAG to BLA neurotransmission. Of relevance, the study helps to validate an important concept that dPAG mediates primal fear emotion and may engage upstream amygdalar targets to evoke defensive responses. The series of experiments provide a compelling case for supporting their conclusions. The study brings important concepts revealing dynamics of fear-related circuits particularly attractive to a broad audience, from basic scientists interested in neural circuits to psychiatrists.

Reviewer #3 (Public Review):

Summary:

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 must 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 neural activity. Comparison across brain areas shows that this contextual signal is stronger in frontal cortical regions than 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.

Strengths:

This is a carefully done study with solid results and thorough controls. The authors identify a contextual signal in baseline neural activity that predicts rule-dependent decision-related activity. The comprehensive characterization across a sensorimotor pathway is another strength. Analyses and perturbation experiments link this contextual signal to animals' behavior. The results provide a neural substrate that will surely inspire follow-up mechanistic investigations.

Weaknesses:

None. The authors have further improved the manuscript during the revision with additional analyses.

Impact:

This study reports an important neural signature for context-dependent decision-making that has important implications for mechanisms of context-dependent neural computation in general.

Reviewer #3 (Public Review):

Summary and overall evaluation:

Human vision is inherently limited so that only a small part of a visual scene can be perceived at a given moment. To address this limitation, the visual system has evolved a number of strategies and mechanisms that work in concert. First, humans move their eyes using saccadic eye movements. This allows us to place the high-resolution region in the center of the eye's retina (the fovea centralis) on objects of interest so that these are sampled with high acuity. Second, salient, conspicuous stimuli that appear abruptly and/or differ strongly from the other stimuli in the scene, seem to automatically attract ("exogenous") attention, so that a large share of the neuronal "resources" for visual processing is devoted to the stimuli, which improves the perception of the stimuli. Third, stimuli that are important for the current task and the current behavioral goals can be prioritized by attention mechanisms ("endogenous" attention), which also secures their allocated share of processing resources and helps them be perceived. It is well-established that eye movements are closely linked to the mechanisms of attention (for a review, see Carrasco, 2011, cited in the manuscript). However, it is still unclear what role voluntary, endogenous attention plays in the control of saccadic eye movements.

The present study used an experimental procedure involving time-pressure for responding, in order to uncover how the control of saccades by exogenous and endogenous attention unfolds over time. The findings of the study indicate that saccade planning was indeed influenced by the locus of endogenous attention, but that this influence was short-lasting and could be overcome quickly. Taken together, the present findings reveal new dynamics between endogenous attention and eye movement control, and lead the way for studying them using experiments under time pressure.

The results provided by the present study advance our understanding of vision, eye movements, and their control by brain mechanisms for attention. In addition, they demonstrate how tasks involving time pressure can be used to study the dynamics of cognitive processes. Therefore, the present study seems highly important not only for vision science, but also for psychology, (cognitive) neuroscience, and related research fields more generally.

Strengths:

The experiments of the study are performed with great care and rigor and the data is analyzed thoroughly and comprehensively. Overall, the results support the authors' conclusions, so I have only minor comments (see below). Taken together, the findings seem important for a wide community of researchers in vision science, psychology, and neuroscience.

Weaknesses (minor points):

(1) In this experimental paradigm, participants must decide where to saccade based on the color of the cue in the visual periphery (they should have made a prosaccade toward a green cue and an antisaccade away from a magenta cue). Thus, irrespective of whether the cue signaled that a prosaccade or an antisaccade was to be made, the identity of the cue was always essential for the task (as the authors explain on p. 5, lines 129-138). Also, the location where the cue appeared was blocked, and thus known to the participants in advance, so that endogenous attention could be directed to the cue at the beginning of a trial (e.g., p. 5, lines 129-132). These aspects of the experimental paradigm differ from the classic prosaccade/antisaccade paradigm (e.g. Antoniades et al., 2013, Vision Research). In the classic paradigm, the identity of the cues does not have to be distinguished to solve the task, since there is only one stimulus that should be looked at (prosaccade) or away from (antisaccade), and whether a prosaccade or antisaccade was required is constant across a block of trials. Thus, in contrast to the present paradigm, in the classic paradigm, the participants do not know where the cue is about to appear, but they know whether to perform a prosaccade or an antisaccade based on the location of the cue.

The present paradigm keeps the location of the cue constant in a block of trials by intention, because this ensures that endogenous attention is allocated to its location and is not overpowered by the exogenous capture of attention that would happen when a single stimulus appeared abruptly in the visual field. Thus, the reason for keeping the location of the cue constant seems convincing. However, I wondered what consequences the constant location would have for the task representations that persist across the task and govern how attention is allocated. In the classic paradigm, there is always a single stimulus that captures attention exogenously (as it appears abruptly). In a prosaccade block, participants can prioritize the visual transient caused by the stimulus, and follow it with a saccade to its coordinates. In an antisaccade block, following the transient with a saccade would always be wrong, so that participants could try to suppress the attention capture by the transient, and base their saccade on the coordinates of the opposite location. Thus, in prosaccade and antisaccade blocks, the task representations controlling how visual transients are processed to perform the task differ. In the present task, prosaccades and antisaccades cannot be distinguished by the visual transients. Thus, such a situation could favor endogenous attention and increase its influence on saccade planning, even though saccade planning under more naturalistic conditions would be dominated by visual transients. I suggest discussing how this (and vice versa the emphasis on visual transients in the classic paradigm) could affect the generality of the presented findings (e.g., how does this relate to the interpretation that saccade plans are obligatorily coupled to endogenous attention? See, Results, p. 10, lines 306-308, see also Deubel & Schneider, 1996, Vision Research).

(2) Discussion (p. 16, lines 472-475): The authors suppose that "It is as if the exogenous response was automatically followed by a motor bias in the opposite direction. Perhaps the oculomotor circuitry is such that an exogenous signal can rapidly trigger a saccade, but if it does not, then the corresponding motor plan is rapidly suppressed regardless of anything else.". I think this interesting point should be discussed in more detail. Could it also be that instead of suppression, other currently active motor plans were enhanced? Would this involve attention? Some attention models assume that attention works by distributing available (neuronal) processing resources (e.g., Desimone & Duncan, 1995, Annual Review of Neuroscience; Bundesen, 1990, Psychological Review; Bundesen et al., 2005, Psychological Review) so that the information receiving the largest share of resources results in perception and is used for action, but this happens without the active suppression of information.

(3) Methods, p. 19, lines 593-596: It is reported that saccades were scored based on their direction. I think more information should be provided to understand which eye movements entered the analysis. Was there a criterion for saccade amplitude? I think it would be very helpful to provide data on the distributions of saccade amplitudes or on their accuracy (e.g. average distance from target) or reliability (e.g. standard deviation of landing points). Also, it is reported that some data was excluded from the analysis, and I suggest reporting how much of the data was excluded. Was the exclusion of the data related to whether participants were "reliable" or "unreliable" performers?

(4) Results, p. 9, lines 262-266: Some data analyses are performed on a subset of participants that met certain performance criteria. The reasons for this data selection seem convincing (e.g. to ensure empirical curves were not flat, line 264). Nevertheless, I suggest to explain and justify this step in more detail. In addition, if not all participants achieved an acceptable performance and data quality, this could also speak to the experimental task and its difficulty. Thus, I suggest discussing the potential implications of this, in particular, how this could affect the studied mechanisms, and whether it could limit the presented findings to a special group within the studied population.

Reviewer #3 (Public Review):

Hall et al. benchmarked different variant calling methods on Nanopore reads of bacterial samples and compared the performance of Nanopore to short reads produced with Illumina sequencing. To establish a common ground for comparison, the authors first generated a variant truth set for each sample and then projected this set to the reference sequence of the sample to obtain a mutated reference. Subsequently, Hall et al. called SNPs and small indels using commonly used deep learning and conventional variant callers and compared the precision and accuracy from reads produced with simplex and duplex Nanopore sequencing to Illumina data. The authors did not investigate large structural variation, which is a major limitation of the current manuscript. It will be very interesting to see a follow-up study covering this much more challenging type of variation.

In their comprehensive comparison of SNPs and small indels, the authors observed superior performance of deep learning over conventional variant callers when Nanopore reads were basecalled with the most accurate (but also computationally very expensive) model, even exceeding Illumina in some cases. Not surprisingly, Nanopore underperformed compared to Illumina when basecalled with the fastest (but computationally much less demanding) method with the lowest accuracy. The authors then investigated the surprisingly higher performance of Nanopore data in some cases and identified lower recall with Illumina short read data, particularly from repetitive regions and regions with high variant density, as the driver. Combining the most accurate Nanopore basecalling method with a deep learning variant caller resulted in low error rates in homopolymer regions, similar to Illumina data. This is remarkable, as homopolymer regions are (or, were) traditionally challenging for Nanopore sequencing.

Lastly, Hall et al. provided useful information on the required Nanopore read depth, which is surprisingly low, and the computational resources for variant calling with deep learning callers. With that, the authors established a new state-of-the-art for Nanopore-only variant, calling on bacterial sequencing data. Most likely these findings will be transferred to other organisms as well or at least provide a proof-of-concept that can be built upon.

As the authors mention multiple times throughout the manuscript, Nanopore can provide sequencing data in nearly real-time and in remote regions, therefore opening up a ton of new possibilities, for example for infectious disease surveillance.

However, the high-performing variant calling method as established in this study requires the computationally very expensive sup and/or duplex Nanopore basecalling, whereas the least computationally demanding method underperforms. Here, the manuscript would greatly benefit from extending the last section on computational requirements, as the authors determine the resources for the variant calling but do not cover the entire picture. This could even be misleading for less experienced researchers who want to perform bacterial sequencing at high performance but with low resources. The authors mention it in the discussion but do not make clear enough that the described computational resources are probably largely insufficient to perform the high-accuracy basecalling required.

Reviewer #3 (Public Review):

Summary:

The rete ovarii (RO) has long been disregarded as a non-functional structure within the ovary. In their study, Anbarci and colleagues have delineated the markers and developmental dynamics of three distinct regions of the RO - the intraovarian rete (IOR), the extraovarian rete (EOR), and the connecting rete (CR). Notably focusing on the EOR, the authors presented evidence illustrating that the EOR forms a convoluted tubular structure culminating in a dilated tip. Intriguingly, microinjections into this tip revealed luminal flow towards the ovary containing potentially secreted functional proteins. Additionally, the EOR cells exhibit associations with vasculature, macrophages, and neuronal projections, proposing the notion that the RO may play a functional role in ovarian development during critical ovariogenesis stages. By identifying marker genes within the RO, the authors have also suggested that the RO could serve as a potential structure linking the ovary with the neuronal system.

Strengths:

Overall, the reviewer commends the authors for their systematic research on the RO, shedding light on this overlooked structure in developing ovaries. Furthermore, the authors have proposed a series of hypotheses that are both captivating and scientifically significant, with the potential to reshape our understanding of ovarian development through future investigations.

Weaknesses:

There is a lack of conclusive data supporting many conclusions in the manuscript. Therefore, the paper's overall conclusions should be moderated until functional validations are conducted.

Reviewer #3 (Public Review):

Summary:

The authors aimed to establish a faster and more efficient method of tracking steps of T-cell extravasation across the blood brain barrier. The authors developed a framework to visualize, recognize and track the movement of different immune cells across primary human and mouse brain microvascular endothelial cells without the need for fluorescence-based imaging. The authors succinctly describe the basic requirements for tracking in the introduction followed by an in-depth account of the execution.

Weaknesses and Strengths:

Materials & methods and results:

(1) The methods section also lacks details of the microfluidic device that the authors talk about in the paper. Under physiological sheer stress, the T-cells detach from the pMBMEC monolayer, and are hence unable to be detected; however, this observation requires an explanation pertaining to the reason of occurrence and potential solutions to circumvent it to ensure physiologically relevant experimental parameters.

(2) The author describes a method for debris exclusion using UFMTrack that eliminates objects of <30 pixels in size from analysis based on a mean pixel size of 400 for T lymphocytes. However, this mean pixel size appears to stem from in-vitro activated CD8 T cells, which rapidly grow and proliferate upon stimulation. In line with this, activated lymphocytes exhibit increased cytoplasmic area, making them appear less dense or "brighter" by phase microscopy compared to naïve lymphocytes, which are relatively compact and subsequently appear dimmer. Given this, it is not clear whether UFMTrack is sufficiently trained to identify naïve human lymphocytes in circulating blood, nor smaller, murine lymphocytes. Analysis of each lymphocyte subtype in terms of pixel size and intensity would be beneficial to strengthen the claim that UFMTrack can identify each of these populations. Additionally, demonstrating that UFMTrack can correctly characterize the behavior of naïve versus activated lymphocytes isolated from murine and human sources would strengthen the claim that UFMTrack can be broadly applied to study lymphocyte dynamics in diverse models without additional training

(3) Average precision was compared to the analysis of UFMTrack but it is unclear how average precision was calculated. This information should have been included in the methods section

(4) CD4 and CD8 T cells exhibit distinct biology and interaction kinetics driven in part by their MHC molecule affinity and distinct receptor expression profiles. Thus, it is unclear why two distinct mechanisms of endothelial cell activation are needed to see differences between the populations.

(5) The BMECs are barrier tissues but were cultured on µdishes in this study. To study the transmigration of T-cells across the endothelium, the model would have been more relevant on a semi-permeable membrane instead of a closed surface.

(6) Methods are provided for the isolation and expansion of human effector and memory CD4+ T cells. However, there is no mention of specific CD4+ T cell populations used for analysis with UFMTrack, nor a clear breakdown of tracking efficiency for each subpopulation. Further, there is no similar method for the isolation of CD8+ T cell compartments. A clear breakdown of the performance efficiency of UFMTrack with each cell population investigated in this study would provide greater insight into the software's performance with regard to tracking the behavior and movement of distinct immune populations.

(7) The results section is quite extensive and discusses details of establishment of the framework while highlighting both the pros and cons of the different aspects of the process, for example the limitation of the two models, 2D and 2D+T were highlighted well. However, the results section includes details which may be more fitting in the methods section.

(8) A few statements in the results section lacked literary support, which was not provided in the discussion either, such as support for increased variance of T-cell instantaneous speed on stimulated vs non-stimulated pMBMECs. Another example is the enhancement of cytokine stimulation directed T-cell movement on the pMBMECs that the authors observed but failed to relay the physiological relevance of it. The authors don't provide enough references for developments in the field prior to their work which form the basis and need for this technology.

(9) The rationale for use of OT-1 and 2D2-derived murine lymphocytes is unclear here. The OT-1 model has been generated to study antigen-specific CD8+ T cell responses, while the 2D2 model has been generated to recapitulate CD4 T cell-specific myelin oligodendrocyte glycoprotein (MOG) responses.

Figures and text:

(1) There are certain discrepancies and misarrangement of figures and text. For example, discussion of the effect of sheer flow on T cell attachment as part of the introduction in figure 1 and then mentioning it in the text again in the results section as part of figure 4 is repetitive.

(2) Section IV, subsection 1 of the results section, refers to 'data acquisition section above' in line 279, however the said section is part of materials and methods which is provided towards the end of the manuscript.

(3) There are figures in the manuscript that have not been referenced in the results section, for example, figure 3A and B. Figure 1 hasn't been addressed until subsection 7 of materials and methods

(4) A lack of significance but an observed trend of increased variance of T cell instantaneous speed is reported in line 296-298; however, the graph (figure 4G) shows a significant change in instantaneous speed between non-stimulated and TNFα-stimulated systems. This is misleading to the readers.

(5) The authors talk about three beginner experimentors testing the manual T cell tracking process but figure 5 only showcases data from two experimentors without stating the reason for excluding experimentor 1.

Discussion:

(1) While the discussion captures the major takeaways from the paper, it lacks relevant supporting references to relate the observation to physiological conditions and applicability.

(2) The discussion lacks connection to the results since the figures were not referenced while discussing an observed trend

(3) The authors briefly looked into mouse and human BMECs and their individual interaction with T-cells, but don't discuss the differences between the two, if any, that challenged their framework.

(4) Even though though the imaging tool relies on difference in appearance for detection, the authors talk about lack of feasibility in detecting transmigration of BMDMs due to their significantly different appearance. The statement lacks a problem solving approach to discuss how and why this was the case.

Relevance to the field:

Utilizing the framework provided by the authors, the application can be adapted and/or utilized for visualizing a range of different cell types, provided they are different in appearance. However, this would require extensive changes to the script and won't be adaptable in its current form.

DOI: 10.1038/s44319-024-00152-3

Resource: (CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)

Curator: @AniH

SciCrunch record: RRID:CVCL_0063

What is this?

DOI: 10.1038/s44319-024-00152-3

Resource: (IMSR Cat# JAX_002014,RRID:IMSR_JAX:002014)

Curator: @AniH

SciCrunch record: RRID:IMSR_JAX:002014

What is this?

DOI: 10.1038/s44319-024-00152-3

Resource: (IMSR Cat# JAX_007676,RRID:IMSR_JAX:007676)

Curator: @AniH

SciCrunch record: RRID:IMSR_JAX:007676

What is this?

Reviewer #3 (Public Review):

I remain enthusiastic about this study. The manuscript is well-written, logical, and conceptually clear. To my knowledge, no prior modeling study has tackled the question of 'why prepare before executing, why not just execute?' Prior studies have simply assumed, to emulate empirical findings, that preparatory inputs precede execution. They never asked why. The authors show that, when there are constraints on inputs, preparation becomes a natural strategy. In contrast, with no constraint on inputs, there is no need for preparation as one could get anything one liked just via the inputs during movement. For the sake of tractability, the authors use a simple magnitude constraint: the cost function punishes the integral of the squared inputs. Thus, if small inputs before movement can reduce the size of the inputs needed during movement, preparation is a good strategy. This occurs if (and only if) the network has strong dynamics (otherwise feeding it preparatory activity would not produce anything interesting). All of this is sensible and clarifying.

As discussed in the prior round of reviews, the central constraint that the authors use is a mathematically tractable stand-in for a range of plausible (but often trickier to define and evaluate) constraints, such as simplicity of inputs (or inputs being things that other areas could provide). The manuscript now embraces this fact more explicitly, and also gives some results showing that other constraints (such as on the derivative of activity, which is one component of complexity) can have the same effect. The manuscript also now discusses and addresses a modest weakness of the previous manuscript: the preparatory activity in their simulations is often overly complex temporally, lacking the (rough) plateau typically seen for data. Depending on your point of view, this is simply 'window dressing', but from my perspective it was important to know that their approach could yield more realistic-looking preparatory activity. Both these additions (the new constraint, and the more realistic temporal profile of preparatory activity) are added simply as supplementary figures rather than in the main text, and are brought up only in the Discussion. At first this struck me as slightly odd, but in the end I think this is appropriate. These are really Discussion-type issues, and dealing with them there makes sense. The 'different constraints' issue in particular is deep, tricky to explore for technical reasons, and could thus support a small research program. I think it is fair to talk about it thoughtfully (as the Discussion now does) and then just mention some simple results.

My remaining comments largely pertain to some subtle (but to me important) nuances at a few locations in the text. These should be easy for the authors to address, in whatever way they see fit.

Specific comments:

(1) The authors state the following on line 56: "For preparatory processes to avoid triggering premature movement, any pre-movement activity in the motor and dorsal pre-motor (PMd) cortices must carefully exclude those pyramidal tract neurons."<br /> This constraint is overly restrictive. PT neurons absolutely can change their activity during preparation in principle (and appear to do so in practice). The key constraint is looser: those changes should have no net effect on the muscles. E.g., if d is the vector of changes in PT neuron firing rates, and b is the vector of weights, then the constraint is that b'd = 0. d = 0 is one good way of doing this, but only one. Half the d's could go up and half could go down. Or they all go up, but half the b's are negative. Put differently, there is no reason the null space has to be upstream of the PT neurons. It could be partly, or entirely, downstream.<br /> In the end, this doesn't change the point the authors are making. It is still the case that d has to be structured to avoid causing muscle activity, which raises exactly the point the authors care about: why risk this unless preparation brings benefits? However, this point can be made with a more accurate motivation. This matters, because people often think that a null-space is a tricky thing to engineer, when really it is quite natural. With enough neurons, preparing in the null space is quite simple.

(2) Line 167: 'near-autonomous internal dynamics in M1'.<br /> It would be good if such statements, early in the paper, could be modified to reflect the fact that the dynamics observed in M1 may depend on recurrence that is NOT purely internal to M1. A better phrase might be 'near-autonomous dynamics that can be observed in M1'. A similar point applies on line 13. This issue is handled very thoughtfully in the Discussion, starting on line 713. Obviously it is not sensible to also add multiple sentences making the same point early on. However, it is still worth phrasing things carefully, otherwise the reader may have the wrong impression up until the Discussion (i.e. they may think that both the authors, and prior studies, believe that all the relevant dynamics are internal to M1). If possible, it might also be worth adding one sentence, somewhere early, to keep readers from falling into this hole (and then being stuck there till the Discussion digs them out).

(3) The authors make the point, starting on line 815, that transient (but strong) preparatory activity empirically occurs without a delay. They note that their model will do this but only if 'no delay' means 'no external delay'. For their model to prepare, there still needs to be an internal delay between when the first inputs arrive and when movement generating inputs arrive.

This is not only a reasonable assumption, but is something that does indeed occur empirically. This can be seen in Figure 8c of Lara et al. Similarly, Kaufman et al. 2016 noted that "the sudden change in the CIS [the movement triggering event] occurred well after (~150 ms) the visual go cue... (~60 ms latency)" Behavioral experiments have also argued that internal movement-triggering events tend to be quite sluggish relative to the earliest they could be, causing RTs to be longer than they should be (Haith et al. Independence of Movement Preparation and Movement Initiation). Given this empirical support, the authors might wish to add a sentence indicating that the data tend to justify their assumption that the internal delay (separating the earliest response to sensory events from the events that actually cause movement to begin) never shrinks to zero.

While on this topic, the Haith and Krakauer paper mentioned above good to cite because it does ponder the question of whether preparation is really necessary. By showing that they could get RTs to shrink considerably before behavior became inaccurate, they showed that people normally (when not pressured) use more preparation time than they really need. Given Lara et al, we know that preparation does always occur, but Haith and Krakauer were quite right that it can be very brief. This helped -- along with neural results -- change our view of preparation from something more cognitive that had to occur, so something more mechanical that was simply a good network strategy, which is indeed the authors current point. Working a discussion of this into the current paper may or may not make sense, but if there is a place where it is easy to cite, it would be appropriate.

Reviewer #3 (Public Review):

The data reported here demonstrate that Sema7a defines the local behavior of growing axons in the developing zebrafish lateral line. The analysis is sophisticated and convincingly demonstrates effects on axon growth and synapse architecture. Collectively, the findings point to the idea that the diffusible form of sema7a may influence how axons grow within the neuromast and that the GPI-linked form of sema7a may subsequently impact how synapses form, though additional work is needed to strongly link each form to its' proposed effect on circuit assembly.

Comments on latest version:

The authors comprehensively and appropriately addressed most of the reviewers' concerns. In particular, they added evidence that hair cells express both Sema7A isoforms, showed that membrane bound Sema7A does not have long range effects on guidance, demonstrated how axons behave close to ectopic Sema7A, and analyzed other features of the hair cells that revealed no strong phenotypes. The authors also softened the language in many, but not all places. Overall, I am satisfied with the study as a whole.

Reviewer #3 (Public Review):

In this work, Jarc et al. describe a method to decouple the mechanisms supporting progenitor self-renewal and expansion from feed-forward mechanisms promoting their differentiation.

The authors aimed at expanding pancreatic progenitor (PP) cells, strictly characterized as PDX1+/SOX9+/NKX6.1+ cells, for several rounds. This required finding the best cell culture conditions that allow sustaining PP cell proliferation along cell passages while avoiding their further differentiation. They achieve this by comparing the transcriptome of PP cells that can be expanded for several passages against the transcriptome of unexpanded (just differentiated) PP cells.

The optimized culture conditions enabled the selection of PDX1+/SOX9+/NKX6.1+ PP cells and their consistent, 2000-fold, expansion over ten passages and 40-45 days. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. These optimized culture conditions consisted in substituting the Vitamin A containing B27 supplement with a B27 formulation devoid of vitamin A (to avoid retinoic acid (RA) signaling from an autocrine feed-forward loop), substituting A38-01 with the ALK5 II inhibitor (ALK5i II) that targets primarily ALK5, supplementation of medium with FGF18 (in addition to FGF2) and the canonical Wnt inhibitor IWR-1, and cell culture on vitronectin-N (VTN-N) as a substrate instead of Matrigel.

The strength of this work relies on a clever approach to identify cell culture modifications that allow expansion of PP cells (once differentiated) while maintaining, if not reinforcing, PP cell identity. Along the work, it is emphasized that PP cell identity is associated to the co-expression of PDX1, SOX9 and NKX6.1. The optimized protocol is unique (among the other datasets used in the comparison shown here) at inducing a strong upregulation of GP2, a unique marker of human fetal pancreas progenitors. Importantly GP2+ enriched hPS cell-derived PP cells are more efficiently differentiating into pancreatic endocrine cells (Aghazadeh et al., 2022; Ameri et al., 2017).

The unlimited expansion of PP cells reported here would allow scaling-up the generation of beta cells, for the cell therapy of diabetes, by eliminating a source of variability derived from the number of differentiation procedures to be carried out when starting at the hPS cell stage each time. The approach presented here would allow selection of the most optimally differentiated PP cell population for subsequent expansion and storage. Among other conditions optimized, the authors report a role for Vitamin A in activating retinoic acid signaling in an autocrine feed-forward loop, and the supplementation with FGF18 to reinforce FGF2 signaling.

This is a relevant topic in the field of research, and some of the cell culture conditions reported here for PP expansion might have important implications in cell therapy approaches. Thus, the approach and results presented in this study could be of interest for researchers working in the field of in vitro pancreatic beta cell differentiation from hPSCs. Table S1 and Table S4 are clearly detailed and extremely instrumental to this aim.

Reviewer #3 (Public Review):

This manuscript extends previous research by this group by relating variation in pupil size to the endpoints of saccades produced by human participants under various conditions including trial-based choices between pairs of spots and search for small items in natural scenes. Based on the premise that pupil size is a reliable proxy of "effort", the authors conclude that less costly saccade targets are preferred. Finding that this preference was influenced by the performance of a non-visual, attention-demanding task, the authors conclude that a common source of effort animates gaze behavior and other cognitive tasks.

Strengths:

Strengths of the manuscript include the novelty of the approach, the clarity of the findings, and the community interest in the problem.

Weaknesses:

Enthusiasm for this manuscript is reduced by the following weaknesses:

(1) A relationship between pupil size and saccade production seems clear based on the authors' previous and current work. What is at issue is the interpretation. The authors test one, preferred hypothesis, and the narrative of the manuscript treats the hypothesis that pupil size is a proxy of effort as beyond dispute or question. The stated elements of their argument seem to go like this:<br /> PROPOSITION 1: Pupil size varies systematically across task conditions, being larger when tasks are more demanding.<br /> PROPOSITION 2: Pupil size is related to the locus coeruleus.<br /> PROPOSITION 3: The locus coeruleus NE system modulates neural activity and interactions.<br /> CONCLUSION: Therefore, pupil size indexes the resource demand or "effort" associated with task conditions.<br /> How the conclusion follows from the propositions is not self-evident. Proposition 3, in particular, fails to establish the link that is supposed to lead to the conclusion.

(2) The authors test one, preferred hypothesis and do not consider plausible alternatives. Is "cost" the only conceivable hypothesis? The hypothesis is framed in very narrow terms. For example, the cholinergic and dopamine systems that have been featured in other researchers' consideration of pupil size modulation are missing here. Thus, because the authors do not rule out plausible alternative hypotheses, the logical structure of this manuscript can be criticized as committing the fallacy of affirming the consequent.

(3) The authors cite particular publications in support of the claim that saccade selection is influenced by an assessment of effort. Given the extensive work by others on this general topic, the skeptic could regard the theoretical perspective of this manuscript as too impoverished. Their work may be enhanced by consideration of other work on this general topic, e.g, (i) Shenhav A, Botvinick MM, Cohen JD. (2013) The expected value of control: an integrative theory of anterior cingulate cortex function. Neuron. 2013 Jul 24;79(2):217-40. (ii) Müller T, Husain M, Apps MAJ. (2022) Preferences for seeking effort or reward information bias the willingness to work. Sci Rep. 2022 Nov 14;12(1):19486. (iii) Bustamante LA, Oshinowo T, Lee JR, Tong E, Burton AR, Shenhav A, Cohen JD, Daw ND. (2023) Effort Foraging Task reveals a positive correlation between individual differences in the cost of cognitive and physical effort in humans. Proc Natl Acad Sci U S A. 2023 Dec 12;120(50):e2221510120.

(4) What is the source of cost in saccade production? What is the currency of that cost? The authors state (page 13), "... oblique saccades require more complex oculomotor programs than horizontal eye movements because more neuronal populations in the superior colliculus (SC) and frontal eye fields (FEF) [76-79], and more muscles are necessary to plan and execute the saccade [76, 80, 81]." This statement raises questions and concerns. First, the basis of the claim that more neurons in FEF and SC are needed for oblique versus cardinal saccades is not established in any of the publications cited. Second, the authors may be referring to the fact that oblique saccades require coordination between pontine and midbrain circuits. This must be clarified. Second, the cost is unlikely to originate in extraocular muscle fatigue because the muscle fibers are so different from skeletal muscles, being fundamentally less fatigable. Third, if net muscle contraction is the cost, then why are upward saccades, which require the eyelid, not more expensive than downward? Thus, just how some saccades are more effortful than others is not clear.

(5) The authors do not consider observations about variation in pupil size that seem to be incompatible with the preferred hypothesis. For example, at least two studies have described systematically larger pupil dilation associated with faster relative to accurate performance in manual and saccade tasks (e.g., Naber M, Murphy P. Pupillometric investigation into the speed-accuracy trade-off in a visuo-motor aiming task. Psychophysiology. 2020 Mar;57(3):e13499; Reppert TR, Heitz RP, Schall JD. Neural mechanisms for executive control of speed-accuracy trade-off. Cell Rep. 2023 Nov 28;42(11):113422). Is the fast relative to the accurate option necessarily more costly?

(6) The authors draw conclusions based on trends across participants, but they should be more transparent about variation that contradicts these trends. In Figures 3 and 4 we see many participants producing behavior unlike most others. Who are they? Why do they look so different? Is it just noise, or do different participants adopt different policies?

Reviewer #3 (Public Review):

Summary:

In this paper, Chikermane et al. leverages a large open dataset of intracranial recordings (sEEG or ECoG) to analyze resting state (eyes closed) oscillatory activity from a variety of human brain areas. The authors identify a dominant proportion of channels in which beta band activity (12-30Hz) is most prominent and subsequently seek to relate this to anatomical connectivity data by using the sEEG/ECoG electrodes as seeds in a large set of MRI data from the human connectome project. This reveals separate regions and white matter tracts for alpha (primarily occipital) and beta (prefrontal cortex and basal ganglia) oscillations. Finally, using a third available dataset of PET imaging, the authors relate the parcellated signals to dopamine signaling as estimated by spatial uptake patterns of dopamine, and reveal a significant correlation between the functional connectivity maps and the dopamine reuptake maps, suggesting a functional relationship between the two.

Strengths:

Overall, I found the paper well justified, focused on an important topic, and interesting. The authors' use of 3 different open datasets was creative and informative, and it significantly adds to our understanding of different oscillatory networks in the human brain, and their more elusive relation with neuromodulator signaling networks by adding to our knowledge of the association between beta oscillations and dopamine signaling. Even my main comments about the lack of a theta network analysis and discussion points are relatively minor, and I believe this paper is valuable and informative.

Weaknesses:

The analyses were adequate, and the authors cleverly leveraged these different datasets to build an interesting story. The main aspect I found missing (in addition to some discussion items, see below) was an examination of the theta network. Theta oscillations have been involved in a number of cognitive processes including spatial navigation and memory, and have been proposed to have different potential originating brain regions, and it would be informative to see how their anatomical networks (e.g. as in Figure 2) look like under the author's analyses.

The authors devote a significant portion of the discussion to relating their findings to a popular hypothesis for the function of beta oscillations, the maintenance of the "status quo", mostly in the context of motor control. As the authors acknowledge, given the static nature of the data and lack of behavior, this interpretation remains largely speculative and I found it a bit too far-reaching given the data shown in the paper. In contrast, I missed a more detailed discussion on the growing literature indicating a role for beta in mood (e.g. in Kirkby et al. 2018), especially given the apparent lack of hippocampal and amygdala involvement in the paper, which was surprising.

Major comment:

• Although the proportion of electrodes with theta-dominant oscillations was lower (~15%) than alpha (~22%) or beta (~57%), it would be very valuable to also see the same analyses the authors carried out in these frequency bands extended to theta oscillations.

Reviewer #3 (Public Review):

Summary:

This manuscript aims to understand the role of GABA-ergic inhibition in the human MT+ region in predicting visuo-spatial intelligence through a combination of behavioral measures, fMRI (for functional connectivity measurement), and MRS (for GABA/glutamate concentration measurement). While this is a commendable goal, it becomes apparent that the authors lack fundamental understanding of vision, intelligence, or the relevant literature. As a result, the execution of the research is less coherent, dampening the enthusiasm of the review.

Strengths:

(1) Comprehensive Approach: The study adopts a multi-level approach, i.e., neurochemical analysis of GABA levels, functional connectivity, and behavioral measures to provide a holistic understanding of the relationship between GABA-ergic inhibition and visuo-spatial intelligence.

(2) Sophisticated Techniques: The use of ultra-high field magnetic resonance spectroscopy (MRS) technology for measuring GABA and glutamate concentrations in the MT+ region is a recent development.

Weaknesses:

Study Design and Hypothesis<br /> (1) The central hypothesis of the manuscript posits that "3D visuo-spatial intelligence (the performance of BDT) might be predicted by the inhibitory and/or excitation mechanisms in MT+ and the integrative functions connecting MT+ with the frontal cortex." However, several issues arise:<br /> 1.1 The Suppression Index depicted in Figure 1a, labeled as the "behavior circle," appears irrelevant to the central hypothesis.<br /> 1.2 The construct of 3D visuo-spatial intelligence, operationalized as the performance in the Block Design task, is inconsistently treated as another behavioral task throughout the manuscript, leading to confusion.<br /> 1.3 The schematics in Figure 1a and Figure 6 appear too high-level to be falsifiable. It is suggested that the authors formulate specific and testable hypotheses and preregister them before data collection.

(2) Central to the hypothesis and design of the manuscript is a misinterpretation of a prior study by Melnick et al. (2013). While the original study identified a strong correlation between WAIS (IQ) and the Suppression Index (SI), the current manuscript erroneously asserts a specific relationship between the block design test (from WAIS) and SI. It should be noted that in the original paper, WAIS comprises Similarities, Vocabulary, Block design, and Matrix reasoning tests in Study 1, while the complete WAIS is used in Study 2. Did the authors conduct other WAIS subtests other than the block design task?

(3) Additionally, there are numerous misleading references and unsubstantiated claims throughout the manuscript. As an example of misleading reference, "the human MT ... a key region in the multiple representations of sensory flows (including optic, tactile, and auditory flows) (Bedny et al., 2010; Ricciardi et al., 2007); this ideally suits it to be a new MD core." The two references in this sentence are claims about plasticity in the congenitally blind with sensory deprivation from birth, which is not really relevant to the proposal that hMT+ is a new MD core in healthy volunteers.<br /> Another example of unsubstantiated claim: the rationale for selecting V1 as the control region is based on the assertion that "it mediates the 2D rather than 3D visual domain (Born & Bradley, 2005)". That's not the point made in the Born & Bradley (2005) paper on MT. It's crucial to note that V1 is where the initial binocular convergence occurs in cortex, i.e., inputs from both the right and left eyes to generate a perception of depth.

Results & Discussion<br /> (1) The missing correlation between SI and BDT is crucial to the rest of the analysis. The authors should discuss whether they replicated the pattern of results from Melnick et al. (2013) despite using only one WAIS subtest.

(2) ROIs: can the authors clarify if the results are based on bilateral MT+/V1 or just those in the left hemisphere? Can the authors plot the MRS scan area in V1? I would be surprised if it's precise to V1 and doesn't spread to V2/3 (which is fine to report as early visual cortex).

(3) Did the authors examine V1 FC with either the frontal regions and/or whole brain, as a control analysis? If not, can the author justify why V1 serves as the control region only in the MRS but not in FC (Figure 4) or the mediation analysis (Figure 5)? That seems a little odd given that control analyses are needed to establish the specificity of the claim to MT+.

(4) It is not clear how to interpret the similarity or difference between panels a and b in Figure 4.

(5) SI is not relevant to the authors' priori hypothesis, but is included in several mediation analyses. Can the authors do model comparisons between the ones in Figure 5c, d, and Figure S6? In other words, is SI necessary in the mediation model? There seem discrepancies between the necessity of SI in Figures 5c/S6 vs. Figure 5d.

(6) The sudden appearance of "efficient information" in Figure 6, referring to the neural efficiency hypothesis, raises concerns. Efficient visual information processing occurs throughout the visual cortex, starting from V1. Thus, it appears somewhat selective to apply the neural efficiency hypothesis to MT+ in this context.

Transparency Issues:<br /> (1) Don't think it's acceptable to make the claim that "All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary information". It is the results or visualizations of data analysis, rather than the raw data themselves, that are presented in the paper/supp info.

(2) No GitHub link has been provided in the manuscript to access the source data, which limits the reproducibility and transparency of the study.

Minor:<br /> "Locates" should be replaced with "located" throughout the paper. For example: "To investigate this issue, this study selects the human MT complex (hMT+), a region located at the occipito-temporal border, which represents multiple sensory flows, as the target brain area."

Use "hMT+" instead of "MT+" to be consistent with the term in the literature.

"Green circle" in Figure 1 should be corrected to match its actual color.

The abbreviation for the Wechsler Adult Intelligence Scale should be "WAIS," not "WASI."

Reviewer #3 (Public Review):

Summary:

-The paper offers a systematic and rigorous description of the layer-and sublayer specific outputs of the somatosensory cortex using a modern toolbox for the analysis of brain connectivity which combines: 1) Layer-specific genetic drivers for conditional viral tracing; 2) whole brain analyses of axon tracts using tissue clearing and imaging; 3) Segmentation and quantification of axons with normalization to the number of transduced neurons; 4) registration of connectivity to a widely used anatomical reference atlas; 5) functional validation of the connectivity using optogenetic approaches in vivo.

Strengths:

- Although the connectivity of the somatosensory cortex is already known, precise data are dispersed in different accounts (papers, online resources,) using different methods. So the present account has the merit of condensing this information in one very precisely documented report. It also brings new insights on the connectivity, such as the precise comparison of layer specific outputs, and of the primary and secondary somatosensory areas. It also shows a topographic organization of the circuits linking the somatosensory and motor cortices. The paper also offers a clear description of the methodology and of a rigorous approach to quantitative anatomy.

Weaknesses:

The weakness relates to the intrinsic limitations of the in toto approaches, that currently lack the precision and resolution allowing to identify single axons, axon branching or synaptic connectivity. These limitations are identified and discussed by the authors.

Reviewer #3 (Public Review):

In the current paper, Abbasi et al. aimed to characterize and compare the patterns of functional connectivity across frequency bands (1 Hz - 90 Hz) between regions of a speech network derived from an online meta-analysis tool (Neurosynth.org) during speech production and perception. The authors present evidence for complex neural dynamics from which they highlight directional connectivity from the right cerebellum to left superior temporal areas in lower frequency bands (up to beta) and between the same regions in the opposite direction in the (lower) high gamma range (60-90 Hz). Abbasi et al. interpret their findings within the predictive coding framework, with the cerebellum and other "higher-order" (motor) regions transmitting top-down sensory predictions to "lower-order" (sensory) regions in the lower frequencies and prediction errors flowing in the opposite direction (i.e., bottom-up) from those sensory regions in the gamma band. They also report a negative correlation between the strength of this top-down functional connectivity and the alignment of superior temporal regions to the syllable rate of one's speech.

Strengths:

(1) The comprehensive characterization of functional connectivity during speaking and listening to speech may be valuable as a first step toward understanding the neural dynamics involved.

(2) The inclusion of subcortical regions and connectivity profiles up to 90Hz using MEG is interesting and relatively novel.

(3) The analysis pipeline is generally adequate for the exploratory nature of the work.

Weaknesses:

(1) The work is framed as a test of the predictive coding theory as it applies to speech production and perception, but the methodological approach is not suited to this endeavor.

(2) Because of their theoretical framework, the authors readily attribute roles or hierarchy to brain regions (e.g., higher- vs lower-order) and cognitive functions to observed connectivity patterns (e.g., feedforward vs feedback, predictions vs prediction errors) that cannot be determined from the data. Thus, many of the authors' claims are unsupported.

(3) The authors' theoretical stance seems to influence the presentation of the results, which may inadvertently misrepresent the (otherwise perfectly valid; cf. Abbasi et al., 2023) exploratory nature of the study. Thus, results about specific regions are often highlighted in figures (e.g., Figure 2 top row) and text without clear reasons.

(4) Some of the key findings (e.g., connectivity in opposite directions in distinct frequency bands) feature in a previous publication and are, therefore, interesting but not novel.

(5) The quantitative comparison between speech production and perception is interesting but insufficiently motivated.

(6) Details about the Neurosynth meta-analysis and subsequent selection of brain regions for the functional connectivity analyses are incomplete. Moreover, the use of the term 'Speech' in Neurosynth seems inappropriate (i.e., includes irrelevant works, yielding questionable results). The approach of using separate meta-analyses for 'Speech production' and 'Speech perception' taken by Abbasi et al. (2023) seems more principled. This approach would result, for example, in the inclusion of brain areas such as M1 and the BG that are relevant for speech production.

(7) The results involving subcortical regions are central to the paper, but no steps are taken to address the challenges involved in the analysis of subcortical activity using MEG. Additional methodological detail and analyses would be required to make these results more compelling. For example, it would be important to know what the coverage of the MEG system is, what head model was used for the source localization of cerebellar activity, and if specific preprocessing or additional analyses were performed to ensure that the localized subcortical activity (in particular) is valid.

(8) The results and methods are often detailed with important omissions (a speech-brain coupling analysis section is missing) and imprecisions (e.g., re: Figure 5; the Connectivity Analysis section is copy-pasted from their previous work), which makes it difficult to understand what is being examined and how. (It is also not good practice to refer the reader to previous publications for basic methodological details, for example, about the experimental paradigm and key analyses.) Conversely, some methodological details are given, e.g., the acquisition of EMG data, without further explanation of how those data were used in the current paper.

(9) The examination of gamma functional connectivity in the 60 - 90 Hz range could be better motivated. Although some citations involving short-range connectivity in these frequencies are given (e.g., within the visual system), a more compelling argument for looking at this frequency range for longer-range connectivity may be required.

(10) The choice of source localization method (linearly constrained minimum variance) could be explained, particularly given that other methods (e.g. dynamic imaging of coherent sources) were specifically designed and might potentially be a better alternative for the types of analyses performed in the study.

(11) The mGC analysis needs to be more comprehensively detailed for the reader to be able to assess what is being reported and the strength of the evidence. Relatedly, first-level statistics (e.g., via estimation of the noise level) would make the mGC and DAI results more compelling.

(12) Considering the exploratory nature of the study, it is essential for other researchers to continue investigating and validating the results presented in the current manuscript. Thus, it is concerning that data and scripts are not fully and openly available. Data need not be in its raw state to be shared and useful, which circumvents the stated data privacy concerns.

Reviewer #3 (Public Review):

Summary:

The authors presented data that linked vitamin B12, S-adenosyl methionine (SAM), and phosphatidylcholine (PC) synthesis to lipid homeostasis in C. elegans. They confirmed mechanisms previously shown by other labs, including the regulation of FAT-7 expression by SBP-1, and the targeting of SEIP-1 by PC levels. The authors also attempted to link the synthesis of phospho-choline by the ASM-3 sphingomyelinase to PC synthesis and lipid homeostasis. However, the relative contribution of phospho-choline by ASM-3 versus the canonical Kennedy pathway was not elucidated. Therefore, the significance of the ASM-3-dependent mechanism to PC synthesis requires further investigation.

Strengths:

The authors used a wide range of biochemical and cell biological methods to measure fatty acid composition, neutral lipid levels, and lipid droplet dynamics in C. elegans. The quality of the data is generally high.

Weaknesses:

Data interpretation and the construction of the working model did not seem to take into account the two well-established pathways for PC synthesis. The Kennedy pathway generates PC from phospho-choline and DAG via a cytidine-based intermediate. The second PC synthesis pathway entails the methylation of PE by PEMT, with the donor methyl groups provided by the vitamin B12-dependent 1-carbon cycle. The authors' model seemed to overlook part of the Kennedy pathway that involves choline kinase (and not ASM-3) as the canonical enzyme that generates phospho-choline. The authors also did not explicitly consider DAG as a precursor of triacylglycerol (TAG), which was directly or indirectly measured as a readout of organismal fat content in the paper. Therefore, alternative models should be entertained. For example, the proposed genetic and dietary effects on lipid homeostasis could stem from the competition for a limiting pool of precursors that were shared by PC and TAG synthesis. PC itself may not have a deterministic role, as depicted by the authors' model. Finally, the claim that "coelomocytes regulate diets-induced lipid homeostasis through asm-3" was not well supported. In the absence of quantitative analysis of phospho-choline in mutants, it was unclear how much ASM-3 contributed to the overall phospho-choline, and ultimately PC level. The proposed inter-tissue regulation of PC synthesis also requires coelomocytes-specific knock-down/depletion of asm-3 for verification.

Reviewer #3 (Public Review):

Summary:

Prior research on SCC3, a cohesin subunit protein, in yeast and Arabidopsis has underscored its vital role in cell division. This study investigated into the specific functions of SCC3 in rice mitosis and meiosis. In a weakened SCC3 mutant, sister chromatids separating was observed in anaphase I, resulting in 24 univalents and subsequent sterility. The authors meticulously documented SCC3's loading and degradation dynamics on chromosomes, noting its impact on DNA replication. Despite the loss of homologous chromosome pairing and synapsis in the mutant, chromosomes retained double-strand breaks without fragmenting. Consequently, the authors inferred that in the scc3 mutant, DNA repair more frequently relies on sister chromatids as templates compared to the wild type.

Strengths:

The study presents exceptionally well-executed research in the field of rice cytogenetics.

Weaknesses:

While the paper's conclusions are generally well-supported, further substantiation is needed for the claim that SCC3 inhibits template choice for sister chromatids. To bolster this conclusion, I recommend that the authors perform whole-genome sequencing on parental and F1 individuals from two rice variants, subsequently calculating the allele frequencies at heterozygous sites in the F1 individuals. If SCC3 indeed inhibits inter-sister chromatid repair in the wild type, we would anticipate a higher frequency of inter-homologous chromosome repair (i.e., gene conversion). This should be manifested as a bias away from the Mendelian inheritance ratio (50:50) in the offspring of the wild type compared to the offspring of the scc3+/- mutant.

Reviewer #3 (Public Review):

Summary:

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 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 of 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:

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

Manuscript revision:

The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.

Reviewer #3 (Public Review):

Summary:

This study aims to demonstrate that cortical feedback is not necessary to signal behavioral outcome to shell neurons of the inferior colliculus during a sound detection task. The demonstration is achieved in a very clear manner by the observation of the activity of cortico-recepient neurons in animals which have received lesions of the auditory cortex. The experiment shows that neither behavior performance nor neuronal responses are significantly impacted by cortical lesions except for the case of partial lesions which seem to have a disruptive effect on behavioral outcome signaling.

Strengths:

The demonstration of the main conclusions is based on state-of-the-art, carefully controlled methods and is highly convincing. There is an in depth discussion of the different effects of auditory cortical lesions on sound detection behavior.

Weaknesses:

The description of feedback signals could be more detailed although it is difficult to achieve good temporal resolution with the calcium imaging technique necessary for targeting cortico-recipient neurons.

Reviewer #3 (Public Review):

Summary:

Leveraging zebra fish as a research model, Wang et al identified "cytoneme-like structures" as a mechanism for mediating cell-cell communications among skin epidermal cells. The authors further demonstrated that the "cytoneme-like structures" can mediate Notch signaling, and the "cytoneme-like structures" are influenced by IL17 signaling.

Strengths:

Elegant zebrafish genetics, reporters, and live imaging.

Weaknesses: (minor)<br /> This paper focused on characterizing the "cytoneme-like structures" between different layers and the NOTCH signaling. However, these "cytoneme-like structures" observed in undifferentiated KC (Figure 2B), although at a slightly lower frequency, were not interpreted. In addition, it is unclear if these "cytoneme-like structures" can mediate other signaling pathways than NOTCH.

Overall, this is a solid paper with convincing data reporting the "cytoneme-like structures" in vivo, and with compelling data demonstrating the roles in NOTCH signaling and the regulation by IL17.

These findings provide a foundation for future work exploring the "cytoneme-like structures" in the mammalian system and other epithelial tissue types. This paper also suggests a potential connection between the "cytoneme-like structures" and psoriasis, which needs to be further explored in clinical samples.

Reviewer #3 (Public Review):

Summary:

In this study, Li et al. identified CAD96CA and FGF1 among 20 receptor tyrosine kinase receptors as mediators of JH signaling. By performing a screen in HaEpi cells with overactivated JH signaling, the authors pinpointed two main RTKs that contribute to the transduction of JH. Using the CRISPR/Cas9 system to generate mutants, the authors confirmed that these RTKs are required for normal JH activation, as precocious pupariation was observed in their absence. Additionally, the authors demonstrated that both CAD96CA and FGF1 exhibit a high affinity for JH, and their activation is necessary for the proper phosphorylation of Tai and Met, transcription factors that promote the transcriptional response. Finally, the authors provided evidence suggesting that the function of CAD96CA and FGF1 as JH receptors is conserved across insects.

Strengths:

The data provided by the authors are convincing and support the main conclusions of the study, providing ample evidence to demonstrate that phosphorylation of the transducers Met and Tai mainly depends on the activity of two RTKs. Additionally, the binding assays conducted by the authors support the function of CAD96CA and FGF1 as membrane receptors of JH. The study's results validate, at least in H. amigera, the predicted existence of membrane receptors for JH.

Weaknesses:

The study has several weaknesses that need to be addressed. Firstly, it is not clear what criteria were used by the authors to discard several other RTKs that were identified as repressors of JH signaling. For example, while NRK and Wsck may not fulfill all the requirements to become JH receptors, other evidence, such as depletion analysis and target gene expression, suggests they are involved in proper JH signaling activation.

Secondly, the expression of the six RTKs, which, when knocked down, were able to revert JH signaling activation, was mainly detected in the last larval stage of H. amigera. However, since JH signaling is active throughout larval development, it is unclear whether these RTKs are completely required for pathway activation or only needed for high activation levels at the last larval stage.<br /> Additionally, the mechanism by which different RTKs exert their functions in a specific manner is not clear. According to the expression profile of the different RTKs, one might expect some redundant role of those receptors. In fact the no reversion of phosphorilation of tai and met upon depletion of Wsck in cells with overactivated JH signalling seems to support this idea.

Nevertheless, and despite the overlapping expression of the different receptors, all RTKs seem to be required for proper pathway activation, even in the case of FGF1 which seems to be only expressed in the midgut. This is an intriguing point unresolved in the study.

Finally, the study does not explain how RTKs with known ligands could also bind JH and contribute to JH signaling activation. in Drosophila, FGF1 is activated by pyramus and thisbe for mesoderm development, while CAD96CA is activated by collagen during wound healing. Now the authors claim that in addition to these ligands, the receptors also bind to JH. However, it is unclear whether these RTKs are activated by JH independently of their known ligands, suggesting a specific binding site for JH, or if they are only induced by JH activation when those ligands are present in a synergistic manner. Alternatively, another explanation could be that the RTK pathways by their known ligands activation may induce certain levels of JH transducer phosphorylation, which, in the presence of JH, contributes to the full pathway activation without JH-RTK binding being necessary.

DOI: 10.21203/rs.3.rs-3592641/v1

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @maulamb

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.21203/rs.3.rs-3592641/v1

Resource: (BDSC Cat# 8121,RRID:BDSC_8121)

Curator: @maulamb

SciCrunch record: RRID:BDSC_8121

What is this?

DOI: 10.21203/rs.3.rs-3592641/v1

Resource: (BDSC Cat# 458,RRID:BDSC_458)

Curator: @maulamb

SciCrunch record: RRID:BDSC_458

What is this?

Reviewer #3 (Public Review):

Summary:

The work by Kalita et al. reports regulation of RecB expression by Hfq protein in E.coli cell. RecBCD is an essential complex for DNA repair and chromosome maintenance. The expression level needs to be regulated at low level under regular growth conditions but upregulated upon DNA damage. Through quantitative imaging, the authors demonstrate that recB mRNAs and proteins are expressed at low level under regular conditions. While the mRNA copy number demonstrates high noise level due to stochastic gene expression, the protein level is maintained at a lower noise level compared to expected value. Upon DNA damage, the authors claim that the recB mRNA concentration is decreased, however RecB protein level is compensated by higher translation efficiency. Through analyzing CLASH data on Hfq, they identified two Hfq binding sites on RecB polycistronic mRNA, one of which is localized at the ribosome binding site (RBS). Through measuring RecB mRNA and protein level in the ∆hfq cell, the authors conclude that binding of Hfq to the RBS region of recB mRNA suppresses translation of recB mRNA. This conclusion is further supported by the same measurement in the presence of Hfq sequestrator, the sRNA ChiX, and the deletion of the Hfq binding region on the mRNA.

Strengths:

(1) The manuscript is well-written and easy to understand.<br /> (2) While there are reported cases of Hfq regulating translation of bound mRNAs, its effect on reducing translation noise is relatively new.<br /> (3) The imaging and analysis are carefully performed with necessary controls.

Weaknesses:

The major weaknesses include a lack of mechanistic depth, and part of the conclusions are not fully supported by the data.

(1) Mechanistically, it is still unclear why upon DNA damage, translation level of recB mRNA increases, which makes the story less complete. The authors mention in the Discussion that a moderate (30%) decrease in Hfq protein was observed in previous study, which may explain the loss of translation repression on recB. However, given that this mRNA exists in very low copy number (a few per cell) and that Hfq copy number is on the order of a few hundred to a few thousand, it's unclear how 30% decrease in the protein level should resides a significant change in its regulation of recB mRNA.<br /> (2) Based on the experiment and the model, Hfq regulates translation of recB gene through binding to the RBS of the upstream ptrA gene through translation coupling. In this case, one would expect that the behavior of ptrA gene expression and its response to Hfq regulation would be quite similar to recB. Performing the same measurement on ptrA gene expression in the presence and absence of Hfq would strengthen the conclusion and model.<br /> (3) The authors agree that they cannot exclude the possibility of sRNA being involved in the translation regulation. However, this can be tested by performing the imaging experiments in the presence of Hfq proximal face mutations, which largely disrupt binding of sRNAs.<br /> (4) The data on construct with a long region of Hfq binding site on recB mRNA deleted is less convincing. There is no control to show that removing this sequence region itself has no effect on translation, and the effect is solely due to the lack of Hfq binding. A better experiment would be using a Hfq distal face mutant that is deficient in binding to the ARN motifs.<br /> (5) Ln 249-251: The authors claim that the stability of recB mRNA is not changed in ∆hfq simply based on the steady-state mRNA level. To claim so, the lifetime needs to be measured in the absence of Hfq.<br /> (6) What's the labeling efficiency of Halo-tag? If not 100% labeled, is it considered in the protein number quantification? Is the protein copy number quantification through imaging calibrated by an independent method? Does Halo tag affect the protein translation or degradation?<br /> (7) Upper panel of Fig S8a is redundant as in Fig 5B. Seems that Fig S8d is not described in the text.

Reviewer #3 (Public Review):

Strength:

The development of an automated Barnes maze allows for more naturalistic and uninterrupted behavior, facilitating the study of spatial learning and memory, as well as the analysis of the brain's neural networks during behavior when combined with neurophysiological techniques. The system's design has been thoughtfully considered, encompassing numerous intricate details. These details include the incorporation of flexible options for selecting start, goal, and proximal landmark positions, the inclusion of a rotating platform to prevent the accumulation of olfactory cues, and careful attention given to atomization, taking into account specific considerations such as the rotation of the maze without causing wire shortage or breakage. When combined with neurophysiological manipulations or recordings, the system provides a powerful tool for studying spatial navigation system.<br /> The behavioral experiment protocols, along with the analysis of animal behavior, are conducted with care, and the development of behavioral modeling to capture the animal's search strategy is thoughtfully executed. It is intriguing to observe how the integration of these innovative stochastic models can elucidate the evolution of mice's search strategy within a variant of the Barnes maze.

Comments on revised version:

The authors have addressed all the points I outlined in the previous round of review, resulting in significant improvements to the manuscript. However, I have one remaining comment. Given the updated inter-animal analysis (Supplementary Figure 8), it appears that male and female mice develop strategies differently across days. Male mice seem to progressively increase their employment of spatial strategy across days, at the expense of the random strategy. Conversely, female mice exhibit both spatial and serial strategies at their highest levels on day 2, with minimal changes observed on the subsequent days.<br /> These findings could alter the interpretation of Figure 5 and the corresponding text in the section "Evolution of search strategy across days".<br /> For instance, this statement on page 6 doesn't hold for female mice: "The spatial strategy was increased across days, ... largely at the expense of the random strategy."

Reviewer #3 (Public Review):

Summary:

The authors investigate the hypothesis that neurexins serve a crucial role as regulators of the synaptic strength and timing at the glycinergic synapse between neurons of the medial nucleus of the trapezoid body (MNTB) and the lateral superior olivary complex (LSO). It is worth mentioning that LSO neurons are an integration station of the auditory brainstem circuit displaying high reliability and temporal precision. These features are necessary for computing interaural cues to derive sound source location from comparing the intensities of sounds arriving at the two ears. In this context, the authors' findings build up according to the hypothesis first by displaying that neurexins were expressed in the MNTB at varying levels. They followed this up with deletion of all neurexins in the MNTB through the employment of a triple knock-out (TKO). Using electrophysiological recordings in acute brainstem slices of these TKO mice, they gathered solid evidence for the role of neurexins in synaptic transmission at this glycinergic synapse primarily by ensuring tight coupling of Ca2+ channels and vesicular release sites. Additionally, the authors uncovered a connection between the deletion of neurexins and a higher number of glycinergic synapses of TKO mice, for which they provided evidence in the form of immunostainings and related it to electrophysiological data on spontaneous release. Consequently, this investigation expands our knowledge on the molecular regulation of synaptic transmission at glycinergic synapses, as well as on the auditory processing at the level of the brainstem.

Strengths:

The authors demonstrate substantial results in support of the hypothesis of a critical role of neurexins for regulating glycinergic transmission in the LSO using various techniques. They provide evidence for the expression of neurexins in the MNTB and consecutively successfully generate and characterize the neurexin TKO. For their study on LSO IPSCs the authors transduced MNTB neurons by co-injection of virus carrying Cre and ChR2 and subsequently optogenetically evoke release of glycine. As a result, they observed a significant reduction in amplitude and significantly slower rise and decay times of the IPSCs of the TKO in comparison with control mice in which MNTB neurons were only transduced with ChR2. Furthermore, they observed an increased paired pulse ratio (PPR) of LSO IPSCs in the TKO mice, indicating lower release probability. Elaborating on the hypothesis that neurexins are essential for the coupling of synaptic vesicles to Ca2+ channels, the authors show lowered Ca2+ sensitivity in the TKO mice. Additionally, they reveal convincing evidence for the connection between the increased frequency of spontaneous IPSC and the higher number of glycinergic synapses of the LSO in the TKO mice, revealed by immunolabeling against the glycinergic presynaptic markers GlyT2 or VGAT.

Weaknesses:

A concern is on novelty as this work on the effects of pan-neurexin deletion in a glycinergic synapse is quite consistent with the authors prior work on glutamatergic synapses (Luo et al., 2020).

Reviewer #3 (Public Review):

Summary

In this manuscript, Weng et al. identify the neuron specific transcriptome that impacts age dependent cognitive decline. The authors design a pipeline to profile neurons from wild type and long-lived insulin receptor/IGF-1 mutants using timepoints when memory functions are declining. They discover signatures unique to neurons which validates their approach. The authors identify that genes related to neuronal identity are lost with age in wild type worms. For example, old neurons reduce the expression of genes linked to synaptic function and neuropeptide signaling and increase the expression of chromatin regulators, insulin peptides and glycoproteins. Depletion of selected genes which are upregulated in old neurons (utx-1, ins-19 and nmgp-1) leads to improved short memory function. This indicates that some genes that increase with age have detrimental effects on learning and memory. The pipeline is then used to test neuronal profiles of long-lived insulin/IGF-1 daf-2 mutants. Genes related to stress response pathways are upregulated in long lived daf-2 mutants (e.g. dod-24, F08H9.4) and those genes are required for improved neuron function.

Strengths

The manuscript is well written, and the experiments are well described. The authors take great care to explain their reasoning for performing experiments in a specific way and guide the reader through the interpretation of the results, which makes this manuscript an enjoyable and interesting read. The authors discover novel regulators of learning and memory using neuron-specific transcriptomic analysis in aged animals, which underlines the importance of cell specific deep sequencing. The timepoints of the transcriptomic profiling are elegantly chosen, as they coincide with the loss of memory and can be used to specifically reveal gene expression profiles related to neuron function. The authors discuss on the dod-24 example how powerful this approach is. In daf-2 mutants whole-body dod-24 expression differs from neuron specific profiles, which underlines the importance of precise cell specific approaches. This dataset will provide a very useful resource for the C. elegans and aging community as it complements existing datasets with additional time points and neuron specific deep profiling.

Weakness

This study nicely describes the neuron specific profiles of aged long-lived daf-2 mutants. Selected neuronal genes that were upregulated in daf-2 mutants (e.g. F08H9.4, mtl-1, dod-24, alh-2, C44B7.5) decreased learning/memory when knocked down. However, the knock down of these genes was not specific to neurons. The authors use a neuron-sensitive RNAi strain to address this concern and acknowledge this caveat in the text. While it is likely that selected candidates act only in neurons it is possible that other tissues participate as well.

Reviewer #3 (Public Review):

Summary:

This is a well prepared manuscript which presented interesting research result.

Strengths:

The omics method produced unbiased results.

Weaknesses:

LPS neutralization is not new method for treating leptospiral infection.

Reviewer #4 (Public Review):

Summary:

In the present study, Spikol et al. explore the projection patterns and functional characteristics of two distinct and genetically defined populations in the larval zebrafish Nucleus Incertus (NI), expressing the transcription factor gsc2 or the neuropeptide rln3a. To label in vivo these neurons two transgenic lines were generated by CRISPR/Cas9 mediated Knock-in. These genetic tools allowed the analysis of the projection patterns of these neuronal populations showing that the NI neurons expressing gsc2 and rln3a exhibit markedly different projection patterns, targeting separate subregions within the midbrain interpeduncular nucleus (IPN).<br /> Functional imaging and behavioral analysis revealed that while gsc2 neurons respond to electric shock stimuli, rln3a neurons show high spontaneous activity and play a role in regulating locomotor activity.

Strengths:

The paper relies on a series of rigorous experimental approaches including molecular genetic, neuroanatomical, functional and behavioral analysis. The resources generated including the two knock-in transgenic reporter lines will be of great value for the zebrafish neurobiology community as well as inspire further studies of the NI in other model systems.

Weaknesses:

Technical weaknesses present in the first version of the manuscript have largely been addressed in the present revision.

Reviewer #3 (Public Review):

Summary:

This manuscript describes RNAi depletion of isp-1 or spg-7 in the GABAergic neurons of C. elegans leads to: lifespan extension; increased resistance to paraquat oxidative stress and heat stress; decreased brood size and mitotic germ cell numbers in the gonad and increased DNA aggregates in the oocytes; increased mitochondrial membrane potential, ATP levels, mitochondrial mass, mitochondrial DNA copies, mitochondrial DNA polymerase gamma polg-1 levels, and decreased ROS levels. The authors further show that daf-16 is necessary for GABAergic depletion of isp-1 mediated lifespan extension, stress resistance, increased mitochondrial membrane potential, mitochondrial mass and DNA copies, and decreased brood size. Unc-25 for GABA synthesis, unc-31 for neuropeptide secretion, and flp-13 neuropeptide are all in the same pathway of isp-1 RNAi in GABAergic neurons for lifespan extension and stress resistance.

Strengths:

The topic is interesting and relatively novel in terms of GABAergic mitochondrial dysfunction. The data provided support the conclusions well.

Weaknesses:

The mechanistic evidence needs to be improved substantially.

Reviewer #3 (Public Review):

Summary:

Mou and Ji investigated neuro-computational mechanisms behind observational spatial learning in rats and reported several signs of functional coupling between the ACC and CA1 at the single neuron level. Using multi-site tetrode recording, they found that ACC cells encoding a path on a maze were activated while a rat observed another rat took that path. This activation was also correlated with the activation of CA1 cells encoding the same path and facilitated their replay during sharp-wave ripples (SWRs) before the recording rat ran on the maze by itself. These activity patterns were associated with correct path choice during self-running and were absent in control conditions where the recording rat did not learn the correct choice through observations. Based on these findings, the authors argue that ACC cells capture the critical information during observation to organize hippocampal cell activity for subsequent spatial decisions.

Strengths:

The authors used multiple outcome measures to build a strong case for path-specific spike coordination between ACC and CA1 cells. The analyses were conducted carefully, and proper control measures were used to establish the statistical significance of the detected effects. The authors also demonstrated tight correlations between the spike coordination patterns and the successful use of observed information for future decisions.

Weaknesses:

(1) As evidence for the activation of path information in the ACC during observation, the authors showed positive correlations between firing rates during observation and those during self-running. This argument will be solidified if the authors use a decoding approach to demonstrate the activation of path-selective ACC ensemble activity patterns during observation. This approach will also open the door to uncovering how the activation of ACC path representation is related to the moment-to-moment position of the demonstrator rat and whether it is coupled with the timing of SWRs.

(2) The authors argued that the ACC biases the content of awake replay in CA1 during SWRs in the observation period. The reviewer wonders if a similar bias also occurs during SWRs in the self-run period (i.e., water consumption after the correct choice). This analysis will be helpful in testing if the biased replay occurs due to the need to convert observed information into future choices.

(3) Although the authors demonstrated the necessity of the ACC for the task, it still remains to be determined firing coordination between the ACC and CA1 during observation is necessary for the correct path choice during self-runs. Some discussion on this point, along with future direction, would be beneficial for readers.

Reviewer #3 (Public Review):

Summary:

In the present work, Deganutti et al. report a structural study on GPCR functional dynamics using a computational approach called supervised molecular dynamics.

Strengths:

The study has the potential to provide novel insight into GPCR functionality. An example is the interaction between loops of GPCR and G proteins, which are not resolved experimentally, or the interaction between D344 and R385 identified during the Gs coupling by GLP-1R. However, validation of the findings, even computationally through for instance in silico mutagenesis study, is advisable.

Weaknesses:

In its current form, the manuscript seems immature and in particular, the described results grasp only the surface of the complex molecular mechanisms underlying GPCR activation. No significant advance of the existing structural data on GPCR and GPCR/G protein coupling is provided. Most of the results are a reproduction of the previously reported structures.

Reviewer #3 (Public Review):

Summary:

The study presented by Leitao et al., represents an important advancement in comprehending the social learning processes of sperm whales across various communicative and socio-cultural contexts. The authors introduce the concept of "vocal style" as an addition to the previously established notion of "vocal repertoire," thereby enhancing our understanding of sperm whale vocal identity.

Strengths:

A key finding of this research is the correlation between the similarity of clan vocal styles for non-ID codas and spatial overlap (while no change occurs for ID codas), suggesting that social learning plays a crucial role in shaping symbolic cultural boundaries among sperm whale populations. This work holds great appeal for researchers interested in animal cultures and communication. It is poised to attract a broad audience, including scholars studying animal communication and social learning processes across diverse species, particularly cetaceans.

Weaknesses:

In terms of terminology, while the authors use the term "saying" to describe whale vocalizations, it may be more conservative to employ terms like "vocalize" or "whale speech" throughout the manuscript. This approach aligns with the distinction between human speech and other forms of animal communication, as outlined in prior research (Hockett, 1960; Cheney & Seyfarth, 1998; Hauser et al., 2002; Pinker & Jackendoff, 2005; Tomasello, 2010).

Reviewer #3 (Public Review):

Summary:

This paper describes a new mechanism of clearance of protein aggregates occurring during mitosis.

The authors have observed that animal cells can clear misfolded aggregated proteins at the end of mitosis. The images and data gathered are solid, convincing, and statistically significant. However, there is a lack of insight into the underlying mechanism. They show the involvement of the ER, ATPase-dependent, BiP chaperone, and the requirement of Cdk1 inactivation (a hallmark of mitotic exit) in the process. They also show that the mechanism seems to be independent of the APC/C complex (anaphase-promoting complex). Several points need to be clarified regarding the mechanism that clears the aggregates during mitosis:

• What happens in the cell substructure during mitosis to explain the recruitment of BiP towards the aggregates, which seem to be relocated to the cytoplasm surrounded by the ER membrane.

• How the changes in the cell substructure during mitosis explain the relocation of protein aggregates during mitosis.

• Why BiP seems to be the main player of this mechanism and not the cyto Hsp70 first described to be involved in protein disaggregation.

Strengths:

Experimental data showing clearance of protein aggregates during mitosis is solid, statistically significant, and very interesting.

Weaknesses:

Weak mechanistic insight to explain the process of protein disaggregation, particularly the interconnection between what happens in the cell substructure during mitosis to trigger and drive clearance of protein aggregates.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Boudjerna and Balagé et al. aim to elucidate the spatial origin of centriole amplification and the mechanisms behind the formation of an apical-basal body patch in multiciliated cells (MCCs). To this end, they focused on the role of microtubules and developed new tools for spatiotemporal and high-resolution analysis of different stages of centriole amplification, including the centrosome stages, A-stage, G-stage, and MCC-stage. Among these tools, the MEF-MCC cells grown on micropatterns stands out for its versatility as it is not tissue-specific and does not require epithelial cell-to-cell contact for differentiation. Additionally, the Cen2-GFP; mRuby-Deup1 knock-in mouse model was used to study different stages of centriole amplification in physiological brain MCCs. This model offers an advantage over the previously described Cen2-GFP model by enabling the resolution of early events in centriole amplification through the visualization of Deup1-positive structures and their dynamics. Finally, the authors leveraged powerful imaging techniques, including super-resolution microscopy, the U-ExM, and high-resolution live cell imaging in order to detect and track centriole amplification, elongation, disengagement, and migration.

By combining the MEF-MCC and knock-in mouse model with spatiotemporal imaging in control and nocodazole-treated cells(treated acutely or chronically), the authors define the sequence of events during centriole amplification, revealing the critical roles of microtubules for the first time. Initially, the centrosome-mediated microtubule network forms, organizing a pericentrosomal nest from which procentrioles and deuterosomes emerge. Their findings indicate the importance of microtubules in recruiting and maintaining pericentriolar material clouds that contain DEUP1, PCNT, SAS6, PLK1, PLK4, and tubulins. Following the amplification stage, the procentrioles mature, leading to cells displaying numerous MTOCs, as demonstrated by regrowth experiments. Mature centrioles then disengage from deuterosomes, attach to the nuclear envelope, and migrate to the apical surface facilitated by microtubules.

Strengths:

The manuscript provides new insights into the regulatory function of microtubules in centriole amplification. Addressing the role of microtubules during different stages of centriole amplification required the development of new tools to study brain MCCs, which will be useful in future studies of MCCs. A notable strength of this manuscript is the authors' thorough and quantitative analysis of highly dynamic processes in MCCs. The precision and detail in describing these dynamic events are impressive. This comprehensive analysis advances our understanding of MCC biology.

Weaknesses:

The role of microtubules and other molecular players during different stages of centriole amplification in brain MCCs can be further studied and strengthened using the tools developed in the manuscript. A more quantitative description of some of the analysis performed in the manuscript is required to strengthen the conclusions.

Reviewer #3 (Public Review):

Summary:

The authors want to prove that there is a redox potential between germline stem cells (GSCs) and somatic cyst stem cells (CySCs) in the Drosophila testis, with ROS being higher in the former compared to the latter. They also want to prove that ROS travels from CySCs to GSCs. Finally, they begin to characterize the phenotypes caused by loss of SOD (which normally lowers ROS levels) in the tj- lineage and how this impacts the germline.

Strengths:

The role of SOD in somatic support cells is an under-explored area.

Weaknesses:

The authors fall short of accomplishing their goals. There are issues with the concept of the paper (ROS gradient between cells that causes a transfer of ROS across membranes for homeostasis), the data, the figures, and the scholarship of the testis. I have discussed each of the points in detail below. These weaknesses negatively impact the conclusions put forward by the authors. In short, their data is not compelling: there is no evidence provided by the authors that ROS diffuses from CySCs to GSCs as most of the claims about stem cells are founded on data about differentiating germ and somatic cells. The somatic SOD depletion phenotype is incompletely characterized and several pathways appear to change in these cells, including reduced Egfr signaling, increased Tor signaling, and increased Hh signaling. None of these results are sufficiently followed up on. And none of them are considered relative to their known roles in the testis. For example, high Hh signaling in CySCs increases their competitiveness with GSCs. Increased Tor signaling in all CySCs does not affect the CySC lineage. Reduced Egfr signaling in CySCs reduces the number of CySCs and reduces/inhibits abscission between GSCs-gonialblasts.

Major issues:

(1) Data<br /> a. Problems proving which mitochondria are associated with which lineage.<br /> b. There is no evidence that ROS diffuses from CySCs into GSCs.<br /> c. The changes in gst-GFP (redox readout) are possibly seen in differentiating germ cells (i.e., spermatogonia) but not in GSCs. This weakens their model that ROS in CySC is transferred to GSCs.<br /> d. Most of the paper examines the effect of SOD depletion (which should increase ROS) on the CySC lineage and GSC lineage. One big caveat is that tj-Gal4 is expressed in hub cells (Fairchild, 2016) so the loss of SOD from hub cells may also contribute to the phenotype. In fact, the niche in Figure 2D looks larger than the niche in the control in Figure 2C, arguing that the expression of Tj in niche cells may be contributing to the phenotype. The authors need to better characterize the niche in tj>SOD-RNAi testes.<br /> e. The tj>SOD-RNAi phenotype is an expansion of the Zfh1+ CySC pool, expansion of the Tj+ Zfh1- cyst cells (both due to increased somatic proliferation) and a non-autonomous disruption of the germline.<br /> f. I am not convinced that MAPK signaling is decreased in tj>SOD-i testes. Not only is this antibody finicky, but the authors don't have any follow-up experiments to see if they can restore SOD-depleted CySCs by expressing an Egfr gain of function. Additionally, reduced Egfr activity causes fewer somatic cells (not more) (Amoyel, 2016) and also inhibits abscission between GSCs and gonialblasts (Lenhart 2015), which causes interconnected cysts of 8- to 16 germ cells with one GSC emanating from the hub.<br /> g. The increase in Hh signaling in SOD-depleted CySCs would increase their competitiveness against GSCs and GSCs would be lost (Amoyel 2014). The authors need to validate that Hh protein expression is indeed increased in SOD-depleted CySCs/cyst cells and which cells are producing this Hh. Normally, only hub cells produce Hh (Michel, 2012; Amoyel 2013) to promote self-renewal in CySCs.<br /> h. The increase in p4E-BP is an indication that Tor signaling is increased, but an increase in Tor in the CySC lineage does not significantly affect the number of CySCs or cyst cells (Chen, 2021). So again I am not sure how increased Tor factors into their phenotype.<br /> i. The over-expression of SOD in CySCs part is incomplete. The authors would need to monitor ROS in these testes. They would also need to examine with tj>SOD affects the size of the hub.

(2) Concept<br /> Why would it be important to have a redox gradient across adjacent cells? The authors mention that ROS can be passed between cells, but it would be helpful for them to provide more details about where this has been documented to occur and what biological functions ROS transfer regulates.

(3) Issues with scholarship of the testis<br /> a. Line 82 - There is no mention of BMPs, which are the only GSC-self-renewal signal. Upd/Jak/STAT is required for adhesion of GSCs to the niche but not self-renewal (Leatherman and Dinardo, 2008, 2010). The author should read a review about the testis. I suggest Greenspan et al 2015. The scholarship of the testis should be improved.<br /> b. Line 82-84 - BMPs are produced by both hub cells and CySCs. BMP signaling in GSCs represses bam. So it is not technically correct to say the CySCs repress bam expression in GSCs.<br /> c. Throughout the figures the authors score Vasa+ cells for GSCs. This is technically not correct. What they are counting is single, Vasa+ cells in contact with the niche. All graphs should be updated with the label "GSCs" on the Y-axis.

(4) Issues with the text<br /> a. Line 1: multi-lineage is not correct. Multi-lineage refers to stem cells that produce multiple types of daughter cells. GSCs produce only one type of offspring and CySCs produce only one type of offspring. So both are uni-lineage. Please change accordingly.<br /> b. Lines 62-75 - Intestinal stem cells have constitutively high ROS (Jaspar lab paper) so low ROS in stem cell cells is not an absolute.<br /> c. Line 79: The term cystic is not used in the Drosophila testis. There are cyst stem cells (CySCs) that produce cyst cells. Please revise.<br /> d. Line 90 - perfectly balanced is an overstatement and should be toned down.<br /> e. Line 98 - division of labour is not supported by the data and should be rephrased.<br /> f. Line 200 - the authors provide no data on BMPs - the GSC self-renewal cue - so they should avoid discussing an absence of self-renewal cues.

(5) Issues with the figures<br /> a. The images are too small to appreciate the location of mitochrondria in GSCs and CySCs.<br /> b. Figure 1<br /> i. cell membranes are not marked, reducing the precision of assigning mitochondria to GSC or CySCs. It would be very helpful if the authors depleted ATP5A from GSCs and showed that the puncta are reduced in these cells and did a similar set of experiments for the tj-Gal4 lineage. It would also be very helpful if the authors expressed membrane markers (like myr-GFP) in the GSC and then in the CySC lineage and then stained with ATP5A. This would pinpoint in which cells ATP5A immunoreactivity is occurring.<br /> ii. The presumed changes in gst-GFP (redox readout) are possibly seen in differentiating germ cells (i.e., spermatogonia) but not in GSC.<br /> iii. Panels F, Q, and S are not explained and currently are irrelevant.<br /> c. Figure 3K - The evidence to support less Ecad in GSCs in tj>SOD-i testes is not compelling as the figure is too small and the insets show changes in Ecad in somatic cells, not GSC.<br /> d. Figure 4:<br /> i. Panel A, B The apparent decline (not quantified) may not contribute to the phenotype.<br /> ii. dpERK is a finicky antibody and the authors are showing a single example of each genotype. This is an important experiment because the authors are going to use it to conclude that MAPK is decreased in the tj>SOD-i samples. However, the authors don't have any positive (dominant-active Egfr) or negative (tj>mapk-i). As is standing the data are not compelling. The graph in F does not convey any useful information.<br /> e. Figure S1D - cannot discern green on black. It is critical for the authors to show monochromes (gray scale) for the readouts that they want to emphasize. I cannot see the green on black in Figure S1D.<br /> f. Figure S4 - there is no quantification of the number of Tj cells in K-N.

(6) Issues with Methods<br /> a. Materials and Methods are not described in sufficient depth - please revise.<br /> b. Note that tj-Gal4 has real-time expression in hub cells and this is not considered by the authors. The ideal genotype for targeting CySCs is tjGal4, Gal80TS, hh-Gal80. Additionally, the authors do not mention whether they are depleting throughout development into adulthood or only in adults. If the latter, then they must have used a temperature shift like growing the flies at 18C and then upshifting to 25C or 29C during adult stages.<br /> c. The authors need to show data points in all of the graphs. Some graphs do this but others do not.<br /> d. The authors state that all data points are from three biological replicates. This is not sufficient for GSC and CySC counts. Most labs count GSCs and CySCs from at least 10 testes of the correct genotype.

Reviewer #3 (Public Review):

The authors tested a dietary intervention focused on improving meal regularity in this interesting paper. The study, a two-group, single-center, randomized, controlled, single-blind trial, utilized a smartphone application to track participants' meal frequencies and instructed the experimental group to confine their eating to these times for six weeks. The authors concluded that improving meal regularity reduced excess body weight despite food intake not being altered and contributed to overall improvements in well-being.

The concept is interesting, but the need for more rigor is of concern.

A notable limitation is the reliance on self-reported food intake, with the primary outcome being self-reported body weight/BMI, indicating an average weight loss of 2.62 kg. Despite no observed change in caloric intake, the authors assert weight loss among participants.

The trial's reliance on self-reported caloric intake is problematic, as participants tend to underreport intake; for example, in the NEJM paper (DOI: 10.1056/NEJM199212313272701), some participants underreported caloric intake by approximately 50%, rendering such data unreliable and hence misleading. More rigorous methods for assessing food intake are available and should have been utilized. Merely acknowledging the unreliability of self-reported caloric intake is insufficient as it would still leave the reader with the impression that there is no change in food intake when we actually have no idea if food intake was altered. A more robust approach to assessing food intake is imperative. Even if a decrease in caloric intake is observed through rigorous measurement, as I am convinced a more rigorous study would unveil testing this paradigm, this intervention may merely represent another short-term diet among countless others that show that one may lose weight by going on a diet, principally due to heightened dietary awareness.

Furthermore, the assessment of circadian rhythm using the MCTQ, a self-reported measure of chronotype, may not be as reliable as more objective methods like actigraphy.

Given the potential limitations associated with self-reported data in both dietary intake and circadian rhythm assessment, the overall impact of this manuscript is low. Increasing rigor by incorporating more objective and reliable measurement techniques in future studies could strengthen the validity and impact of the findings.

Reviewer #3 (Public Review):

Summary:

In this study, the authors have started off using an immortalized human cell line and then gene-edited it to decrease the levels of VEGF1 (in order to influence vascularization), and the levels of Runx2 (to decrease chondro/osteogenesis). They first transplanted these cells with a collagen scaffold. The modified cells showed a decrease in vascularization when VEGF1 was decreased, and suggested an increase in cartilage formation.

In another study, the matrix generated by these cells was subsequently remodeled into a bone marrow organ. When RUNX2 was decreased, the cells did not mineralize in vitro, and their matrices expressed types I and II collagen but not type X collagen in vitro, in comparison with unedited cells. In vivo, the author claims that remodeling of the matrices into bone was somewhat inhibited. Lastly, they utilized matrices generated by RUNX2 edited cells to regenerate chondro-osteal defects. They suggest that the edited cells regenerated cartilage in comparison with unedited cells.

Strengths:

-The notion that inducing changes in the ECM by genetically editing the cells is a novel one, as it has long been thought that ECM composition influences cell activity.

-If successful, it may be possible to make off-the-shelf ECMS to carry out different types of tissue repair.

Weaknesses:

-The authors have not generated histologically identifiable cartilage or bone in their transplants of the cells with a type I scaffold.

-In many cases, they did not generate histologically identifiable cartilage with their cell-free-edited scaffold. They did generate small amounts of bone but this is most likely due to BMPs that were synthesized by the cells and trapped in the matrix.

-There is a great deal of missing detail in the manuscript.

-The in vivo study is underpowered, the results are not well documented pictorially, and are not convincing.

-Given the fact that they have genetically modified cells, they could have done analyses of ECM components to determine what was different between the lines, both at the transcriptome and the protein level. Consequently, the study is purely descriptive and does not provide any mechanistic understanding of what mixture of matrix components and growth factors works best for cartilage or bone. But this presupposes that they actually induced the formation of bona fide cartilage, at least.

DOI: 10.21203/rs.3.rs-4166090/v1

Resource: Fiji (RRID:SCR_002285)

Curator: @evieth

SciCrunch record: RRID:SCR_002285

What is this?

DOI: 10.21203/rs.3.rs-4166090/v1

Resource: ImageJ (RRID:SCR_003070)

Curator: @evieth

SciCrunch record: RRID:SCR_003070

What is this?