Reviewer #3 (Public Review):

Summary:

The paper attempts to model the functional significance of variants of PLCG2 in a set of patients with variable clinical manifestations.

Strengths:

A study attempting to use the Drosophila system to test the function of variants reported from human patients.

Weaknesses:

Additional experiments are needed to shore up the claims in the paper. These are listed below.

Major Comments:

(1) Does the pLI/ missense constraint Z score prediction algorithm take into consideration whether the gene exhibits monoallelic or biallelic expression?

(2) Figure 1B: Include human PLCG2 in the alignment that displays the species-wide conserved variant residues.

(3) Figure 4A:<br /> Given that<br /> (i) sl is predicted to be the fly ortholog for both mammalian PLCγ isozymes: PLCG1 and PLCG2 [Line 62]<br /> (ii) they are shown to have non-redundant roles in mammals [Line 71] and<br /> (iii) reconstituting PLCG1 is highly toxic in flies, leading to increased lethality.<br /> This raises questions about whether sl mutant phenotypes are specifically caused by the absence of PLG1 or PLCG2 functions in flies. Can hPLCG2 reconstitution in sl mutants be used as a negative control to rule out the possibility of the same?

(4) Do slT2A/Y; UAS-PLCG1Reference flies survive when grown at 22{degree sign}C? Since transgenic fly expressing PLCG1 cDNA when driven under ubiquitous gal4s, Tubulin and Da, can result in viable progeny at 22{degree sign}C, the survival of slT2A/Y; UAS-PLCG1Reference should be possible.<br /> and similarly<br /> Does slT2A flies exhibit the phenotypes of (i) reduced eclosion rate (ii) reduced wing size and ectopic wing veins and (iii) extra R7 photoreceptor in the fly eye at 22{degree sign}C?<br /> If so, will it be possible to get a complete rescue of the slT2A mutant phenotypes with the hPLCG1 cDNA at 22{degree sign}C? This dataset is essential to establish Drosophila as an ideal model to study the PLCG1 de novo variants.

(5) Localisation and western blot assays to check if the introduction of the de novo mutations can have an impact on the sub-cellular targeting of the protein or protein stability respectively.

(6) Analysing the nature of the reported gain of function (experimental proof for the same is missing in the manuscript) variants:<br /> Instead of directly showing the effect of introducing the de novo variant transgenes in the Drosophila model especially when the full-length PLCG1 is not able to completely rescue the slT2A phenotype;<br /> (i) Show that the gain-of-function variants can have an impact on the protein function or signalling via one of the three signalling outputs in the mammalian cell culture system: (i) inositol-1,4,5-trisphosphate production, (ii) intracellular Ca2+ release or (iii) increased phosphorylation of extracellular signal-related kinase, p65, and p38.<br /> OR<br /> (ii) Run a molecular simulation to demonstrate how the protein's auto-inhibited state can be disrupted and basal lipase activity increased by introducing D1019G and D1165G, which destabilise the association between the C2 and cSH2 domains. The H380R variant may also exhibit characteristics similar to the previously documented H335A mutation which leaves the protein catalytically inactive as the residue is important to coordinate the incoming water molecule required for PIP2 hydrolysis.

(7) Clarify the reason for carrying out the wing-specific and eye-specific experiments using nub-gal4 and eyless-gal4 at 29˚C despite the high gal4 toxicity at this temperature.

(8) For the sake of completeness the authors should also report other variants identified in the genomes of these patients that could also contribute to the clinical features.

Reviewer #3 (Public Review):

In this manuscript, Casas-Tintó et al. explore the role of glial cells in the response to a neurodegenerative injury in the adult brain. They used Drosophila melanogaster as a model organism and found that glial cells are able to generate new neurons through the mechanism of transdifferentiation in response to injury.

This paper provides a new mechanism in regeneration and gives an understanding of the role of glial cells in the process.

Now it got it wrong

Reviewer #3 (Public Review):

Summary:

Based on their observation that tumor has an iron-deficient microenvironment, and the assumption that nutritional immunity is important in bacteria-mediated tumor modulation, the authors postulate that manipulation of iron homeostasis can affect tumor growth. This paper uses straightforward in vitro and in vivo techniques to examine a specific and important question of nutritional immunity in bacteria-mediated tumor therapy. They are successful in showing that manipulation of iron regulation during nutritional immunity does affect the virulence of the bacteria, and in turn the tumor. These findings open future avenues of investigation, including the use of different bacteria, different delivery systems for therapeutics, and different tumor types. The authors were also successful in addressing the reviewer's concerns adequately.

Reviewer #3 (Public Review):

Summary:

In the 'bCFS' paradigm, a monocular target gradually increases in contrast until it breaks interocular suppression by a rich monocular suppressor in the other eye. The present authors extend the bCFS paradigm by allowing the target to reduce back down in contrast until it becomes suppressed again. The main variable of interest is the contrast difference between breaking suppression and (re) entering suppression. The authors find this difference to be constant across a range of target types, even ones that differ substantially in the contrast at which they break interocular suppression (the variable conventionally measured in bCFS). They also measure how the difference changes as a function of other manipulations. Interpretation is in terms of the processing of unconscious visual content, as well as in terms of the mechanism of interocular suppression.

Strengths:

Interpretation of bCFS findings is mired in controversy, and this is an ingenuous effort to move beyond the paradigm's exclusive focus on breaking suppression. The notion of using the contrast difference between breaking and entering suppression as an index of suppression depth is interesting. The finding that this difference is similar for a range of target types that do differ in the contrast at which they break suppression, suggests a common mechanism of suppression across those target types.

Reviewer #3 (Public Review):

Summary:

The authors image dopamine axons in medial prefrontal cortex (mPFC) using microprism-mediated two-photon calcium imaging. They image these axons as mice learn that two auditory cues predict two distinct outcomes, tailshock, or water delivery. They find that some axons show a preference for encoding of the shock and some show a preference for encoding of water. The authors report a greater number of dopamine axons in mPFC that respond to shock. Across time, the shock-preferring axons begin to respond preferentially to the cue predicting shock, while there is a less pronounced increase in the water-responsive axons that acquire a response to the water-predictive cue (these axons also increase non-significantly to the shock-predictive cue). These data lead the authors to argue that dopamine axons in mPFC preferentially encode aversive stimuli.

Strengths:

The experiments are beautifully executed and the authors have mastered an impressively complex technique. Specifically, they are able to image and track individual dopamine axons in mPFC across days of learning. And this technique is used the way it should be: the authors isolate distinct dopamine axons in mPFC and characterize their encoding preferences and how this evolves across learning of cue-shock and cue-water contingencies. Thus, these experiments are revealing novel information about how aversive and rewarding stimuli is encoded at the level of individual axons, in a way that has not been done before. This is timely and important.

Weaknesses:

The overarching conclusion of the paper is that dopamine axons preferentially encode aversive stimuli. However, this is confounded by differences in the strength of the aversive and appetitive outcomes. As the authors point out, the axonal response to stimuli is sensitive to outcome magnitude (Supp Fig 3). That is, if you increase the magnitude of water or shock that is delivered, you increase the change in fluorescence that is seen in the axons. Unsurprisingly, the change in fluorescence that is seen to shock is considerably higher than water reward. Further, over 40% of the axons respond to water early in training [yet just a few lines below the authors write: "Previous studies have demonstrated that the overall dopamine release at the mPFC or the summed activity of mPFC dopamine axons exhibits a strong response to aversive stimuli (e.g., tail shock), but little to rewards", which seems inconsistent with their own data]. Given these aspects of the data, it could be the case that the dopamine axons in mPFC encodes different types of information and delegates preferential processing to the most salient outcome across time. The use of two similar sounding tones (9Khz and 12KHz) for the reward and aversive predicting cues are likely to enhance this as it requires a fine-grained distinction between the two cues in order to learn effectively. That is not to say that the mice cannot distinguish between these cues, rather that they may require additional processes to resolve the similarity, which are known to be dependent on the mPFC.

There is considerable literature on mPFC function across species that would support such a view. Specifically, theories of mPFC function (in particular prelimbic cortex, which is where the axon images are mostly taken) generally center around resolution of conflict in what to respond, learn about, and attend to. That is, mPFC is important for devoting the most resources (learning, behavior) to the most relevant outcomes in the environment. This data then, provides a mechanism for this to occur in mPFC. That is, dopamine axons signal to the mPFC the most salient aspects of the environment, which should be preferentially learnt about and responded towards. This is also consistent with the absence of a negative prediction error during omission: the dopamine axons show increases in responses during receipt of unexpected outcomes but do not encode negative errors. This supports a role for this projection in helping to allocate resources to the most salient outcomes and their predictors, and not learning per se. Below are a just few references from the rich literature on mPFC function (some consider rodent mPFC analogous to DLPFC, some mPFC), which advocate for a role in this region in allocating attention and cognitive resources to most relevant stimuli, and do not indicate preferential processing of aversive stimuli.

References:<br /> 1. Miller, E. K., & Cohen, J. D. (2001). An integrative theory of prefrontal cortex function. Annual review of neuroscience, 24(1), 167-202.<br /> 2. Bissonette, G. B., Powell, E. M., & Roesch, M. R. (2013). Neural structures underlying set-shifting: roles of medial prefrontal cortex and anterior cingulate cortex. Behavioural brain research, 250, 91-101.<br /> 3. Desimone, R., & Duncan, J. (1995). Neural mechanisms of selective visual attention. Annual review of neuroscience, 18(1), 193-222.<br /> 4. Sharpe, M. J., Stalnaker, T., Schuck, N. W., Killcross, S., Schoenbaum, G., & Niv, Y. (2019). An integrated model of action selection: distinct modes of cortical control of striatal decision making. Annual review of psychology, 70, 53-76.<br /> 5. Ridderinkhof, K. R., Ullsperger, M., Crone, E. A., & Nieuwenhuis, S. (2004). The role of the medial frontal cortex in cognitive control. science, 306(5695), 443-447.<br /> 6. Nee, D. E., Kastner, S., & Brown, J. W. (2011). Functional heterogeneity of conflict, error, task-switching, and unexpectedness effects within medial prefrontal cortex. Neuroimage, 54(1), 528-540.<br /> 7. Isoda, M., & Hikosaka, O. (2007). Switching from automatic to controlled action by monkey medial frontal cortex. Nature neuroscience, 10(2), 240-248.

Reviewer #3 (Public Review):

Summary:

The authors note that negative ruminations can lead to pathological brain states and mood/anxiety dysregulation. They test this idea by using mouse engram-tagging technology to label dentate gyrus ensembles activated during a negative experience (fear conditioning). They show that chronic chemogenetic activation of these ensembles leads to behavioral (increased anxiety, increased fear generalization, reduced fear extinction) and neural (increases in neuroinflammation, microglia, and astrocytes).

Strengths:

The question the authors ask here is an intriguing one, and the engram activation approach is a powerful way to address the question. Examination of a wide range of neural and behavioral dependent measures is also a strength.

Weaknesses:

The major weakness is that the authors have found a range of changes that are correlates of chronic negative engram reactivation. However, they do not manipulate these outcomes to test whether microglia, astrocytes, or neuroinflammation are causally linked to the dysregulated behaviors.

Reviewer #3 (Public Review):

Summary:

Here Li et al. examine pup-directed behavior in virgin Mandarin voles. Some males and females tend towards infanticide, others tend towards pup care. c-Fos staining showed more oxytocin cells activated in the paraventricular nucleus (PVN) of the hypothalamus in animals expressing pup care behaviors than in infanticidal animals. Optogenetic stimulation of PVN oxytocin neurons (with an oxytocin-specific virus to express the opsin transgene) increased pup-care, or in infanticidal voles increased latency towards approach and attack.

Suppressing the activity of PVN oxytocin neurons promoted infanticide. The use of a recent oxytocin GRAB sensor (OT1.0) showed changes in medial prefrontal cortex (mPFC) signals as measured with photometry in both sexes. Activating mPFC oxytocin projections increased latency to approach and attack in infanticidal females and males (similar to the effects of peripheral oxytocin injections), whereas in pup-caring animals only males showed a decrease in approach. Inhibiting these projections increased infanticidal behaviors in both females and males and had no effect on pup caretaking.

Strengths:

Adopting these methods for Mandarin voles is an impressive accomplishment, especially the valuable data provided by the oxytocin GRAB sensor. This is a major achievement and helps promote systems neuroscience in voles.

Weaknesses:

The study would be strengthened by an initial figure summarizing the behavioral phenotypes of voles expressing pup care vs infanticide: the percentages and behavioral scores of individual male and female nulliparous animals for the behaviors examined here. Do the authors have data about the housing or life history/experiences of these animals? How bimodal and robust are these behavioral tendencies in the population?

Optogenetics with the oxytocin promoter virus is a nice advance here. More details about their preparation and methods should be in the main text, and not simply relegated to the methods section. For optogenetic stimulation in Figure 2, how were the stimulation parameters chosen? There is a worry that oxytocin neurons can co-release other factors- are the authors sure that oxytocin is being released by optogenetic stimulation as opposed to other transmitters or peptides, and acting through the oxytocin receptor (as opposed to a vasopressin receptor)?

Given that they are studying changes in latency to approach/attack, having some controls for motion when oxytocin neurons are activated or suppressed might be nice. Oxytocin is reported to be an anxiolytic and a sedative at high levels.

The OT1.0 sensor is also amazing, these data are quite remarkable. However, photometry is known to be susceptive to motion artifacts and I didn't see much in the methods about controls or correction for this. It's also surprising to see such dramatic, sudden, and large-scale suppression of oxytocin signaling in the mPFC in the infanticidal animals - does this mean there is a substantial tonic level of oxytocin release in the cortex under baseline conditions?

Figure 5 is difficult to parse as-is, and relates to an important consideration for this study: how extensive is the oxytocin neuron projection from PVN to mPFC?

In Figures 6 and 7, the authors use the phrase 'projection terminals'; however, to my knowledge, there have not been terminals (i.e., presynaptic formations opposed to a target postsynaptic site) observed in oxytocin neuron projections into target central regions.

Projection-based inhibition as in Figure 7 remains a controversial issue, as it is unclear if the opsin activation can be fast enough to reduce the fast axonal/terminal action potential. Do the authors have confirmation that this works, perhaps with the oxytocin GRAB OT sensor?

As females and males had similar GRAB OT1.0 responses in mPFC, why would the behavioral effects of increasing activity be different between the sexes?

Local Only website possible?

Reviewer #3 (Public Review):

The authors delved into an important aspect of abortifacient diseases of livestock in Tanzania. The thoughts of the authors on the topic and its significance are implied, and the methodological approach needs further clarity. The number of wards in the study area, statistical selection of wards, type of questionnaire ie open or close-ended. Statistical analyses of outcomes were not clearly elucidated in the manuscript. Fifteen wards were mentioned in the text but 13 used what were the exclusion criteria. Observations were from pastoral, agropastoral, and smallholder agroecological farmers. No sample numbers or questionnaires were attributed to the above farming systems to correlate findings with management systems. The impacts of the research investigation output are not clearly visible as to warrant intervention methods. What were the identified pathogens from laboratory investigation, particularly with the use of culture and PCR not even mentioning the zoonotic pathogens encountered if any? The public health importance of any of the abortifacient agents was not highlighted.

In conclusion, based on the intent of the authors and the content of this research, and the weight of the research topic, there are obvious weaknesses in the critical data analysis to demonstrate cause, effect, and impact.

Reviewer #3 (Public Review):

A classic method to detect recessive disease variants is homozygosity mapping, where affected individuals in a pedigree are scanned for the presence of runs of homozygosity (ROH) intersecting in a given region. The method could in theory be extended to biobanks with large samples of unrelated individuals; however, no efficient method was available (to the best of my knowledge) for detecting overlapping clusters of ROH in such large samples. In this paper, the authors developed such a method based on the PBWT data structure. They applied the method to the UK biobank, finding a number of associations, some of them not discovered in single SNP associations.

Major strengths:<br /> • The method is innovative and algorithmically elegant and interesting. It achieves its purpose of efficiently and accurately detecting ROH clusters overlapping in a given region. It is therefore a major methodological advance.<br /> • The method could be very useful for many other researchers interested in detecting recessive variants associated with any phenotype.<br /> • The statistical analysis of the UK biobank data is solid and the results that were highlighted are interesting and supported by the data.

Major weaknesses:<br /> • The positions and IDs of the ROH clusters in the UK biobank are not available for other researchers. This means that other researchers will not be able to follow up on the results of the present paper.<br /> • The vast majority of the discoveries were in regions already known to be associated with their respective phenotypes based on standard GWAS.<br /> • The running time seems rather long (at least for the UK biobank), and therefore it will be difficult for other researchers to extensively experiment with the method in very large datasets. That being said, the method has a linear running time, so it is already faster than a naïve algorithm.

Reviewer #3 (Public Review):

Summary:

In this article, the authors provide an inventory of the 5' spliced leader sequences, cap structures, and eIF4E isoforms present in the model dinoflagellate species A. carterae. They provide evidence that the 5' cap structure is m7G, as it is in most characterized eukaryotes that do not employ trans-splicing for mRNA maturation, and that there are additional methylated nucleotides throughout the spliced leader RNAs. They then show that of the 8 different eIF4E species in A. carterae, only a subset of eIF4E1 and eIF4E2 proteins are detected and that the levels change according to time of day. Interestingly, while the eIF4E1 proteins bind a canonical cap nucleotide and are able to complement eIF4E-deficiency in yeast, an eIF4E2 paralog does not bind the traditional cap.

Strengths:

A strength of the article is that the authors have clearly presented the findings and by straying away from traditional model organisms, they have highlighted unique and interesting features of an understudied system for translational control. They provide complementary evidence for most findings using multiple techniques. E.g. the evidence that eIF4E1A binds m7GTP is supported by both pulldowns using m7GTP sepharose as well as SPR experiments to directly monitor binding of recombinant protein with affinity measurements. The methods are extremely detailed noting cell numbers, volumes, concentrations, etc. used in the experiments to be easily replicated.

Weaknesses:

While not necessary to support the author's conclusions, the significance of the work would be further enhanced by additional experiments to gain insights into mechanisms for translational control and to link specific SLs to organismal functions or mechanisms of mRNA recruitment.

-Monitoring diel expression of SLs and direct sequencing of mature mRNA would yield insights into whether there is regulated expression of RNAs with different SLs or the SLs themselves. This would also allow the authors to perform gene ontology to link SL expression at different points in the diel cycle to related functions, e.g. photosynthesis.

-In addition, the work would be strengthened by polysome sequencing or ribosome profiling as a function of the diel cycle, with analyses of when various spliced leader sequences are recruited to ribosomes in parallel with western blotting of polysome fractions to determine when various eIF4E isoforms are present on polysomes. This is a substantial expansion though from what the authors focused on in this manuscript, and not having these experiments does not undermine the findings presented. Alternatively, they could attempt to make bioinformatic comparisons with existing ribosome profiling datasets from a related dinoflagellate, Lingulodinium polyedrum, discussed briefly, if there were sufficient overlap between SL RNAs in these organisms.

Reviewer #3 (Public Review):

Summary:

Lichtinger et al. have used an extensive set of molecular dynamics (MD) simulations to study the conformational dynamics and transport cycle of an important member of the proton-coupled oligopeptide transporters (POTs), namely SLC15A2 or PepT2. This protein is one of the most well-studied mammalian POT transporters that provides a good model with enough insight and structural information to be studied computationally using advanced enhanced sampling methods employed in this work. The authors have used microsecond-level MD simulations, constant-PH MD, and alchemical binding free energy calculations along with cell-based transport assay measurements; however, the most important part of this work is the use of enhanced sampling techniques to study the conformational dynamics of PepT2 under different conditions.

The study attempts to identify links between conformational dynamics and chemical events such as proton binding, ligand-protein interactions, and intramolecular interactions. The ultimate goal is of course to understand the proton-coupled peptide and drug transport by PepT2 and homologous transporters in the solute carrier family.

Some of the key results include<br /> (1) Protonation of H87 and D342 initiate the occluded (Occ) to the outward-facing (OF) state transition.

(2) In the OF state, through engaging R57, substrate entry increases the pKa value of E56 and thermodynamically facilitates the movement of protons further down.

(3) E622 is not only essential for peptide recognition but also its protonation facilitates substrate release and contributes to the intracellular gate opening. In addition, cell-based transport assays show that mutation of residues such as H87 and D342 significantly decreases transport activity as expected from simulations.

Strengths:

(1) This is an extensive MD-based study of PepT2, which is beyond the typical MD studies both in terms of the sheer volume of simulations as well as the advanced methodology used. The authors have not limited themselves to one approach and have appropriately combined equilibrium MD with alchemical free energy calculations, constant-pH MD, and geometry-based free energy calculations. Each of these 4 methods provides a unique insight regarding the transport mechanism of PepT2.

(2) The authors have not limited themselves to computational work and have performed experiments as well. The cell-based transport assays clearly establish the importance of the residues that have been identified as significant contributors to the transport mechanism using simulations.

(3) The conclusions made based on the simulations are mostly convincing and provide useful information regarding the proton pathway and the role of important residues in proton binding, protein-ligand interaction, and conformational changes.

Weaknesses:

(1) Some of the statements made in the manuscript are not convincing and do not abide by the standards that are mostly followed in the manuscript. For instance, on page 4, it is stated that "the K64-D317 interaction is formed in only ≈ 70% of MD frames and therefore is unlikely to contribute much to extracellular gate stability." I do not agree that 70% is negligible. Particularly, Figure S3 does not include the time series so it is not clear whether the 30% of the time where the salt bridge is broken is in the beginning or the end of simulations. For instance, it is likely that the salt bridge is not initially present and then it forms very strongly. Of course, this is just one possible scenario but the point is that Figure S3 does not rule out the possibility of a significant role for the K64-D317 salt bridge.

(2) Similarly, on page 4, it is stated that "whether by protonation or mutation - the extracellular gate only opens spontaneously when both the H87 interaction network and D342-R206 are perturbed (Figure S5)." I do not agree with this assessment. The authors need to be aware of the limitations of this approach. Consider "WT H87-prot" and "D342A H87-prot": when D342 residue is mutated, in one out of 3 simulations, we see the opening of the gate within 1 us. When D342 residue is not mutated we do not see the opening in any of the 3 simulations within 1 us. It is quite likely that if rather than 3 we have 10 simulations or rather than 1 us we have 10 us simulations, the 0/3 to 1/3 changes significantly. I do not find this argument and conclusion compelling at all.

(3) While the MEMENTO methodology is novel and interesting, the method is presented as flawless in the manuscript, which is not true at all. It is stated on Page 5 with regards to the path generated by MEMENTO that "These paths are then by definition non-hysteretic." I think this is too big of a claim to say the paths generated by MEMENTO are non-hysteretic by definition. This claim is not even mentioned in the original MEMENTO paper. What is mentioned is that linear interpolation generates a hysteresis-free path by definition. There are two important problems here: (a) MEMENTO uses the linear interpolation as an initial step but modifies the intermediates significantly later so they are no longer linearly interpolated structures and thus the path is no longer hysteresis-free; (b) a more serious problem is the attribution of by-definition hysteresis-free features to the linearly interpolated states. This is based on conflating the hysteresis-free and unique concepts. The hysteresis in MD-based enhanced sampling is related to the presence of barriers in orthogonal space. For instance, one may use a non-linear interpolation of any type and get a unique pathway, which could be substantially different from the one coming from the linear interpolation. None of these paths will be hysteresis-free necessarily once subjected to MD-based enhanced sampling techniques.

Reviewer #3 (Public Review):

Summary:

The authors were trying to elucidate the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of "abnormal" enlarged vesicles when USP8 function was lost. At this specific point, it's not clear what the objective of the authors was. What would have been their hypothesis addressing whether the reduced lysosomal structures in USP8 (-) animals were linked to MVB formation? Then they observed that the abnormally enlarged vesicles, marked by the PI3P biosensor YFP-2xFYVE, are bigger but in the same number in USP8 (-) compared to wild-type animals, suggesting homotypic fusion. They confirmed this result by knocking down USP8 in a human cell line, and they observed enlarged vesicles marked by YFP-2xFYVE as well. At this point, there is quite an important issue. The use of YFP-2xFYVE to detect early endosomes requires the transfection of the cells, which has already been demonstrated to produce differences in the distribution, number, and size of PI3P-positive vesicles (doi.org/10.1080/15548627.2017.1341465). The enlarged vesicles marked by YFP-2xFYVE would not necessarily be due to the loss of UPS8. In any case, it appears relatively clear that USP8 localizes to early endosomes, and the authors claim that this localization is mediated by Rabex-5 (or Rabx-5). They finally propose that USP8 dissociates Rabx-5 from early endosomes facilitating endosome maturation.

Weaknesses:

The weaknesses of this study are, on one side, that the results are almost exclusively dependent on the overexpression of fusion proteins. While useful in the field, this strategy does not represent the optimal way to dissect a cell biology issue. On the other side, the way the authors construct the rationale for each approximation is somehow difficult to follow. Finally, the use of two models, C. elegans and a mammalian cell line, which would strengthen the observations, contributes to the difficulty in reading the manuscript.

The findings are useful but do not clearly support the idea that USP8 mediates Rab5-Rab7 exchange and endosome maturation, In contrast, they appear to be incomplete and open new questions regarding the complexity of this process and the precise role of USP8 within it.

Reviewer #3 (Public Review):

Summary:

Loss of cell attachment to extracellular matrix (ECM) triggers aniokis (a type of programmed cell death), and resistance to aniokis plays a role in cancer development. However, mechanisms underlying anoikis resistance, and the precise role of F-actin, are not fully known.

Here the authors describe the formation of a new organelle, giant unilocular vacuole (GUVac), in cells whose F-actin is disrupted during loss of matrix attachment. GUVac formation (diameter >500 nm) resulted from a previously unrecognised macropinocytosis-like process, characterized by inwardly curved micron-sized plasma membrane invaginations, dependent on F-actin depolymerization, septin recruitment, and PI(3)P. Finally, the authors show GUVac formation after loss of matrix attachment promotes resistance to anoikis.

From these results, the authors conclude that GUVac formation promotes cell survival in environments where F-actin is disrupted and conditions of cell stress.

Strengths:

The manuscript is clear and well-written, figures are all presented at a very high level.

A variety of cutting-edge cell biology techniques (eg time-lapse imaging, EM, super-resolution microscopy) are used to study the role of the cytoskeleton in GUVac formation. It is discovered that: (i) a macropinocytosis-like process dependent on F-actin depolymerisation, SEPT6 recruitment, and PI(3)P contributes to GUVac formation, and (ii) GUVac formation is associated with resistance to cell death.

Weaknesses:

The manuscript is highly reliant on the use of drugs, or combinations of drugs, for long periods of time (6hr, 18hr..). Wherever possible the authors should test conclusions drawn from experiments involving drugs also using other canonical cell biology approaches (eg siRNA, Crispr). Although suggestive as a first approach, it is not reliable to draw conclusions from experiments where only drug combinations are being advanced (eg LatB + FCF).

F-actin is well known to play a wide variety of roles in cell death and other canonical cell death pathways (PMID: 26292640). The authors show using pharmacological inhibition that F-actin is key for GUVac formation. However, especially when testing for physiological relevance, how can these other roles for F-actin be ruled out?

To test the role of septins in GUVac formation only recruitment studies and no direct functional work is performed. A drug forchlofeneuron (FCF) is used, but this is well known to have off-target effects (PMID: 27473917).

Cells that possess GUVac are resistant to aniokis, but how are these cells resistant? This report is focused on mechanisms underlying GUVac formation and does not directly test for mechanisms underlying aniokis resistance.

Reviewer #3 (Public Review):

Summary:

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

Strength:

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

Weaknesses:

While the structure of the TM2-TM3 loop may suggest a mechanism for stabilizing the closed conformation, the EM density is not strong enough to support direct interaction with carbamylated or mutated K125.

Overall, the cryo-EM structures presented in this study support their proposing mechanism in which carbamylation at K125 promotes Cx26 gap junction closure. Through careful control of the pH and PCO2 for each cryo-EM sample, the current study substantiated that the more closed conformation observed in high PCO2 is independent of pH but likely triggered by carbamylation. This was unclear from their prior cryo-EM map of wildtype Cx26 at high PCO2.

While the new structures successfully visualize the TM2-TM3 loop, which likely plays significant roles in CO2-regulated Cx26 activity, further studies are necessary to understand the underlying mechanism. For instance, the current study lacks explanation regarding what propels the movement of the N-terminal helix, how carbamylated K125 interacts with the TM2-TM3 loop, the importance of the lipids visualized in the map, or the reason why gap junctions are constitutively open while hemichannels are closed under normal PCO2 levels

Reviewer #3 (Public Review):

Granule cells' axons bifurcate to form parallel fibers (PFs) and ascending axons (AAs). While the significance of PFs on cerebellar plasticity is widely acknowledged, the importance of AAs remains unclear. In the current paper, Conti and Auger conducted electrophysiological experiments in rat cerebellar slices and identified a new form of synaptic plasticity in the AA-Purkinje cell (PC) synapses. Upon simultaneous stimulation of AAs and PFs, AA-PC EPSCs increased, while PFs-EPSCs decreased. This suggests that synaptic responses to AAs and PFs in PCs are jointly regulated, working as an additional mechanism to integrate motor/sensory input. This finding may offer new perspectives in studying and modeling cerebellum-dependent behavior. Overall, the experiments are performed well. However, there are two weaknesses. First, the baseline of electrophysiological recordings is influenced significantly by run-down, making it difficult to interpret the data quantitatively. The amplitude of AA-EPSCs is relatively small and the run-down may mask the change. The authors should carefully reexamine the data with appropriate controls and statistics. Second, while the authors show AA-LTP depends on mGluR, NMDA receptors, and GABA-A receptors, which cell types express these receptors and how they contribute to plasticity is not clarified. The recommended experiments may help to improve the quality of the manuscript.

Reviewer #3 (Public Review):

Summary

The L114P gain of function mutation in the K2P channel TALK-1 encoded by KCNJ16 has been associated with maturity-onset diabetes of the young (MODY). In this study, Nakhe et al. generated mice carrying L114P TALK-1 and evaluated the impact of the mutation on pancreatic islet functions and glucose homeostasis. The authors report that the mutation increases neonatal lethality, owing to hyperglycemia caused by a lack of glucose-stimulated Ca2+ influx and insulin secretion. Adult mutant mice showed glucose intolerance and fasting hyperglycemia, which is attributed to blunted glucose-stimulated insulin secretion as well as increased glucagon secretion. Interestingly, male mice were more affected than female mice. Islets from adult mutant mice were found to have reduced Ca2+ entry upon glucose stimulation but also enhanced IP3-induced ER Ca2+ release, consistent with previous studies from the group showing a role of TALK-1 in ER Ca2+ homeostasis. Finally, comparison of bulk RNA sequencing results from WT and mutant islets revealed altered expression of genes involved in β-cell identify, function and signaling, which also contributes to the observed islet dysfunction.

Strengths

This is a well-executed and rigorous study that will be of great interest to the diabetes and islet biology communities. The findings provide convincing evidence supporting a causal role of the L114P gain of function TALK-1 mutation in glucose-stimulated insulin secretion defects and diabetes. The neonatal diabetes phenotype and the gender difference uncovered by the study have important clinical implications. The complexity of TALK-1 expression and hormone secretion in different endocrine cell types and how it impacts glucose homeostasis is elegantly illustrated in the L114P TALK-1 mouse model. The authors carefully and thoroughly addressed limitations of their study and discussed future directions. The importance of TALK-1 in β-cell and islet function demonstrated by this study will prompt future efforts targeting this important channel for diabetes treatment.

Reviewer #3 (Public Review):

Summary:

This manuscript reports the discovery of new compounds that selectively inhibit SMARCA4/SMARCA2 ATPase activity that work through a different mode as previously developed SMARCA4/SMARCA2 inhibitors. They also demonstrate the anti-tumor effects of the compounds on uveal melanoma cell proliferation and tumor growth. The findings indicate that the drugs exert their effects by altering chromatin accessibility at binding sites for lineage-specific transcription factors within gene enhancer regions. In uveal melanoma, altered expression of the transcription factor, SOX10, and SOX10 target gene underlies the anti-proliferative effects of the compounds. This study is significant because the discovery of new SMARCA4/SMARCA2 inhibitory compounds that can abrogate uveal melanoma tumorigenicity has therapeutic value. In addition, the findings provide evidence for the therapeutic use of these compounds in other transcription factor-dependent cancers.

Strengths:

The strengths of this manuscript include biochemical evidence that the new compounds are selective for SMARCA4/SMARCA2 over other ATPases and that the mode of action is distinct from a previously developed compound, BRM014, which binds the RecA lobe of SMARCA2. There is also strong evidence that FHT1015 suppresses uveal melanoma proliferation by inducing apoptosis. The in vivo suppression of tumor growth without toxicity validates the potential therapeutic utility of one of the new drugs. The conclusion that FHT1015 primarily inhibits SMARCA4 activity and thereby suppresses chromatin accessibility at lineage-specific enhancers is substantiated by ATAC-seq and ChIP-seq studies.

Weaknesses:

The weaknesses include a lack of more precise information on which SMARCA4/SMARCA2 residues the drugs bind. Although the I1173M/I1143M mutations are evidence that the critical residues for binding reside outside the RecA lobe, this site is conserved in CHD4, which is not affected by the compounds. Hence, this site may be necessary but not sufficient for drug binding or specifying selectivity. A more precise evaluation of the region specifying the effect of the new compounds would strengthen the evidence that they work through a novel mode and that they are selective. Another concern is that the mechanisms by which FHT1015 promotes apoptosis rather than simply cell cycle arrest are not clear. Does SOX10 or another lineage-specific transcription factor underlie the apoptotic effects of the compounds?

Reviewer #3 (Public Review):

Summary:

This study presents a new approach of combining two measurements (pHLA binding and pHLA-TCR binding) in order to refine predictions of which patient mutations are likely presented to and recognized by the immune system. Improving such predictions would play an important role in making personalized anti-cancer vaccinations more effective.

Strengths:

The study combines data from pre-existing tools pVACseq and pMTNet and applies them to a CRC patient population, which the authors show may improve the chance of identifying immunogenic, cancer-derived neoepitopes. Making the datasets collected publicly available would expand beyond the current datasets that typically describe caucasian patients.

Weaknesses:

It is unclear whether the pNetMHCpan and pMTNet tools used by the authors are entirely independent, as they appear to have been trained on overlapping datasets, which may explain their similar scores. The pHLA-TCR score seems to be driving the effects, but this not discussed in detail.

Due to sample constraints, the authors were only able to do a limited amount of experimental validation to support their model; this raises questions as to how generalisable the presented results are. It would be desirable to use statistical thresholds to justify cutoffs in ELISPOT data.

Some of the TCR repertoire metrics presented in Figure 2 are incorrectly described as independent variables and do not meaningfully contribute to the paper. The TCR repertoires may have benefitted from deeper sequencing coverage, as many TCRs appear to be supported only by a single read.

Reviewer #3 (Public Review):

The mechanical properties of DNA wrapped in nucleosomes affect the stability of nucleosomes and may play a role in the regulation of DNA accessibility in eukaryotes. In this manuscript, Ngo and coworkers study how the stability of a nucleosome is affected by the introduction of a CC mismatched base pair, which has been reported to increase the flexibility of DNA. Previously, the group has used a sophisticated combination of single-molecule FRET and force spectroscopy with an optical trap to show that the more flexible half of a 601 DNA segment provides for more stable wrapping as compared to the other half. Here, it is confirmed with a single-molecule cyclization essay that the introduction of a CC mismatch increases the flexibility of a DNA fragment. Consistent with the previous interpretation, it also increased the unwrapping force for the half of the 601 segment in which the CC mismatch was introduced, as measured with single-molecule FRET and force spectroscopy. Enhanced stability was found up to 56 bp into the nucleosome. The intricate role of mechanical stability of nucleosomes was further investigated by comparing force-induced unwrapping profiles of yeast and Xenopus histones. Intriguingly, asymmetric unwrapping was more pronounced for yeast histones.

Note from Reviewing Editor:

The authors addressed the points in the reviews by making appropriate text additions and clarifications.

Reviewer #3 (Public Review):

Summary:

Asabuki and Clopath study stochastic sequence learning in recurrent networks of Poisson spiking neurons that obey Dale's law. Inspired by previous modeling studies, they introduce two distinct learning rules, to adapt excitatory-to-excitatory and inhibitory-to-excitatory synaptic connections. Through a series of computer experiments, the authors demonstrate that their networks can learn to generate stochastic sequential patterns, where states correspond to non-overlapping sets of neurons (cell assemblies) and the state-transition conditional probabilities are first-order Markov, i.e., the transition to a given next state only depends on the current state. Finally, the authors use their model to reproduce certain experimental songbird data involving highly-predictable and highly-uncertain transitions between song syllables.

Strengths:

This is an easy-to-follow, well-written paper, whose results are likely easy to reproduce. The experiments are clear and well-explained. The study of songbird experimental data is a good feature of this paper; finches are classical model animals for understanding sequence learning in the brain. I also liked the study of rapid task-switching, it's a good-to-know type of result that is not very common in sequence learning papers.

Weaknesses:

While the general subject of this paper is very interesting, I missed a clear main result. The paper focuses on a simple family of sequence learning problems that are well-understood, namely first-order Markov sequences and fully visible (no-hidden-neuron) networks, studied extensively in prior work, including with spiking neurons. Thus, because the main results can be roughly summarized as examples of success, it is not entirely clear what the main point of the authors is.

Going into more detail, the first major weakness I see in this paper is the heuristic choice of learning rules. The paper studies Poisson spiking neurons (I return to this point below), for which learning rules can be derived from a statistical objective, typically maximum likelihood. For fully-visible networks, these rules take a simple form, similar in many ways to the E-to-E rule introduced by the authors. This more principled route provides quite a lot of additional understanding on what is to be expected from the learning process. For instance, should maximum likelihood learning succeed, it is not surprising that the statistics of the training sequence distribution are reproduced. Moreover, given that the networks are fully visible, I think that the maximum likelihood objective is a convex function of the weights, which then gives hope that the learning rule does succeed. And so on. This sort of learning rule has been studied in a series of papers by David Barber and colleagues [refs. 1, 2 below], who applied them to essentially the same problem of reproducing sequence statistics in recurrent fully-visible nets. It seems to me that one key difference is that the authors consider separate E and I populations, and find the need to introduce a balancing I-to-E learning rule.

Because the rules here are heuristic, a number of questions come to mind. Why these rules and not others - especially, as the authors do not discuss in detail how they could be implemented through biophysical mechanisms? When does learning succeed or fail? What is the main point being conveyed, and what is the contribution on top of the work of e.g. Barber, Brea, et al. (2013), or Pfister et al. (2004)?

The use of a Poisson spiking neuron model is the second major weakness of the study. A chief challenge in much of the cited work is to generate stochastic transitions from recurrent networks of deterministic neurons. The task the authors set out to do is much easier with stochastic neurons; it is reasonable that the network succeeds in reproducing Markovian sequences, given an appropriate learning rule. I believe that the main point comes from mapping abstract Markov states to assemblies of neurons. If I am right, I missed more analyses on this point, for instance on the impact that varying cell assembly size would have on the findings reported by the authors.

Finally, it was not entirely clear to me what the main fundamental point in the HVC data section was. Can the findings be roughly explained as follows: if we map syllables to cell assemblies, for high-uncertainty syllable-to-syllable transitions, it becomes harder to predict future neural activity? In other words, is the main point that the HVC encodes syllables by cell assemblies?

(1) Learning in Spiking Neural Assemblies, David Barber, 2002. URL: https://proceedings.neurips.cc/paper/2002/file/619205da514e83f869515c782a328d3c-Paper.pdf

(2) Correlated sequence learning in a network of spiking neurons usingmaximum likelihood, David Barber, Felix Agakov, 2002. URL: http://web4.cs.ucl.ac.uk/staff/D.Barber/publications/barber-agakov-TR0149.pdf

Reviewer #3 (Public Review):

Summary:

In this paper, the authors sought to evaluate whether the novel TB drug candidate, spectinamide 1599 (S), given via inhalation to mouse TB models, and combined with the drugs B (bedaquiline) and Pa (pretomanid), would demonstrate similar efficacy to that of BPaL regimen (where L is linezolid). Because L is associated with adverse events when given to patients long-term, and one of those is associated with myelosuppression (bone marrow toxicity) the authors also sought to assess blood parameters, effects on bone marrow, immune parameters/cell effects following treatment of mice with BPaS and BPaL. They conclude that BPaL and BPaS have equivalent efficacy in both TB models used and that BPaL resulted in weight loss and anemia (whereas BPaL did not) under the conditions tested, as well as effects on bone marrow.

Strengths:

The authors used two mouse models of TB that are representative of different aspects of TB in patients (which they describe well), intending to present a fuller picture of the activity of the tested drug combinations. They conducted a large body of work in these infected mice to evaluate efficacy and also to survey a wide range of parameters that could inform the effect of the treatments on bone marrow and on the immune system. The inclusion of BPa controls (in most studies) and also untreated groups led to a large amount of useful data that has been collected for the mouse models per se (untreated) as well as for BPa - in addition to the BPaS and BPaL combinations which are of particular interest to the authors. Many of these findings related to BPa, BPaL, untreated groups, etc corroborate earlier findings and the authors point this out effectively and clearly in their manuscript. To go further, in general, it is a well-written and cited article with an informative introduction.

Weaknesses:

The authors performed a large amount of work with the drugs given at the doses and dosing intervals started, but at present, there is no exposure data available in the paper. It would be of great value to understand the exposures achieved in plasma at least (and in the lung if more relevant for S) in order to better understand how these relate to clinical exposures that are observed at marketed doses for B, Pa, and L as well as to understand the exposure achieved at the doses being evaluated for S. If available as historical data this could be included/cited. Considering the great attempts made to evaluate parameters that are relevant to clinical adverse events, it would add value to understand what exposures of drug effects such as anemia, weight loss, and bone marrow effects, are being observed.

It would also be of value to add an assessment of whether the weight loss, anemia, or bone marrow effects observed for BPaL are considered adverse, and the extent to which we can translate these effects from mouse to patient (i.e. what are the limitations of these assessments made in a mouse study?). For example, is the small weight loss seen as significant, or is it reversible? Is the magnitude of the changes in blood parameters similar to the parameters seen in patients given L?

In addition, it is always challenging to interpret findings for combinations of drugs, so the addition of language to explain this would add value: for example, how confident can we be that the weight loss seen for only the BPaL group is due to L as opposed to a PK interaction leading to an elevated exposure and weight loss due to B or Pa?

Turning to the evaluations of activity in mouse TB models, unfortunately, the evaluations of activity in the BALB/c mouse model as well as the spleens of the Kramnik model resulted in CFU below/at the limit of detection and so, to this reviewer's understanding of the data, comparisons between BPaL and BPaS cannot be made and so the conclusion of equivalent efficacy in BALB/c is not supported with the data shown. There is no BPa control in the BALB/c study, therefore it is not possible to discern whether L or S contributed to the activity of BPaL or BPaS; it is possible that BPa would have shown the same efficacy as the 3 drug combinations. It would be valuable to conduct a study including a BPa control and with a shorter treatment time to allow comparison of BPa, BPaS, and BPaL. In the Kramnik lungs, as the authors rightly note, the studies do not support any contribution of S or L to BPa - i.e. the activity observed for BPa, BPaL, and BPaS did not significantly differ. Although the conclusions note equivalency of BPaL and BPaS, which is correct, it would be helpful to also include BPa in this statement; it would be useful to conduct a study dosing for a longer period of time or assessing a relapse endpoint, where it is possible that a contribution of L and/or S may be seen - thus making a stronger argument for S contributing an equivalent efficacy to L. The same is true for the assessment of lesions - unfortunately, there was no BPa control meaning that even where equivalency is seen for BPaL and BPaS, the reader is unable to deduce whether L or S made a contribution to this activity.

Reviewer #3 (Public Review):

Summary:

The article by Huang et.al. presents an in-depth study on the role of DNA methylation in regulating virulence and metabolism in Pseudomonas syringae, a model phytopathogenic bacterium. This comprehensive research utilized single-molecule real-time (SMRT) sequencing to profile the DNA methylation landscape across three model pathovars of P. syringae, identifying significant epigenetic mechanisms through the Type-I restriction-modification system (HsdMSR), which includes a conserved sequence motif associated with N6-methyladenine (6mA). The study provides novel insights into the epigenetic mechanisms of P. syringae, expanding the understanding of bacterial pathogenicity and adaptation. The use of SMRT sequencing for methylome profiling, coupled with transcriptomic analysis and in vivo validation, establishes a robust evidence base for the findings

Strengths:

The results are presented clearly, with well-organized figures and tables that effectively illustrate the study's findings.

Weaknesses:

It would be helpful to add more details, especially in the methods, which make it easy to evaluate and enhance the manuscript's reproducibility.

Reviewer #3 (Public Review):

Summary:

Pal et al. provide valuable evidence supporting distinct vascular bed-specific VEGF-A mediated vascular permeability function of Neuropilin-1 (NRP1) in adult mice. Using a suite of genetic mice models and state-of-the-art vascular permeability assays the authors demonstrate that ear skin vasculature of EC-specific NRP1 adult knockout mice is hypersensitive to VEGF-A mediated high-molecular weight dye leakage from venules, as opposed to back skin and tracheal vasculature where EC-specific NRP1 loss had a more classical negative effect on permeability. Interestingly, both whole organism KO of NRP1 and a blocking antibody treatment, attenuated VEGF-A mediated permeability in ear skin and had the usual attenuation of permeability phenotype in back skin and tracheal vasculature. Using a pericyte promoter specific reporter mice line, the authors characterize NRP1 expression in the vascular beds of the ear dermis and back skin and conclude that NRP1 expression is higher in perivascular cells in the ear dermis as opposed to back skin vasculature, thus indicating a juxtracrine NRP1-VEGFR2 signaling model in adult mice. Further, they use a Vegfr2 phosphosite mutant homozygous mice model in the background of NRP1 iECKO to find the hypersensitivity to VEGF-A stimulation in ear skin is abrogated and therefore, prove the juxtracrine NRP1 control of VEGFR2 mediated downstream signaling leading to vascular permeability. Further, they successfully show distinctive vascular bed-specific results as above using a well-characterized VE-Cadherin Y685 antibody staining which corresponds to vascular leakage downstream of VEGF-A/VEGFR2 signaling in ear dermis and back skin vascular beds.

Strengths:

The question of the in vivo role of NRP1 in VEGF-A-induced hyper-permeability is an unresolved one and the elegant use of genetic mice models to demonstrate the phenotypes is valuable to the field. The organotypic differences observed in vascular permeability upon VEGF-A treatment in ear skin versus back skin and tracheal vasculature are solid. The subsequent investigation to validate heightened VEGFR2 signaling in ear dermis downstream of VEGF-A stimulation using Vegfr2 Y949F mice, VEC Y685 antibody, and pPLCγ antibody is also very convincing.

Weaknesses:

The mechanism proposed by the authors by which EC-specific loss of NRP1 caused hypersensitivity to VEGF-A in ear dermis is through elevated juxtracrine signaling of NRP1 expressed in pericytes in trans binding and retaining VEGFR2 on the cell surface of ECs to sustain downstream signaling for longer time, in corroboration to earlier findings in Koch et al., 2014, where NRP1 was studied in the context of tumor angiogenesis. To support their claim, the authors stain the ear dermis and back skin vasculature of Pdgfrb-GFP reporter mice, with NRP1 and CD31 antibodies and find out that ear skin vasculature has higher perivascular cells as opposed to back skin vasculature. While this is a good experiment to prove the above point, there are no functional experiments to support this model.

Overall, although the paper presents very useful findings in the field of NRP1-VEGFR2 biology, and most of the conclusions are well supported by the data, there are a few points if addressed can significantly substantiate the model of juxtracrine signaling proposed by the authors. They are:

(1) It will be important to know if the perivascular to vascular NRP1 expression (such as in Figure 3B) increases further in ear skin vasculatures of NRP1 iECKO mice compared to otherwise WT mice.

(2) Does knocking out NRP1 in pericytes attenuate the VEGF-A mediated hyperpermeability observed in ear skin of NRP1 iECKO mice (similar to experiments in 1C, 2C)?

(3) What is the status of VEGFR2 expression in ECs of ear skin and back skin of NRP1 iECKO and NRP1 iKO mice? This experiment is a proof-of-concept and is not essential to prove the point of juxtracrine NRP1 signaling since downstream readouts - pPLCγ and VEC Y685 staining have already been shown to correlate in the ear dermis.

Reviewer #3 (Public Review):

Summary:

This paper by Portela Catani et al examines the antigenic relationships (measured using monotypic ferret and mouse sera) across a panel of N2 genes from the past 14 years, along with the underlying sequence differences and phylogenetic relationships. This is a highly significant topic given the recent increased appreciation of the importance of NA as a vaccine target, and the relative lack of information about NA antigenic evolution compared with what is known about HA. Thus, these data will be of interest to those studying the antigenic evolution of influenza viruses. The methods used are generally quite sound, though there are a few addressable concerns that limit the confidence with which conclusions can be drawn from the data/analyses.

Strengths:

-The significance of the work, and the (general) soundness of the methods.<br /> -Explicit comparison of results obtained with mouse and ferret sera

Weaknesses:

- Machine learning analyses neither experimentally validated nor shown to be better than simple, phylogenetic-based inference.

Reviewer #3 (Public Review):

Summary:

Single unit neural activity tuned to environmental or behavioral variables gradually changes over time. This phenomenon, called representational drift, occurs even when all external variables remain constant, and challenges the idea that stable neural activity supports the performance of well-learned behaviors. While a number of studies have described representational drift across multiple brain regions, our understanding of the underlying mechanism driving drift is limited. Ratzon et al. propose that implicit regularization - which occurs when machine learning networks continue to reconfigure after reaching an optimal solution - could provide insights into why and how drift occurs in neurons. To test this theory, Ratzon et al. trained a recurrent neural network (RNN) trained to perform the oft-utilized linear track behavioral paradigm and compare the changes in hidden layer units to those observed in hippocampal place cells recorded in awake, behaving animals.

Ratzon et al. clearly demonstrate that hidden layer units in their model undergo consistent changes even after the task is well-learned, mirroring representational drift observed in real hippocampal neurons. They show that the drift occurs across three separate measures: the active proportion of units (referred to as sparsification), spatial information of units, and correlation of spatial activity. They continue to address the conditions and parameters under which drift occurs in their model to assess the generalizability of their findings to non-spatial tasks. Last, they investigate the mechanism through which sparsification occurs, showing that flatness of the manifold near the solution can influence how the network reconfigures. The authors suggest that their findings indicate a three stage learning process: 1) fast initial learning followed by 2) directed motion along a manifold which transitions to 3) undirected motion along a manifold.

Overall, the authors' results support the main conclusion that implicit regularization in machine learning networks mirrors representational drift observed in hippocampal place cells. Their findings promise to open new fields of inquiry into the connection between machine learning and representational drift in other, non-spatial learning paradigms, and to generate testable predictions for neural data.

Strengths:

(1) Ratzon et al. make an insightful connection between well-known phenomena in two separate fields: implicit regularization in machine learning and representational drift in the brain. They demonstrate that changes in a recurrent neural network mirror those observed in the brain, which opens a number of interesting questions for future investigation.

(2) The authors do an admirable job of writing to a large audience and make efforts to provide examples to make machine learning ideas accessible to a neuroscience audience and vice versa. This is no small feat and aids in broadening the impact of their work.

(3) This paper promises to generate testable hypotheses to examine in real neural data, e.g., that drift rate should plateau over long timescales (now testable with the ability to track single-unit neural activity across long time scales with calcium imaging and flexible silicon probes). Additionally, it provides another set of tools for the neuroscience community at large to use when analyzing the increasingly high-dimensional data sets collected today.

Weaknesses:

The revised manuscript addresses all the weaknesses outlined in my initial review. However, there is one remaining (minor) weakness regarding how "sparseness" is used and defined.

Sparseness can mean different things to different fields. For example, for engram studies, sparseness could be measured at the population level by the proportion of active cells, whereas for a physiology study, sparseness might be measured at the neuron level by the change in peak firing rate of each cell as an animal enters that cell's place field. In this manuscript, the idea of "sparseness" is introduced indirectly in the last paragraph of the introduction as "...changes in activity statistics (sparseness)...", but it is unclear from the preceding text if the referenced "activity statistics" used to define sparseness are the "fraction of active units," or their "tuning specificity," or both. While sparseness is clearly defined in the Methods section for the RNN, there is no mention of how it is defined for neural data, and spatial information is not mentioned at all. For clarity, I suggest explicitly defining sparseness for both the RNN and real neural data early in the main text, e.g. "Here, we measure sparseness in neural data by A and B, and by the analogous metric(s) of X and Y in our RNN..." This is a small but important nuance that will enhance the ease of reading for a broad neuroscience audience.

Reviewer #3 (Public Review):

Summary:

The manuscript by Nagel, et al. describes studies of mouse vomeronasal sensory neuron (VSN) tuning to mouse urine samples across different sexes and strains, including wild mice, alongside mass spectrometry analysis of the same samples. The authors performed live Ca2+ imaging (CAL520 dye) of VSNs in acute vomeronasal organ (VNO) slices to determine how VSNs are tuned to pairs of stimuli that differ in their origin (e.g. male C57BL/6 versus male BALB/c urine, male C57BL/6 versus female C57BL/6, etc.). For each pair of tested odorants, the results measure the proportion of VSNs that respond to both stimuli ("generalists") or just one of the two ("specialists"), as well as metrics of tuning preference and response reliability. The authors find in most cases that generalists make up a larger proportion of responsive VSNs than specialists, but several pairwise comparisons showed a high degree of strain selectivity. Notably, the authors evaluated VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding strain-dependent differences in the representation of mouse urine. Alongside these measurements of VSN tuning, the authors report results of mass spectrometry analyses of volatiles and proteins in the same urine samples. These analyses indicated a number of molecules in each category that vary across sex and strain, and therefore represent candidate vomeronasal ligands. However, this study did not directly test whether any of these candidate molecules drives VSN activity. Overall, this work provides solid information related to mouse vomeronasal chemosensation.

Strengths:

A strength of the current study is its focus on characterizing the neural responses of the VNO to urine derived from wild mice. The majority of existing vomeronasal system research has relied on the use of inbred strains for both neural response recordings and investigations of candidate vomeronasal system ligands. Inbreeding in laboratory environments may alter the chemical composition of bodily secretions, thereby potentially changing the information they contain. Moreover, the more homogeneous nature of inbred strains could be critical when studying the AOS mediated social aspects. If there exist noticeable differences in the chemical composition of secretions from wild animals compared to inbred strains, this would suggest that future research must consider natural sources of candidate ligands outside of inbred strains. This work identifies some intriguing differences, worthy of further exploration, between the urine composition of wild mice versus inbred mice, as well as disparities in how the VNO responds to urine from these different sources. However, the molecular composition and VNO responsiveness to wild mouse urine was found to be highly overlapping with inbred mouse urine, supporting the continued investigation of candidate ligands found in inbred mouse urine.

Another positive aspect of this work is its use of the same set of stimuli as a previous study by the same authors (Bansal et al., 2021) in the downstream accessory olfactory bulb. The consistency in stimulus selection facilitates a comparison of information processing of sex and strain information from the sensory periphery to the brain. Although comparisons between the two connected regions are not a focus of this work, and methodological differences (e.g., Ca2+ imaging versus electrophysiology) may introduce caveats into comparisons, the support of "apples to apples" comparisons across connected circuits is critical to progress in the field.

Finally, this study directly measured VSN tuning in both male C57BL/6 and male BALB/c VNOs, finding subtle but important differences in the representation of mouse urine in these two recipient strains. Given that there is a long history of behavioral research into strain-specific differences in social behavior, this research paves the way for future studies into how different mouse strains detect and process social chemosignals.

Weaknesses:

One of the primary objectives in this study is to ascertain the extent to which the response profiles of VSNs are specific to sex and strain. The design of these Ca2+ imaging experiments uses a simple stimulus design, using two interleaved bouts of stimulation with pairs of urine (e.g., male versus female C57BL/6, male C57BL/6 versus male BALB/c) at a single dilution factor (1:100). This introduces two significant limitations: (1) the "generalist" versus "specialist" descriptors pertain only to the specific pairwise comparisons made and (2) there is no information about the sensitivity/concentration-dependence of the responses.

The functional measurements of VSN tuning to various pairs of urine stimuli are presented alongside mass spectrometry-based comparisons. However, the mass spectrometry-based analysis was performed separately from VSN tuning experiments/analysis. The juxtaposition of these measurements may give some readers the impression that VSN tuning measurements were integrated with molecular profiling (i.e., that the molecular diversity was causally related physiological responses). This is a hypothesis raised by the parallel studies, but not a supported conclusion of the current work.

The impact of mass spectrometry findings is acknowledged to be limited to nonvolatile organic compounds and proteins/peptides, and that it is possible that few of these candidate molecules are active in the VNO. Moreover, it remains possible that the VSN responses are driven mostly by small nonvolatiles (e.g., polar steroids), a class of strong VSN ligands that were excluded from molecular analysis.

Reviewer #3 (Public Review):

A brain region called the retrotrapezoid nucleus (RTN) regulates breathing in response to changes in CO2/H+, a process termed central chemoreception. A transcription factor called PHOX2B is important for RTN development and mutations in the PHOX2B gene result in a severe type of sleep apnea called Congenital Central Hypoventilation Syndrome. PHOX2B is also expressed throughout life, but its postmitotic functions remain unknown. This study shows that knockdown of PHOX2B in the RTN region in adult rats decreased expression of Task2 and Gpr4 in Nmb-expressing RTN chemoreceptors and this corresponded with a diminished ventilatory response to CO2 but did not impact baseline breathing or the hypoxic ventilatory response. These results provide novel insight regarding postmitotic functions of PHOX2B in RTN neurons.

I have two main concerns and several points of clarification.

Main issues:<br /> (1) The experimental approach was not targeted to Nmb+ neurons and since other cells in the area also express Phox2b, conclusions should be tempered to focus on Phox2b expressing parafacial neurons NOT specifically RTN neurons

(2) It's not clear whether PHOX2B is important for transcription of pH sensing machinery, cell health or both. If knockdown of PHOX2B knockdown results in loss of RTN neurons this is also expected to decrease Task2 and Gpr4 levels, albeit by a transcription-independent mechanism.

Other points:

(3) All individual data points should be visible in floating bar graphs in Figs 1 and 4. For example, I don't see any dots for naïve animals in any of the panels in Fig. 1.

(4) the C1 and facial partly overlap with the RTN at this level of the medulla and these cells should appear as Phox2b+/Nmb- cells so it is not clear to me why these cells are not evident in the control tissue in figs 2B and 3B. Also, some of the bregma levels shown in Fig. 5A overlap with Figs 2-3 so again it's not clear to me how this non-cell type specific viral approach was targeted to Nmb cells but not near by TH+ cells. Please clarify.

(5) How do you get a loss of Nmb+ neurons (Figs 2-3) with no change in Nmb fluorescence (Fig. 5B)? In the absence of representative images these results are not compelling and should be substantiated by more readily quantifiable approaches like qPCR.

Reviewer #3 (Public Review):

Summary:

The manuscript addresses a question inspired by the Baroceptor Hypothesis and its links to visual awareness and interoception. Specifically, the reported study aimed to determine if the effects of cardiac contraction (systole) on binocular rivalry (BR) are facilitatory or suppressive. The main experiment - relying on a technically challenging procedure of presenting stimuli synchronised with the heartbeats of participants - has been conducted with great care, and numerous manipulation checks the authors report convincingly show that the methods they used work as intended. Moreover, the control experiment allows for excluding alternative explanations related to participants being aware of their heartbeats. Therefore, the study convincingly shows the effect of cardiac activity on BR - and this is an important finding. The results, however, do not allow for unambiguously determining if this effect is facilitatory or suppressive (see details below), which renders the study not as informative as it could be.

While the authors strongly focus on interoception and awareness, this study will be of interest to researchers studying BR as such. Moreover, the code and the data the authors share can facilitate the adoption of their methods in other labs.

Strengths:

(1) The study required a complex technical setup and the manuscript both describes it well and demonstrates that it was free from potential technical issues (e.g. in section 3.3. Manipulation check).

(2) The sophisticated statistical methods the authors used, at least for a non-statistician like me, appear to be well-suited for their purpose. For example, they take into account the characteristics of BR (gamma distributions of dominance durations). Moreover, the authors demonstrate that at least in one case their approach is more conservative than a more basic one (Binomial test) would be.

(3) Finally, the control experiment, and the analysis it enabled, allow for excluding a multitude of alternative explanations of the main results.

(4) The authors share all their data and materials, even the code for the experiment.

(5) The manuscript is well-written. In particular, it introduces the problem and methods in a way that should be easy to understand for readers coming from different research fields.

Weaknesses:

(1) The interpretation of the main result in the context of the Baroceptor hypothesis is not clear. The manuscript states: The Baroreceptor Hypothesis would predict that the stimulus entrained to systole would spend more time suppressed and, conversely, less time dominant, as cortical activity would be suppressed each time that stimulus pulses. The manuscript does not specify why this should be the case, and the term 'entrained' is not too helpful here (does it refer to neural entrainment? or to 'being in phase with'?). The answer to this question is provided by the manuscript only implicitly, and, to explain my concern, I try to spell it out here in a slightly simplified form.

During systole (cardiac contraction), the visual system is less sensitive to external information, so it 'ignores' periods when the systole-synchronised stimulus is at the peak of its pulse. Conversely, the system is more sensitive during diastole, so the stimulus that is at the peak of its pulse then should dominate for longer, because its peaks are synchronised with the periods of the highest sensitivity of the visual system when the information used to resolve the rivalry is sampled from the environment. This idea, while indeed being a clever test of the hypothesis in question, rests on one critical assumption: that the peak of the stimulus pulse (as defined in the manuscript) is the time when the stimulus is the strongest for the visual system. The notion of 'stimulus strength' is widely used in the BR literature (see Brascamp et al., 2015 for a review). It refers to the stimulus property that, simply speaking, determines its tendency to dominate in the BR. The strength of a stimulus is underpinned by its low-level visual properties, such as contrast and spatial frequency content. Coming back to the manuscript, the pulsing of the stimuli affected at least spatial frequency (and likely other low-level properties), and it is unknown if it was in phase with the pulsing of the stimulus strength, or not. If my understanding of the premise of the study is correct, the conclusions drawn by the authors stand only if it was.

In other words, most likely the strength of one of the stimuli was pulsating in sync with the systole, but is it not clear which stimulus it was. It is possible that, for the visual system, the stimulus meant to pulse in sync with the systole was pulsing strength-wise in phase with the diastole (and the one intended to pulse with in sync with the diastole strength-wise pulsed with the systole). If this is the case, the predictions of the Baroceptor Hypothesis hold, which would change the conclusion of the manuscript.

(2) Using anaglyph goggles necessitates presenting stimuli of a different colour to each eye. The way in which different colours are presented can impact stimulus strength (e.g. consider that different anaglyph foils can attenuate the light they let through to different degrees). To deal with such effects, at least some studies on BR employed procedures of adjusting the colours for each participant individually (see Papathomas et al., 2004; Patel et al., 2015 and works cited there). While I think that counterbalancing applied in the study excludes the possibility that colour-related effects influenced the results, the effects of interest still could be stronger for one of the coloured foils.

(3) Several aspects of the methods (e.g. the stimuli), are not described at the level of detail some readers might be accustomed to. The most important issue here is the task the participants performed. The manuscript says that they pressed a button whenever they experienced a switch in perception, but it is only implied that there were different buttons for each stimulus.

Brascamp, J. W., Klink, P. C., & Levelt, W. J. M. (2015). The 'laws' of binocular rivalry: 50 years of Levelt's propositions. Vision Research, 109, 20-37. https://doi.org/10.1016/j.visres.2015.02.019<br /> Papathomas, T. V., Kovács, I., & Conway, T. (2004). Interocular grouping in binocular rivalry: Basic attributes and combinations. In D. Alais & R. Blake (Eds.), Binocular Rivalry (pp. 155-168). MIT Press<br /> Patel, V., Stuit, S., & Blake, R. (2015). Individual differences in the temporal dynamics of binocular rivalry and stimulus rivalry. Psychonomic Bulletin and Review, 22(2), 476-482. https://doi.org/10.3758/s13423-014-0695-1

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors propose that astrocytic water channel AQP4 represents the dominant pathway for tonic water efflux without which astrocytes undergo cell swelling. The authors measure changes in astrocytic sulforhodamine fluorescence as the proxy for cell volume dynamics. Using this approach, they perform a technically elegant series of ex vivo and in vivo experiments exploring changes in astrocytic volume in response to AQP4 inhibitor TGN-020 and/or neuronal stimulation. The key finding is that TGN-020 produces an apparent swelling of astrocytes and modifies astrocytic cell volume regulation after spreading depolarizations. Additionally, systemic application of TGN-020 produced changes in diffusion-weighted MRI signal, which the authors interpret as cellular swelling. This study is perceived as potentially significant. However, several technical caveats should be strongly considered and perhaps addressed through additional experiments.

Strengths:

(1) This is a technically elegant study, in which the authors employed a number of complementary ex vivo and in vivo techniques to explore functional outcomes of aquaporin inhibition. The presented data are potentially highly significant (but see below for caveats and questions related to data interpretation).

(2) The authors go beyond measuring cell volume homeostasis and probe for the functional significance of AQP4 inhibition by monitoring Ca2+ signaling in neurons and astrocytes (GCaMP6 assay).

(3) Spreading depolarizations represent a physiologically relevant model of cellular swelling. The authors use ChR2 optogenetics to trigger spreading depolarizations. This is a highly appropriate and much-appreciated approach.

Weaknesses:

(1) The main weakness of this study is that all major conclusions are based on the use of one pharmacological compound. In the opinion of this reviewer, the effects of TGN-020 are not consistent with the current knowledge on water permeability in astrocytes and the relative contribution of AQP4 to this process.

Specifically: Genetic deletion of AQP4 in astrocytes reduces plasmalemmal water permeability by ~two-three-fold (when measured a 37oC, Solenov et al., AJP-Cell, 2004). This is a significant difference, but it is thought to have limited/no impact on water distribution. Astrocytic volume and the degree of anisosmotic swelling/shrinkage are unchanged because the water permeability of the AQP4-null astrocytes remains high. This has been discussed at length in many publications (e.g., MacAulay et al., Neuroscience, 2004; MacAulay, Nat Rev Neurosci, 2021) and is acknowledged by Solenov and Verkman (2004).

Keeping this limitation in mind, it is important to validate astrocytic cell volume changes using an independent method of cell volume reconstruction (diameter of sulforhodamine-labeled cell bodies? 3D reconstruction of EGFP-tagged cells? Else?)

(2) TGN-020 produces many effects on the brain, with some but not all of the observed phenomena sensitive to the genetic deletion of AQP4. In the context of this work, it is important to note that TGN-020 does not completely inhibit AQP4 (70% maximal inhibition in the original oocyte study by Huber et al., Bioorg Med Chem, 2009). Thus, besides not knowing TGN-020 levels inside the brain, even "maximal" AQP4 inhibition would not be expected to dramatically affect water permeability in astrocytes.

This caveat may be addressed through experiments using local delivery of structurally unrelated AQP4 blockers, or, preferably, AQP4 KO mice.

(3) This reviewer thinks that the ADC signal changes in Figure 5 may be unrelated to cellular swelling. Instead, they may be a result of the previously reported TGN-020-induced hyphemia (e.g., H. Igarashi et al., NeuroReport, 2013) and/or changes in water fluxes across pia matter which is highly enriched in AQP4. To amplify this concern, AQP4 KO brains have increased water mobility due to enlarged interstitial spaces, rather than swollen astrocytes (RS Gomolka, eLife, 2023). Overall, the caveats of interpreting DW-MRI signal deserve strong consideration.

Reviewer #3 (Public Review):

Summary:

Neurogenesis in the mammalian olfactory epithelium persists throughout the life of the animal. The process replaces damaged or dying olfactory sensory neurons. It has been tacitly that replacement of the OR subtypes is stochastic, although anecdotal evidence has suggested that this may not be the case. In this study, Santoro and colleagues systematically test this hypothesis by answering three questions: is there enrichment of specific OR subtypes associated with neurogenesis? Is the enrichment dependent on sensory stimulus? Is the enrichment the result of differential generation of the OR type or from differential cell death regulated by neural activity? The authors provide some solid evidence indicating that musk odor stimulus selectively promotes the OR types expressing the musk receptors. The evidence argues against a random selection of ORs in the regenerating neurons.

Strengths:

The strength of the study is a thorough and systematic investigation of the expression of multiple musk receptors with unilateral naris occlusion or under different stimulus conditions. The controls are properly performed. This study is the first to formulate the selective promotion hypothesis and the first systematic investigation to test it. The bulk of the study uses in situ hybridization and immunofluorescent staining to estimate the number of OR types. These results convincingly demonstrate the increased expression of musk receptors in response to male odor or muscone stimulation.

Weaknesses:

A major weakness of the current study is the single-cell RNASeq result. The authors use this piece of data as a broad survey of receptor expression in response to unilateral nasal occlusion. However, several issues with this data raise serious concerns about the quality of the experiment and the conclusions. First, the proportion of OSNs, including both the immature and mature types, constitutes only a small fraction of the total cells. In previous studies of the OSNs using the scRNASeq approach, OSNs constitute the largest cell population. It is curious why this is the case. Second, the authors did not annotate the cell types, making it difficult to assess the potential cause of this discrepancy. Third, given the small number of OSNs, it is surprising to have multiple musk receptors detected in the open side of the olfactory epithelium whereas almost none in the closed side. Since each OR type only constitutes ~0.1% of OSNs on average, the number of detected musk receptors is too high to be consistent with our current understanding and the rest of the data in the manuscript. Finally, unlike the other experiments, the authors did not describe any method details, nor was there any description of quality controls associated with the experiment. The concerns over the scRNASeq data do not diminish the value of the data presented in the bulk of the study but could be used for further analysis.

A weakness of the experiment assessing musk receptor expression is that the authors do not distinguish immature from mature OSNs. Immature OSNs express multiple receptor types before they commit to the expression of a single type. The experiments do not reveal whether mature OSNs maintain an elevated expression level of musk receptors.

There are also two conceptual issues that are of concern. The first is the concept of selective neurogenesis. The data show an increased expression of musk receptors in response to male odor stimulation. The authors argue that this indicates selective neurogenesis of the musk receptor types. However, it is not clear what the distinction is between elevated receptor expression and a commitment to a specific fate at an early stage of development. As immature OSNs express multiple receptors, a likely scenario is that some newly differentiated immature OSNs have elevated expression of not only the musk receptors but also other receptors. The current experiments do not distinguish the two alternatives. Moreover, as pointed out above, it is not clear whether mature OSNs maintain the increased expression. Although a scRNASeq experiment can clarify it, the authors, unfortunately, did not perform an in-depth analysis to determine at which point of neurogenesis the cells commit to a specific musk receptor type. The quality of the scRNASeq data unfortunately also does not lend confidence for this type of analysis.

A second conceptual issue, the idea of homeostasis in regeneration, which the authors presented in the Introduction, needs clarification. In its current form, it is confusing. It could mean that a maintenance of the distribution of receptor types, or it could mean the proper replacement of a specific OR type upon the loss of this type. The authors seem to refer to the latter and should define it properly.

Reviewer #3 (Public Review):

In this manuscript, Magnuson and colleagues investigate the meiotic functions of ARID1A, a putative DNA binding subunit of the SWI/SNF chromatin remodeler BAF. The authors develop a germ cell specific conditional knockout (cKO) mouse model using Stra8-cre and observe that ARID1A-deficient cells fail to progress beyond pachytene, although due to inefficiency of the Stra8-cre system the mice retain ARID1A-expressing cells that yield sperm and allow fertility. Because ARID1A was found to accumulate at the XY body late in Prophase I, the authors suspected a potential role in meiotic silencing and by RNAseq observe significant misexpression of sex-linked genes that typically are silenced at pachytene. They go on to show that ARID1A is required for exclusion of RNA PolII from the sex body and for limiting promoter accessibility at sex-linked genes, consistent with a meiotic sex chromosome inactivation (MSCI) defect in cKO mice. The authors proceed to investigate the impacts of ARID1A on H3.3 deposition genome-wide. H3.3 is known be regulated by ARID1A and is linked to silencing, and here the authors find that upon loss of ARID1A, overall H3.3 enrichment at the sex body as measured by IF failed to occur, but H3.3 was enriched specifically at transcriptional start sites of sex-linked genes that are normally regulated by ARID1A. The results suggest that ARID1A normally prevents H3.3 accumulation at target promoters on sex chromosomes and based on additional data, restricts H3.3 to intergenic sites. Finally, the authors present data implicating ARID1A and H3.3 occupancy in DSB repair, finding that ARID1A cKO leads to a reduction in focus formation by DMC1, a key repair protein. Overall the paper provides new insights into the process of MSCI from the perspective of chromatin composition and structure, and raises interesting new questions about the interplay between chromatin structure, meiotic silencing and DNA repair.

In general the data are convincing. The conditional KO mouse model has some inherent limitations due to incomplete recombination and the existence of 'escaper' cells that express ARID1A and progress through meiosis normally. This reviewer feels that the authors have addressed this point thoroughly and have demonstrated clear and specific phenotypes using the best available animal model. The data demonstrate that the mutant cells fail to progress past pachytene, although it is unclear whether this specifically reflects pachytene arrest, as accumulation in other stages of Prophase also is suggested by the data in Table 1.

The revised manuscript more appropriately describes the relationship between ARID1A and DNA damage response (DDR) signaling. The authors don't see defects in a few DDR markers in ARID1A CKO cells (including a low resolution assessment of ATR), suggesting that ARID1A may not be required for meiotic DDR signaling. However, as previously noted the data do not rule out the possibility that ARID1A is downstream of DDR signaling, and the authors note the possibility of a role for DDR signaling upstream of ARID1A.

A final comment relates to the impacts of ARID1A loss on DMC1 focus formation and the interesting observation of reduced sex chromosome association by DMC1. The authors additionally assess the related recombinase RAD51 and suggest that it is unaffected by ARID1A loss. However, only a single image of RAD51 staining in the cKO is provided (Fig. S11) and there are no associated quantitative data provided. The data are suggestive and conclusions about the impacts of ARID1A loss on RAD51 must be considered as preliminary until more rigorously assessed.

Reviewer #3 (Public Review):

Summary:

This work describes a new pathway by which malaria parasites, P. falciparum, may regulate their growth and virulence (i.e. their expression of virulence-linked cytoadhesins). This is a topic of considerable interest in the field - does this important parasite sense factor(s) in its host bloodstream and regulate itself accordingly? Several fragments of evidence have come out on this topic in the past decade, showing, for example, reduced parasite growth under calorie restriction (in mice); parasite dormancy in response to amino acid starvation (in culture and in mice), and also reduced virulence in dry-season, low-parasitaemia infections in humans. The molecular mechanisms that may underlie this interesting biology remain only poorly understood.

Here, the authors show that dry-season P. falciparum parasites have reduced expression of Pol3-transcribed tRNAs and ncRNAs that positively regulate virulence gene expression. They link the level of Pol3 activity to PfMaf1, a remnant of the largely-absent nutrient-sensing TOR pathway in this parasite. They propose that in the dry season, human hosts may be calorie-restricted, leading to Maf1 moving to the nucleus and suppressing Pol3, thus downregulated growth and virulence of parasites. The evidence is intriguing and the idea is conceptually elegant.

Strengths:

The use of dry/wet-season field samples from The Gambia is a strength, showing potential real-world relevance. The generation of an inducible knockdown of Maf1 in lab-cultured parasites is also a strength, allowing this pathway to be studied somewhat in isolation.

Weaknesses:

(1) The signals upstream of Maf1 remain rather a black box. 4 are tested - heatshock and low-glucose, which seem to suppress ALL transcription; low-Isoleucine and high magnesium, which suppress Pol3. Therefore the authors use Mg supplementation throughout as a 'starvation type' stimulus. They do not discuss why they didn't use amino acid limitation, which could be more easily rationalised physiologically. It may for experimental simplicity (no need for dropout media) but this should be discussed, and ideally sample experiments with low-IsoLeu should be done too, to see if the responses (e.g. cytoadhesion) are all the same.

(2) The proteomics, conducted to seek partners of Maf1, is probably the weakest part. From Fig S4 it is clear that the proteins highlighted in the text are highly selected (as ones that might be relevant, e.g. phosphatases), but many others are more enriched. It would be good to see a) the top hits from the whole list provided as a short table within the main proteomics figure, along with the GO terms that actually came top in enrichment; b) the whole list provided as a supp. spreadsheet for easy re-analysis, rather than a PDF which cannot be easily re-used.

(3) Fig 3 shows the Maf1-low line has very poor growth after only 5 days but it is stated that no dead parasites are seen even after 8 cycles and the merozoites number is down only ~18 to 15... is this too small to account for such poor growth (~5-fold reduced in a single cycle, day 3-5)? It would additionally be interesting to see a cell-cycle length assessment and invasion assay, to see of Maf1-low parasite have further defects in growth.

Other weaknesses, which are more restricted but were not addressed in revision, are highlighted below:

Fig S1B - The downregulation of RNAPol3 transcripts caused by a commercial Pol3 inhibitor is pretty weak - mostly non-significant. The authors might comment on why they think this is, when interfering with PfMaf1 evidently has a greater effect.

Fig 2D: the legend states ' Expressed transcripts from three replicates between control and addition of MgCl2 that are significantly up-regulated are highlighted in red while significantly down-regulated RNA Pol III genes are highlighted in blue (FDR corrected p-value of <0.05) and a FC {greater than or equal to}{plus minus} 1.95) with examples listed as text'. This isn't very clear. The authors could clarify whether they took ALL (Pol3 or not) upregulated genes to show in red, but only putative Pol3-regulated genes to show in blue? If so, why? Or did they take all significantly downregulated genes, and found they were all annotated as pol3 transcribed? (I cannot see any dots that are not blue. If there are some, a clearer figure is needed?)

Line 227: 'PfMaf1 levels were shown to decrease by approximately 57% in total extracts after one cycle' - the provenance of this very precise percentage isn't clear (it does not appear on the figure). Is it densitometry of a western blot? And if so, is it an average of the 3 replicates that are stated in the legend (but not shown), or from the single example blot shown in Figure 3?

Fig 4A: the western blot, as shown, lacks controls, both for loading and for completeness of cyto/nuclear fractionation. To avoid confusion, these should be shown in the main figure, as is standard in the field, rather than separately in a supp figure. Ideally, 3 repeats should be done, with densitiometry quantification.

Reviewer #3 (Public Review):

Unfortunately, this study fails to incorporate the most important variable impacting the ability to predict mosaicism, the accuracy of the test. The fact is that most embryos diagnosed as mosaic are not mosaic. There may be 4 cases out of thousands and thousands of transfers where a confirmation was made. Mosaicism has become a category of diagnosis in which embryos with noisy NGS profiles are placed. With VeriSeq NGS it is not possible to routinely distinguish true mosaicism from noise. An analysis of NGS noise levels (MAPD) versus the rate of mosaics by clinic using the registry will likely demonstrate this is the case. Without accounting for the considerable inaccuracy of the method of testing the proposed modeling is meaningless.

Recent data using more accurate methods of identifying mosaicism indicate that the prevalence of true preimplantation embryonic mosaicism is only 2%, which is also consistent with findings made post-implantation. This model fails to account for the possibility that, because so few embryos are actually mosaic, there is actually no relevance to clinical care whatsoever. In fact, differences in clinical outcomes of embryos designated as mosaic could be entirely attributed to poor embryo quality resulting in noise levels that make NGS results fall into the "mosaic" category.

Additional comments:

Indeed, as more data emerges, it appears that the majority of embryos from both healthy and infertile couples are mosaic to some degree (Coticchio et al., 2021; Griffin et al., 2022).

This statement should be softened as all embryos will be considered mosaic when a method with a 10% false positive rate is applied to 10 more parts of the same embryo. The distinction between artifact and true mosaicism cannot be made with nearly all current methods of testing. When virtually no embryos display uniform aneuploidy in a rebiopsy study, there should be great concern over the accuracy of the testing used. The vast majority of aneuploidy is meiotic in origin.

Experimental data provides strong evidence that, for the most part, the biopsy result obtained accurately represents the chromosome constitution of the rest of the embryo (Kim 96 et al., 2022; Navratil et al., 2020; Victor et al., 2019).

This statement is incorrect given published systematic review of the literature indicates a 10% false positive rate based on rebiopsy results.

This shows that accurately classifying a mosaic embryo based on a single biopsy is not robust.

This is exactly why the practice of designating embryo mosaics with intermediate copy numbers should not exist.

Reviewer #3 (Public Review):

The article presents a comprehensive study on the stratification of viral shedding patterns in saliva among COVID-19 patients. The authors analyze longitudinal viral load data from 144 mildly symptomatic patients using a mathematical model, identifying three distinct groups based on the duration of viral shedding. Despite analyzing a wide range of clinical data and micro-RNA expression levels, the study could not find significant predictors for the stratified shedding patterns, highlighting the complexity of SARS-CoV-2 dynamics in saliva. The research underscores the need for identifying biomarkers to improve public health interventions and acknowledges several limitations, including the lack of consideration of recent variants, the sparsity of information before symptom onset, and the focus on symptomatic infections.

The manuscript is well-written, with the potential for enhanced clarity in explaining statistical methodologies. This work could inform public health strategies and diagnostic testing approaches. However, there is a thorough development of new statistical analysis needed, with major revisions to address the following points:

(1) Patient characterization & selection: Patient immunological status at inclusion (and if it was accessible at the time of infection) may be the strongest predictor for viral shedding in saliva. The authors state that the patients were not previously infected by SARS-COV-2. Was Anti-N antibody testing performed? Were other humoral measurements performed or did everything rely on declaration? From Figure 1A, I do not understand the rationale for excluding asymptomatic patients. Moreover, the mechanistic model can handle patients with only three observations, why are they not included? Finally, the 54 patients without clinical data can be used for the viral dynamics fitting and then discarded for the descriptive analysis. Excluding them can create a bias. All the discarded patients can help the virus dynamics analysis as it is a population approach. Please clarify. In Table 1 the absence of sex covariate is surprising.

(2) Exact study timReviewer #3 (Public Review):eline for explanatory covariates: I understand the idea of finding « early predictors » of long-lasting viral shedding. I believe it is key and a great question. However, some samples (Figure 4A) seem to be taken at the end of the viral shedding. I am not sure it is really easier to micro-RNA saliva samples than a PCR. So I need to be better convinced of the impact of the possible findings. Generally, the timeline of explanatory covariate is not described in a satisfactory manner in the actual manuscript. Also, the evaluation and inclusion of the daily symptoms in the analysis are unclear to me.

(3) Early Trajectory Differentiation: The model struggles to differentiate between patients' viral load trajectories in the early phase, with overlapping slopes and indistinguishable viral load peaks observed in Figures 2B, 2C, and 2D. The question arises whether this issue stems from the data, the nature of Covid-19, or the model itself. The authors discuss the scarcity of pre-symptom data, primarily relying on Illinois patients who underwent testing before symptom onset. This contrasts earlier statements on pages 5-6 & 23, where they claim the data captures the full infection dynamics, suggesting sufficient early data for pre-symptom kinetics estimation. The authors need to provide detailed information on the number or timing of patient sample collections during each period.

(4) Conditioning on the future: Conditioning on the future in statistics refers to the problematic situation where an analysis inadvertently relies on information that would not have been available at the time decisions were made or data were collected. This seems to be the case when the authors create micro-RNA data (Figure 4A). First, when the sampling times are is something that needs to be clarified by the authors (for clinical outcomes as well). Second, proper causal inference relies on the assumption that the cause precedes the effect. This conditioning on the future may result in overestimating the model's accuracy. This happens because the model has been exposed to the outcome it's supposed to predict. This could question the - already weak - relation with mir-1846 level.

(5) Mathematical Model Choice Justification and Performance: The paper lacks mention of the practical identifiability of the model (especially for tau regarding the lack of early data information). Moreover, it is expected that the immune effector model will be more useful at the beginning of the infection (for which data are the more parsimonious). Please provide AIC for comparison, saying that they have "equal performance" is not enough. Can you provide at least in a point-by-point response the VPC & convergence assessments?

(6) Selected features of viral shedding: I wonder to what extent the viral shedding area under the curve (AUC) and normalized AUC should be added as selected features.

(7) Two-step nature of the analysis: First you fit a mechanistic model, then you use the predictions of this model to perform clustering and prediction of groups (unsupervised then supervised). Thus you do not propagate the uncertainty intrinsic to your first estimation through the second step, ie. all the viral load selected features actually have a confidence bound which is ignored. Did you consider a one-step analysis in which your covariates of interest play a direct role in the parameters of the mechanistic model as covariates? To pursue this type of analysis SCM (Johnson et al. Pharm. Res. 1998), COSSAC (Ayral et al. 2021 CPT PsP), or SAMBA ( Prague et al. CPT PsP 2021) methods can be used. Did you consider sampling on the posterior distribution rather than using EBE to avoid shrinkage?

(8) Need for advanced statistical methods: The analysis is characterized by a lack of power. This can indeed come from the sample size that is characterized by the number of data available in the study. However, I believe the power could be increased using more advanced statistical methods. At least it is worth a try. First considering the unsupervised clustering, summarizing the viral shedding trajectories with features collapses longitudinal information. I wonder if the R package « LongituRF » (and associated method) could help, see Capitaine et al. 2020 SMMR. Another interesting tool to investigate could be latent class models R package « lcmm » (and associated method), see Proust-Lima et al. 2017 J. Stat. Softwares. But the latter may be more far-reached.

(9) Study intrinsic limitation: All the results cannot be extended to asymptomatic patients and patients infected with recent VOCs. It definitively limits the impact of results and their applicability to public health. However, for me, the novelty of the data analysis techniques used should also be taken into consideration.

Strengths are:<br /> - Unique data and comprehensive analysis.<br /> - Novel results on viral shedding.

Weaknesses are:<br /> - Limitation of study design.<br /> - The need for advanced statistical methodology.

Reviewer #3 (Public Review):

Summary:

This manuscript by Liu et al. presents a case that CAPSL mutations are a cause of familial exudative vitreoretinopathy (FEVR). Attention was initially focused on the CAPSL gene from whole exome sequence analysis of two small families. The follow-up analyses included studies in which CAPSL was manipulated in endothelial cells of mice and multiple iterations of molecular and cellular analyses. Together, the data show that CAPSL influences endothelial cell proliferation and migration. Molecularly, transcriptomic and proteomic analyses suggest that CAPSL influences many genes/proteins that are also downstream targets of MYC and may be important to the mechanisms.

Strengths:

This multi-pronged approach found a previously unknown function for CAPSLs in endothelial cells and pointed at MYC pathways as high-quality candidates in the mechanism.

Weaknesses:

Two issues shape the overall impact for me. First, the unreported population frequency of the variants in the manuscript makes it unclear if CAPSL should be considered an interesting candidate possibly contributing to FEVR, or possibly a cause. Second, it is unclear if the identified variants act dominantly, as indicated in the pedigrees. The studies in mice utilized homozygotes for an endothelial cell-specific knockout, leaving uncertainty about what phenotypes might be observed if mice heterozygous for a ubiquitous knockout had instead been studied.

In my opinion, the following scientific issues are specific weaknesses that should be addressed:

(1) Please state in the manuscript the number of FEVR families that were studied by WES. Please also describe if the families had been selected for the absence of known mutations, and/or what percentage lack known pathogenic variants.

(2) A better clinical description of family 3104 would enhance the manuscript, especially the father. It is unclear what "manifested with FEVR symptoms, according to the medical records" means. Was the father diagnosed with FEVR? If the father has some iteration of a mild case, please describe it in more detail. If the lack of clinical images in the figure is indicative of a lack of medical documentation, please note this in the manuscript.

(3) The TGA stop codon can in some instances also influence splicing (PMID: 38012313). Please add a bioinformatic assessment of splicing prediction to the assays and report its output in the manuscript.

(4) More details regarding utilizing a "loxp-flanked allele of CAPSL" are needed. Is this an existing allele, if so, what is the allele and citation? If new (as suggested by S1), the newly generated CAPSL mutant mouse strain needs to be entered into the MGI database and assigned an official allele name - which should then be utilized in the manuscript and who generated the strain (presumably a core or company?) must be described.

(5) The statement in the methods "All mice used in the study were on a C57BL/6J genetic background," should be better defined. Was the new allele generated on a pure C57BL/6J genetic background, or bred to be some level of congenic? If congenic, to what generation? If unknown, please either test and report the homogeneity of the background, or consult with nomenclature experts (such as available through MGI) to adopt the appropriate F?+NX type designation. This also pertains to the Pdgfb-iCreER mice, which reference 43 describes as having been generated in an F2 population of C57BL/6 X CBA and did not designate the sub-strain of C57BL/6 mice. It is important because one of the explanations for missing heritability in FEVR may be a high level of dependence on genetic background. From the information in the current description, it is also not inherently obvious that the mice studied did not harbor confounding mutations such as rd1 or rd8.

(6) In my opinion, more experimental detail is needed regarding Figures 2 and 3. How many fields, of how many retinas and mice were analyzed in Figure 2? How many mice were assessed in Figure 3?

(7) I suggest adding into the methods whether P-values were corrected for multiple tests.

Control and centralization are noways in university

Reviewer #3 (Public Review):

Summary:

Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), Peters and co-workers showed that the disordered N-terminus of both LRMP and HCN4 are necessary for LRMP to interact with HCN4 and inhibit the cAMP-dependent potentiation of channel opening. Strikingly, they identified two HCN4-specific residues, P545 and T547 in the C-linker of HCN4, that are close in proximity to the cAMP transduction centre (elbow Clinker, S4/S5-linker, HCND) and account for the LRMP effect.

Strengths:

Based on these data, the Authors propose a mechanism in which LRMP specifically binds to HCN4 via its isotype-specific Nterminal sequence and thus prevents the cAMP transduction mechanism by acting at the interface between the elbow Clinker, the S4S5-linker, the HCND.

Weaknesses:

Although the work is interesting, there are some discrepancies between data that need to be addressed.

- I suggest inserting in Table 1 and in the text, the Δ shift values (+cAMP; + LRMP; +cAMP/LRMP). This will help readers.

- Figure 1 is not clear, the distribution of values is anomalously high. For instance, in 1B the distribution of values of V1/2 in the presence of cAMP goes from - 85 to -115. I agree that in the absence of cAMP, HCN4 in HEK293 cells shows some variability in V1/2 values, that nonetheless cannot be so wide (here the variability spans sometimes even 30 mV) and usually disappears with cAMP (here not).<br /> This problem is spread throughout the ms, and the measured mean effects indeed always at the limit of statistical significance. Why so? Is this a problem with the analysis, or with the recordings?<br /> There are several other problems with Figure 1 and in all figures of the ms: the Y scale is very narrow while the mean values are marked with large square boxes. Moreover, the exemplary activation curve of Fig 1A is not representative of the mean values reported in Figure 1B, and the values of 1B are different from those reported in Table 1.<br /> On this ground it is difficult to judge the conclusions and it would also greatly help if exemplary current traces would also be shown.

- "....HCN4-P545A/T547F was insensitive to LRMP (Figs. 6B and 6C; Table 1), indicating that the unique HCN4 C-linker is necessary for regulation by LRMP. Thus, LRMP appears to regulate HCN4 by altering the interactions between the C-linker, S4-S5 linker, and N-terminus at the cAMP transduction centre."

Although this is an interesting theory, there are no data supporting it. Indeed, P545 and T547 at the tip of the C-linker elbow (fig 6A) are crucial for LRMP effect, but these two residues are not involved in the cAMP transduction centre (interface between HCND, S4S5 linker and Clinker elbow), at least for the data accumulated till now in the literature. Indeed, the hypothesis that LRMP somehow inhibits the cAMP transduction mechanism of HCN4 given the fact that the two necessary residues P545 and T547 are close to the cAMP transduction centre, awaits to be proven.

Moreover, I suggest analysing the putative role of P545 and T547 in the light of the available HCN4 structures. In particular, T547 (elbow) point towards the underlying shoulder of the adjacent subunit and, therefore, it is in a key position for the cAMP transduction mechanism. The presence of bulky hydrophobic residues (very different nature compared to T) in the equivalent position of HCN1 and HCN2 is also favouring this hypothesis. In this light, it will also be interesting to see whether single T547F mutation is sufficient to prevent LRMP effect.

Reviewer #3 (Public Review):

Summary:

This research article reports that a greater number of senescent osteoclasts (SnOCs), which produce Netrin-1 and NGF, are responsible for innervation in the LSI and aging animal models.

Strengths:

The research is based on previous findings in the authors' lab and the fact that the IVD structure was restored by treatment with ABT263. The logic is clear and clarifies the pathological role of SnOCs, suggesting the potential utilization of senolytic drugs for the treatment of LBP. Generally, the study is of good quality and the data is convincing.

Weaknesses:

All my concerns have been well addressed, no further comments.

Reviewer #3 (Public Review):

In the manuscript entitled "Embryonic Origins of Forebrain Oligodendrocytes Revisited by Combinatorial Genetic Fate Mapping," Cai et al. used an intersectional/subtractional strategy to genetically fate-map the oligodendrocyte populations (OLs) generated from medial ganglionic eminence (NKX2.1+), lateral ganglionic eminences, and dorsal progenitor cells (EMX1+). Specifically, they generated an OL-expressing reporter mouse line OpalinP2A-Flpo-T2A-tTA2 and bred with region-specific neural progenitor-expressing Cre lines EMX1-Cre for dOL and NKX2.1-Cre for MPOL. They used a subtractional strategy in the OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mouse line to predict the origins of OLs from lateral/caudal ganglionic eminences (LC). With their genetic tools, the authors concluded that neocortical OLs primarily consist of dOLs. Although the populations of OLs (dOLs or MP-OLs) from Emx1+ or Nkx2.1+ progenitors are largely consistent with previous findings, they observed that MP-OLs contribute minimally but persist into adulthood without elimination as in the previous report (PMID: 16388308).

Intriguingly, by using an indirect subtraction approach, they hypothesize that both Emx1-negative and Nkx2.1-negative cells represent the progenitors from lateral/caudal ganglionic eminences (LC), and conclude that neocortical OLs are not derived from the LC region. This is in contrast to the previous observation for the contribution of LC-expressing progenitors (marked by Gsx2-Cre) to neocortical OLs (PMID: 16388308). The authors claim that Gsh2 is not exclusive to progenitor cells in the LC region (PMID: 32234482). However, Gsh2 exhibits high enrichment in the LC during early embryonic development. The presence of a small population of Gsh2-positive cells in the late embryonic cortex could originate/migrate from Gsh2-positive cells in the LC at earlier stages (PMID: 32234482). Consequently, the possibility that cortical OLs derived from Gsh2+ progenitors in LC could not be conclusively ruled out. Notably, a population of OLs migrating from the ventral to the dorsal cortical region was detected after eliminating dorsal progenitor-derived OLs (PMID: 16436615).

The indirect subtraction data for LC progenitors drawn from the OpalinFlp-tdTOM reporter in Emx1-negative and Nkx2.1-negative cells in the OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mouse line present some caveats that could influence their conclusion. The extent of activity from the two Cre lines in the OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mice remains uncertain. The OpalinFlp-tdTOM expression could occur in the presence of either Emx1Cre or Nkx2.1Cre, raising questions about the contribution of the individual Cre lines. To clarify, the authors should compare the tdTOM expression from each individual Cre line, OpalinFlp::Emx1Cre::RC::FLTG or OpalinFlp::Nkx2.1Cre::RC::FLTG, with the combined OpalinFlp::Emx1Cre::Nkx2.1Cre::RC::FLTG mouse line. This comparison is crucial as the results from the combined Cre lines could appear similar to only one Cre line active.

Overall, the authors provided intriguing findings regarding the origin and fate of oligodendrocytes from different progenitor cells in embryonic brain regions. However, further analysis is necessary to substantiate their conclusion about the fate of LC-derived OLs convincingly.

Reviewer #3 (Public Review):

Summary:

The main objective of this work has been to delve into the mechanisms underlying the increment of D-serine in serum, as a marker of renal injury.

Strengths:

With a multi-hierarchical approach, the work shows that Ischemia reperfusion injury in kidney causes a specific increment in renal reabsorption of D-serine that, at least in part, is due to the increased expression of the apical transporter ASCT2. In the way, the authors revealed that SMCT1 also transports D-serine.

The manuscript also supports that increased expression of ASCT2, even together with the parallel decreased expression of SMCT1, in renal proximal tubules underlies the increased reabsorption of D-serine responsible of the increment of this enantiomer in serum in a murine model of ischemia reperfusion injury.

Weaknesses:

Remains to be clarified whether ASCT2 has substantial stereospecificity in favor of D- versus L-serine to sustain a ~10-fold decreased in the ratio D-serine/L-serine in the urine of mouse under ischemia reperfusion injury (IRI).<br /> It is not clear how the increment in the expression of ASCT2, in parallel with the decreased expression of SMCT1, results in increased renal reabsorption of D-serine in IRI.

I am satisfied with the changes the authors have introduced in the text of the revised version of their manuscript.

Reviewer #3 (Public Review):

Summary:

Zhao et al. address the question of whether intermediate states of the epithelial-to-mesenchymal transition (EMT) exist in a natural developmental context as well as in cancer cells. This is important not only for our understanding of these developmental systems but also for their development as resources for new anti-cancer approaches. Guided by single-cell RNA sequencing analysis of delaminating mouse cranial neural crest cells, they identify two distinct populations with transcriptional signatures intermediate between neuroepithelial progenitors and migrating crest. Both clusters are also spatially intermediate and are actively cycling, with one in S-phase and one in G2/M. They show that blocking progression through S phase prior to the onset of delamination and knockdown of intermediate state marker Dlc1 both reduce the number of migratory cells that have completed EMT. Overall, the work provides a modern take and new insights into the classical developmental process of neural crest delamination.

Strengths:

• Deep analysis of the scRNAseq dataset revealed previously unappreciated cell populations intermediate between premigratory and migratory crest.<br /> • The observation that delaminating/intermediate neural crest cells appear to be in S or G2/M phase is interesting and worth reporting, though the ultimate significance remains unclear, given that they do not make distinct derivatives depending on their cycle state.<br /> • The authors employ new methods for multiplex spatial imaging to more accurately define their populations of interest and their relative positions.<br /> • The authors present evidence that intermediate state gene Dlc1 (a Rho GAP) is not just a marker but functionally required for neural crest delamination in mouse, as previously shown in chicken.

Weaknesses:

• Similar experiments involving blockade of cell cycle progression and Dlc1 dose manipulation were previously performed in chick models, as noted in the discussion. The newly-defined intermediate states give added context to the results, but they are not entirely novel.

Reviewer #3 (Public Review):

Summary:

In this study, the authors set out to address the question of how the SNARE protein Syntaxin 17 senses autophagosome maturation by being recruited to autophagosomal membranes only once autophagosome formation and sealing is complete. The authors discover that the C-terminal region of Syntaxin 17 is essential for its sensing mechanism that involves two transmembrane domains and a positively charged region. The authors discover that the lipid PI4P is highly enriched in mature autophagosomes and that electrostatic interaction with Syntaxin 17's positively charged region with PI4P drives recruitment specifically to mature autophagosomes. The temporal basis for PI4P enrichment and Syntaxin 17 recruitment to ensure that unsealed autophagosomes do not fuse with lysosomes is a very interesting and important discovery. Overall, the data are clear and convincing, with the study providing important mechanistic insights that will be of broad interest to the autophagy field, and also to cell biologists interested in phosphoinositide lipid biology. The author's discovery also provides an opportunity for future research in which Syntaxin 17's c-terminal region could be used to target factors of interest to mature autophagosomes.

Strengths:

The study combines clear and convincing cell biology data with in vitro approaches to show how Syntaxin 17 is recruited to mature autophagosomes. The authors take a methodical approach to narrow down the critical regions within Syntaxin 17 required for recruitment and use a variety of biosensors to show that PI4P is enriched on mature autophagosomes.

Weaknesses:

There are no major weaknesses, overall the work is highly convincing. It would have been beneficial if the authors could have shown whether altering PI4P levels would affect Syntaxin 17 recruitment. However, this is understandably a challenging experiment to undertake and the authors outlined their various attempts to tackle this question.

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 revised submission:

The revised manuscript is significantly improved. 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):

Summary:

Authors mapped monosynaptic inputs to dopamine, GABA, and glutamate neurons in VTA under different anesthesia methods, and under drugs (cocaine, morphine, methamphetamine, amphetamine, nicotine, fluoxetine). They found that input patterns under different conditions are separated, and identified some key brain areas to contribute to such separation. They also searched a database for gene expression patterns that are common across input brain areas with some changes by anesthesia or drug administration.

Strengths:

The whole-brain approach to address drug effects is appealing and their conclusion is clear. The methodology and motivation are clearly explained.

Weaknesses:

While gene expression analyses may not be related to their findings on the anatomical effects of drugs, this will be a nice starting point for follow-up studies.

Reviewer #3 (Public Review):

Summary:

The authors aimed to develop an automated tool to easily collect, process, and annotate the biomedical literature for higher efficiency and better reproducibility.

Strengths:

Two charms coming with the efforts made by the team are Pubget (for efficient and reliable grabbing articles from PubMed) and labelbuddy (for annotating text). They make text-mining of the biomedical literature more accessible, effective, and reproducible for streamlined text-mining and meta-science projects. The data were collected and analyzed using solid and validated methodology and demonstrated a very promising direction for meta-science studies.

Weaknesses:

More developments are needed for different resources of literature and strengths of AI-powered functions.

Reviewer #3 (Public Review):

Summary:

The neural retina is one of the most energetically active tissues in the body and research into retinal metabolism has a rich history. Prevailing dogma in the field is that the photoreceptors of the neural retina (rods and cones) are heavily reliant on glycolysis, and as oxygen tension at the level of photoreceptors is very low, these specialized sensory neurons carry out aerobic glycolysis, akin to the Warburg effect in cancer cells. It has been found that this unique metabolism changes in many retinal diseases, and targeting disease-altered retinal metabolism may be a viable treatment strategy. The neural retina is composed of 11 different cell types, and many research groups over the past century have contributed to our current understanding of cell-specific metabolism of retinal cells. More recently, it has been shown in mouse models and co-culture of the mouse neural retina with human RPE cultures that photoreceptors are reliant on the underlying retinal pigment epithelium for supplying nutrients. Chen and colleagues add to this body of work by studying an ex vivo culture of the developing mouse retina that maintained contact with the retinal pigment epithelium. They exposed such ex vivo cultures to small molecule inhibitors of specific metabolic pathways, performing targeted metabolomics on the tissue and staining tissue with key metabolic enzymes to lay the groundwork for what metabolic pathways may be active in particular cell types of the retina. The authors conclude that rod and cone photoreceptors are reliant on different metabolic pathways to maintain their cell viability - in particular, that rods rely on oxidative phosphorylation and cones rely on glycolysis. Further, their data suggest multiple mechanisms whereby glycolysis may occur simultaneously with anapleurosis to provide abundant energy to photoreceptors. The data from metabolomics revealed several novel findings in retinal metabolism, including the use of glutamine to fuel the mini-Krebs cycle, the utilization of the Cahill cycle in photoreceptors, and a taurine/hypotaurine shuttle between the underlying retinal pigment epithelium and photoreceptors to transfer reducing equivalents from the RPE to photoreceptors. In addition, this study provides quantitative metabolomics datasets that can be compared across experiments and groups. The use of this platform will allow for rapid testing of novel hypotheses regarding the metabolic ecosystem in the neural retina.

Strengths:

The data on differences in susceptibility of rods and cones to mitochondrial dysfunction versus glycolysis provides novel hypothesis-generating conjectures that can be tested in animal models. The multiple mechanisms that allow anapleurosis and glycolysis to run side-by-side add significant novelty to the field of retinal metabolism, setting the stage for further testing of these hypotheses as well.

Weaknesses:

Almost all of the conclusions from the paper are preliminary, based on data showing enzymes necessary for a metabolic process are present and the metabolites for that process are also present. However, to truly prove whether these processes are happening (rather than speculation of the possibility they are happening), further experiments are necessary. As it currently stands, results from this study contradict results from other studies - in particular that cones, not rods, are most reliant of glycolysis. The authors attempt to address these contradictions, but without further experimentation, logical arguments carry only so much weight. At a minimum, the authors have argued that the small molecules they use are exquisitely specific for their intended targets, but validating results with a second small molecule that hits the same target but is structurally different would bolster their claims. Genetically knocking down the intended targets with interfering RNA technology would also be possible, as would explant cultures from knock-out animals. Without these studies to confirm target specificity, combined with the fact that conclusions from this study contradict existing studies in the literature, the results have to be categorized as speculative and hypothesis-generating rather than conclusive.

Reviewer #3 (Public Review):

Summary:

This is a conceptually appealing study by Giunti et al in which the authors identify a role for PTEN/daf-18 and daf-16/FOXO in the development of inhibitory GABA neurons, and then demonstrate that a diet rich in ketone body β-hydroxybutyrate partially suppresses the PTEN mutant phenotypes. The authors use three assays to assess their phenotypes: (1) pharmacological assays (with levamisole and aldicarb); (2) locomotory assays and (3) cell morphological assays. These assays are carefully performed and the article is clearly written. While neurodevelopmental phenotypes had been previously demonstrated for PTEN/daf-18 and daf-16/FOXO (in other neurons), and while KB β-hydroxybutyrate had been previously shown to increase daf-16/FOXO activity (in the context of aging), this study is significant because it demonstrates the importance of KB β-hydroxybutyrate and DAF-16 in the context of neurodevelopment. Conceptually, and to my knowledge, this is the first evidence I have seen of a rescue of a developmental defect with dietary metabolic intervention, linking, in an elegant way, the underpinning genetic mechanisms with novel metabolic pathways that could be used to circumvent the defects.

Strengths:

What their data clearly demonstrate, is conceptually appealing, and in my opinion, the biggest contribution of the study is the ability of reverting a neurodevelopmental defect with a dietary intervention that acts upstream or in parallel to DAF-16/FOXO.

Weaknesses:

The model shows AKT-1 as an inhibitor of DAF-16, yet their studies show no differences from wildtype in akt-1 and akt-2 mutants. AKT is not a major protein studied in this paper, and it can be removed from the model to avoid confusion, or the result can be discussed in the context of the model to clarify interpretation.

When testing additional genes in the DAF-18/FOXO pathway, there were no significant differences from wild type in most cases. This should be discussed. Could there be an alternate pathway via DAF-18/DAF-16, excluding the PI3K pathway or are there variations in activity of PI3K genes during a ketogenic diet that are hard to detect with current assays?

The consequence of SOD-3 expression in the broader context of GABA neurons was not discussed. SOD-3 was also measured in the pharynx but measuring it in neurons would bolster the claims.

If they want to include AKT-1, seeing its effect on SOD-3 expression could be meaningful to the model.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

All prior concerns have been addressed.

Reviewer #3 (Public Review):

Summary:

In this study the authors used patch-clamp to characterize the implication of various voltage-gated Na+ channels in the firing properties of mouse nociceptive sensory neurons. They claim that depending on the culture conditions NaV1.3, NaV1.7, and NaV1.8 have distinct contributions to action potential firing and that similar firing patterns can result from distinct relative roles of these channels.

Strengths:

The paper addresses the important issue of understanding the lack of success of therapeutic strategies targeting NaV channels in the context of pain. Specifically, the authors test the hypothesis that different NaV channels contribute in a plastic manner to action potential firing, which may be the reason why it is difficult to target pain by inhibiting these channels.

Weaknesses:

(1) - The main claim of this paper is that "nociceptors can achieve equivalent excitability using different combinations of NaV1.3, NaV1.7, and NaV1.8". From this, they allude to the manifestation of "degeneracy", a concept implying that a biological process can occur via distinct sets of underlying components.<br /> In my opinion, the analyses of the data is biased towards the author's interpretation.<br /> - First, when comparing the excitability across neurons one should relate the response (in this case mean firing frequency) to the absolute size of the stimulus, not to the size of the stimulus normalized to the rheobase (see e.g., Figs. 1A). From this particular figure the authors conclude that the excitability is similar in the culture stages DIV0 and DIV4-7, but these data were not directly compared.<br /> - Second, the authors reach their conclusion from the comparison of the (average) firing rate determined over 1 s current stimulation in distinct conditions. However, this is not the only parameter that determines how sensory neurons might convey information. For instance, the time dependence of the instantaneous frequency, the actual firing pattern, maybe also important.<br /> - Third, the use of 1 s of current stimulation might not be sufficient to characterize the firing pattern if one wants to obtain conclusions that could translate to clinical settings (i.e., sustained pain).<br /> - Fourth, out of principle, the gating properties of NaV1.7 and NaV1.8 channels are not identical, and therefore their contributions to excitability should not be the same. A neuron in which NaV1.7 is the main contributor is expected to have a damping firing pattern due to cumulative channel inactivation, whereas another depending mainly on NaV1.8 is expected to display more sustained firing. This is actually seen in the results of the modelling.

(2) - The quality of some recordings is dubious. The currents shown as TTX-sensitive in Fig. 1D look very strange (not like the ones at Baseline DIV4-7). These traces show abnormally fast inactivation and even transient deflections above zero current line. These are obvious artifacts of the subtraction procedure, probably due to unstable current amplitudes along the recording time. Similar odd-looking traces are shown in Fig. 3A.

(3) - I would like to point out that the main Significance Statement of the manuscript reads "The analgesic efficacy of subtype-selective drugs hinges on which subtype controls excitability". I would like to point out that, in addition of being extremely obvious for anyone knowing a bit about pain signaling, the authors did not test the analgesic efficacy of any drug in this study.

(4) - A critical issue in the manuscript is the unnecessary use of phrases that imply that biological entities have some sort of willpower, flirting with anthropomorphism and teleological language.<br /> Sentences such as "Nociceptive sensory neurons convey pain signals to the CNS using action potentials" (see the Abstract) should be avoided. Neurons do not really "use" action potentials, they have no will to do so. Action potentials are not tools or means to be "used" by neurons. There are many other examples of misuse of the verb "use" in many other sentences. These were pointed out during the revision phase, but unfortunately the authors refused to correct them.

Reviewer #3 (Public Review):

The original research article, titled "mitoBKCa is functionally expressed in murine and human breast cancer cells and promotes metabolic reprogramming" by Bischof et al, has demonstrated the underlying molecular mechanisms of alterations in the function of Ca2+ activated K+ channel of large conductance (BKCa) in the development and progression of breast cancer. The authors also proposed that targeting mitoBKCa in combination with established anti-cancer approaches, could be considered as a novel treatment strategy in breast cancer treatment.

The paper is modified according to the reviewer's comments. Most of the queries raised by this reviewer were answered. However, the preclinical implication of this study can also be manifested in combinatorial treatment with known chemotherapeutic drugs which is lacking in this manuscript. Hopefully, the authors will consider this in their future study.

Reviewer #3 (Public Review):

Astrocyte biology is an active area of research and this study is timely and adds to a growing body of literature in the field. The RNA-seq, Herp expression, and Ca2+ release data across wild-type, Bmal1 knockout, and Herp knockdown cellular models are robust and lend considerable support to the study's conclusions, highlighting their importance. Despite these strengths, the manuscript presents a gap in elucidating the dynamics of HERP and the involvement of ITPR1/2 in modulating Ca2+ release patterns and their circadian variations, which remains insufficiently supported and characterized. While the Connexin data underscore the importance of rhythmic Ca2+ release triggered by ATP, the relationship here appears correlational and the role of HERP and ITPR in Cx function remains to be characterized. Moreover, enhancing the manuscript's clarity and readability could significantly benefit the presentation and comprehension of the findings.

Reviewer #3 (Public Review):

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

Previous studies have shown that DHODH inhibitors can increase expression of innate immunity-related genes but the role of DHODH and pyrimidine nucleotides in antigen presentation has not been previously reported. A strength of the manuscript is the solid in vitro mechanistic data supported by analysis in multiple cell lines. The in vivo data show compelling additive effects of DHODH inhibitors and ICB. However, more controls and experiments would be required to define the nature of these effects and to confirm that the mechanistic in vitro data is conserved in vivo.

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

Comments on revised version:

The authors have addressed my questions regarding validation of gene expression in other cell lines. They have also provided an explanation about why in vivo evaluations could not be performed for the experiment in Figure 5E.

Reviewer #3 (Public Review):

Summary:

This manuscript by Liu et al. presents a case that CAPSL mutations are a cause of familial exudative vitreoretinopathy (FEVR). Attention was initially focused on the CAPSL gene from whole exome sequence analysis of two small families. The follow-up analyses included studies in which CAPSL was manipulated in endothelial cells of mice and multiple iterations of molecular and cellular analyses. Together, the data show that CAPSL influences endothelial cell proliferation and migration. Molecularly, transcriptomic and proteomic analyses suggest that CAPSL influences many genes/proteins that are also downstream targets of MYC and may be important to the mechanisms.

Strengths:

This multi-pronged approach found a previously unknown function for CAPSLs in endothelial cells and pointed at MYC pathways as high-quality candidates in the mechanism.

Weaknesses:

Two issues shape the overall impact for me. First, the unreported population frequency of the variants in the manuscript makes it unclear if CAPSL should be considered an interesting candidate possibly contributing to FEVR, or possibly a cause. Second, it is unclear if the identified variants act dominantly, as indicated in the pedigrees. The studies in mice utilized homozygotes for an endothelial cell-specific knockout, leaving uncertainty about what phenotypes might be observed if mice heterozygous for a ubiquitous knockout had instead been studied.

In my opinion, the following scientific issues are specific weaknesses that should be addressed:

(1) Please state in the manuscript the number of FEVR families that were studied by WES. Please also describe if the families had been selected for the absence of known mutations, and/or what percentage lack known pathogenic variants.

(2) A better clinical description of family 3104 would enhance the manuscript, especially the father. It is unclear what "manifested with FEVR symptoms, according to the medical records" means. Was the father diagnosed with FEVR? If the father has some iteration of a mild case, please describe it in more detail. If the lack of clinical images in the figure is indicative of a lack of medical documentation, please note this in the manuscript.

(3) The TGA stop codon can in some instances also influence splicing (PMID: 38012313). Please add a bioinformatic assessment of splicing prediction to the assays and report its output in the manuscript.

(4) More details regarding utilizing a "loxp-flanked allele of CAPSL" are needed. Is this an existing allele, if so, what is the allele and citation? If new (as suggested by S1), the newly generated CAPSL mutant mouse strain needs to be entered into the MGI database and assigned an official allele name - which should then be utilized in the manuscript and who generated the strain (presumably a core or company?) must be described.

(5) The statement in the methods "All mice used in the study were on a C57BL/6J genetic background," should be better defined. Was the new allele generated on a pure C57BL/6J genetic background, or bred to be some level of congenic? If congenic, to what generation? If unknown, please either test and report the homogeneity of the background, or consult with nomenclature experts (such as available through MGI) to adopt the appropriate F?+NX type designation. This also pertains to the Pdgfb-iCreER mice, which reference 43 describes as having been generated in an F2 population of C57BL/6 X CBA and did not designate the sub-strain of C57BL/6 mice. It is important because one of the explanations for missing heritability in FEVR may be a high level of dependence on genetic background. From the information in the current description, it is also not inherently obvious that the mice studied did not harbor confounding mutations such as rd1 or rd8.

(6) In my opinion, more experimental detail is needed regarding Figures 2 and 3. How many fields, of how many retinas and mice were analyzed in Figure 2? How many mice were assessed in Figure 3?

(7) I suggest adding into the methods whether P-values were corrected for multiple tests.

Reviewer #3 (Public Review):

Summary:

ImmCellTyper is a new toolkit for Cytometry by time-of-flight data analysis. It includes BinaryClust, a semi-supervised clustering tool (which takes into account prior biological knowledge), designed for automated classification and annotation of specific cell types and subpopulations. ImmCellTyper also integrates a variety of tools to perform data quality analysis, batch effect correction, dimension reduction, unsupervised clustering, and differential analysis.

Strengths:

The proposed algorithm takes into account the prior knowledge.<br /> The results on different benchmarks indicate competitive or better performance (in terms of accuracy and speed) depending on the method.

Weaknesses:

The proposed algorithm considers only CyTOF markers with binary distribution.

Reviewer #3 (Public Review):

Ephaptic inhibition between neurons housed in the same sensilla has been long discovered in flies, but the molecular basis underlying this inhibition is underexplored. Specifically, it remains poorly understood which receptors or channels are important for maintaining the transepithelial potential between the sensillum lymph and the hemolymph (known as the sensillum potential), and how this affects the excitability of neurons housed in the same sensilla.

Lee et al. used single-sensillum recordings (SSR) of the labellar taste sensilla to demonstrate that the HCN channel, Ih, is critical for maintaining sensillum potential in flies. Ih is expressed in sugar-sensing GRNs (sGRNs) but affects the excitability of both the sGRNs and the bitter-sensing GRNs (bGRNs) in the same sensilla. Ih mutant flies have decreased sensillum potential, and bGRNs of Ih mutant flies have a decreased response to the bitter compound caffeine. Interestingly, ectopic expression of Ih in bGRNs also increases sGRN response to sucrose, suggesting that Ih-dependent increase in sensillum potential is not specific to Ih expressed in sGRNs. The authors further demonstrated, using both SSR and behavior assays, that exposure to sugars in the food substrate is important for the Ih-dependent sensitization of bGRNs. The experiments conducted in this paper are of interest to the chemosensory field. The observation that Ih is important for the activity in bGRNs albeit expressed in sGRNs is especially fascinating and highlights the importance of non-synaptic interactions in the taste system.

Despite the interesting results, this paper is not written in a clear and easily understandable manner. It uses poorly defined terms without much elaboration, contains sentences that are borderline unreadable even for those in the narrower chemosensory field, and many figures can clearly benefit from more labeling and explanation. It certainly needs a bit of work.

Below are the major points:

(1) Throughout the paper, it is assumed that Ih channels are expressed in sugar-sensing GRNs but not bitter-sensing GRNs. However, both this paper and citation #17, another paper from the same lab, contain only circumstantial evidence for the expression of Ih channels in sGRNs. A simple co-expression analysis, using the Ih-T2A-GAL4 line and Gr5a-LexA/Gr66a-LexA line, all of which are available, could easily demonstrate the co-expression. Including such a figure would significantly strengthen the conclusion of this paper.

(2) Throughout this paper, it is often unclear which class of labellar taste sensilla is being recorded. S-a, S-b, I-a, and I-b sensilla all have different sensitivities to bitters and sugars. Each figure should clearly indicate which sensilla is being recorded. Justification should be provided if recordings from different classes of sensilla are being pooled together for statistics.

(3) In many figures, there is a lack of critical control experiments. Examples include Figures 1C-F (lacking UAS control), Figure 2I-J (lacking UAS control), Figure 4E (lacking the UAS and GAL4 control, and it is also strange to compare Gr64f > RNAi with Gr66a > RNAi, instead of with parental GAL4 and UAS controls.), and Figure 5D (lacking UAS control). Without these critical control experiments, it is difficult to evaluate the quality of the work.

(4) Figure 2A could benefit from more clarification about what exactly is being recorded here. The text is confusing: a considerable amount of text is spent on explaining the technical details of how SP is recorded, but very little text about what SP represents, which is critical for the readers. The authors should clarify in the text that SP is measuring the potential between the sensillar lymph, where the dendrites of GRNs are immersed, and the hemolymph. Adding a schematic figure to show that SP represents the potential between the sensillar lymph and hemolymph would be beneficial.

(5) The sGRN spiking rate in Figure 4B deviates significantly from previous literature (Wang, Carlson, eLife 2022; Jiao, Montell PNAS 2007, as examples), and the response to sucrose in the control flies is not dosage-dependent, which raises questions about the quality of the data. Why are the responses to sucrose not dosage-dependent? The responses are clearly not saturated at these (10 mM to 100 mM) concentrations.

(6) In Figure 4C, instead of showing the average spike rate of the first five seconds and the next 5 seconds, why not show a peristimulus time histogram? It would help the readers tremendously, and it would also show how quickly the spike rate adapts to overexpression and control flies. Also, since taste responses adapt rather quickly, a 500 ms or 1 s bin would be more appropriate than a 5-second bin.

(7) Lines 215 - 220. The authors state that the presence of sugars in the culture media would expose the GRNs to sugar constantly, without providing much evidence. What is the evidence that the GRNs are being activated constantly in flies raised with culture media containing sugars? The sensilla are not always in contact with the food.

(8) Line 223. To show that bGRN spike rates in Ih mutant flies "decreased even more than WT", you need to compare the difference in spike rates between the sorbitol group and the sorbitol + sucrose group, which is not what is currently shown.

(9) To help readers better understand the proposed mechanisms here, including a schematic figure would be helpful. This should show where Ih is expressed, how Ih in sGRNs impacts the sensillum potential, how elevated sensillum potential increases the electrical driving force for the receptor current, and affects the excitability of the bGRNs in the same sensilla, and how exposure to sugar is proposed to affect ion homeostasis in the sensillum lymph.

Reviewer #3 (Public Review):

Summary:

The present study proposes a neural circuit model consisting of coupled sensory and memory networks to explain the circuit mechanism of the cardinal effect in orientation perception which is characterized by the bias towards the oblique orientation and the largest variance at the oblique orientation.

Strengths:

The authors have done numerical simulations and preliminary analysis of the neural circuit model to show the model successfully reproduces the cardinal effect. And the paper is well-written overall. As far as I know, most of the studies on the cardinal effect are at the level of statistical models, and the current study provides one possibility of how neural circuit models reproduce such an effect.

Weaknesses:

There are no major weaknesses and flaws in the present study, although I suggest the author conduct further analysis to deepen our understanding of the circuit mechanism of the cardinal effects. Please find my recommendations for concrete comments.

Reviewer #3 (Public Review):

Summary:

This important study relies on a rare dataset: intracranial recordings within the thalamus and the subthalamic nucleus in awake humans, while they were performing a tactile detection task. This procedure allowed the authors to identify a small but significant proportion of individual neurons, in both structures, whose activity correlated with the task (e.g. their firing rate changed following the audio cue signalling the start of a trial) and/or with the stimulus presentation (change in firing rate around 200 ms following tactile stimulation) and/or with participant's reported subjective perception of the stimulus (difference between hits and misses around 200 ms following tactile stimulation). Whereas most studies interested in the neural underpinnings of conscious perception focus on cortical areas, these results suggest that subcortical structures might also play a role in conscious perception, notably tactile detection.

Strengths:

There are two strongly valuable aspects in this study that make the evidence convincing and even compelling. First, these types of data are exceptional, the authors could have access to subcortical recordings in awake and behaving humans during surgery. Additionally, the methods are solid. The behavioral study meets the best standards of the domain, with a careful calibration of the stimulation levels (staircase) to maintain them around the detection threshold, and an additional selection of time intervals where the behavior was stable. The authors also checked that stimulus intensity was the same on average for hits and misses within these selected periods, which warrants that the effects of detection that are observed here are not confounded by stimulus intensity. The neural data analysis is also very sound and well-conducted. The statistical approach complies with current best practices, although I found that, in some instances, it was not entirely clear which type of permutations had been performed, and I would advocate for more clarity in these instances. Globally the figures are nice, clear, and well presented. I appreciated the fact that the precise anatomical location of the neurons was directly shown in each figure.

Weaknesses:

Some clarification is needed for interpreting Figure 3, top rows: in my understanding the black curve is already the result of a subtraction between stimulus present trials and catch trials, to remove potential drifts; if so, it does not make sense to compare it with the firing rate recorded for catch trials.

I also think that the article could benefit from a more thorough presentation of the data and that this could help refine the interpretation which seems to be a bit incomplete in the current version. There are 8 stimulus-responsive neurons and 8 perception-selective neurons, with only one showing both effects, resulting in a total of 15 individual neurons being in either category or 13 neurons if we exclude those in which the behavior is not good enough for the hit versus miss analysis (Figure S4A). In my opinion, it should be feasible to show the data for all of them (either in a main figure, or at least in supplementary), but in the present version, we get to see the data for only 3 neurons for each analysis. This very small selection includes the only neuron that shows both effects (neuron #001; which is also cue selective), but this is not highlighted in the text. It would be interesting to see both the stimulus-response data and the hit versus miss data for all 13 neurons as it could help develop the interpretation of exactly how these neurons might be involved in stimulus processing and conscious perception. This should give rise to distinct interpretations for the three possible categories. Neurons that are stimulus-responsive but not perception-selective should show the same response for both hits and misses and hence carry out indifferently conscious and unconscious responses. The fact that some neurons show the opposite pattern is particularly intriguing and might give rise to a very specific interpretation: if the neuron really doesn't tend to respond to the stimulus when hits and misses are put together, it might be a neuron that does not directly respond to the stimulus, but whose spontaneous fluctuations across trials affect how the stimulus is perceived when they occur in a specific time window after the stimulus. Finally, neuron #001 responds with what looks like a real burst of evoked activity to stimulation and also shows a difference between hits and misses, but intriguingly, the response is strongest for misses. In the discussion, the interesting interpretation in terms of a specific gating of information by subcortical structures seems to apply well to this last example, but not necessarily to the other categories.

Reviewer #3 (Public Review):

Summary:

Using ex vivo electrophysiology and morphological analysis, Boi et al. investigate the electrophysiological and morphological properties of serotonergic and dopaminergic subpopulations in the dorsal raphe nucleus (DRN). They performed labor-intensive and rigorous electrophysiology with posthoc immunohistochemistry and neuronal reconstruction to delineate the two major cell classes in the DRN: DRN-DA and DRN-5HT, named according to their primary neurotransmitter machinery. They find that the dopaminergic (DRN-DA) and serotonergic (DRN-5HT) neurons are electrophysiologically and morphologically distinct, and are altered following striatal injection of the toxin 6-OHDA. However, these alterations were largely prevented in DRN-5HT neurons by pre-treatment with desipramine. These findings suggest an important interplay between catecholaminergic systems in healthy and parkinsonian conditions, as well as a relationship between neuronal structure and function.

Strengths:

Large, well-validated dataset that will be a resource for others.<br /> Complementary electrophysiological and anatomical characterizations.<br /> Conclusions are justified by the data.<br /> Relevant for basic scientists interested in DRN cell types and physiology<br /> Relevant for those interested in serotonin and/or DRN neurons in Parkinson's Disease

Weaknesses:

Given the scope of the author's questions and hypotheses, I did not identify any major weaknesses.

Reviewer #3 (Public Review):

Summary:

The authors have examined the 5-HT3 receptor using voltage clamp fluorometry, which enables them to detect structural changes at the same time as the state of receptor activation. These are ensemble measurements, but they enable an impressive scheme of the action of different agonists and antagonists to be built up. The growing array of structural snapshots of 5-HT3 receptors is used to good effect to understand the results.

Strengths:

The combination of rigorously tested fluorescence reporters with oocyte electrophysiology across a large panel of ligands is a solid development for this receptor type.

Weaknesses:

In their revision, the authors corrected all the weaknesses of the original submission.

Reviewer #3 (Public Review):

Summary:

Chang et al. investigated the mechanisms governing collagen fibrillogenesis, firstly demonstrating that cells within tail tendons are able to uptake exogenous collagen and use this to synthesize new collagen-1 fibrils. Using an endocytic inhibitor, the authors next showed that endocytosis was required for collagen fibrillogenesis and that this process occurs in a circadian rhythmic manner. Using knockdown and overexpression assays, it was then demonstrated that collagen fibril formation is controlled by vacuolar protein sorting 33b (VPS33b), and this VPS33b-dependent fibrillogenesis is mediated via Integrin alpha-11 (ITGA11). Finally, the authors demonstrated increased expression of VPS33b and ITGA11 at the gene level in fibroblasts from patients with idiopathic pulmonary fibrosis (IPF), and greater expression of these proteins in both lung samples from IPF patients and in chronic skin wounds, indicating that endocytic recycling is disrupted in fibrotic diseases.

Strengths:

The authors have performed a comprehensive functional analysis of the regulators of endocytic recycling of collagen, providing compelling evidence that VPS33b and ITGA11 are crucial regulators of this process.

Weaknesses:

Throughout the study, several different cell types have been used (immortalised tail tendon fibroblasts, NIHT3T cells, and HEK293T cells). In general, it is not clear which cells have been used for a particular experiment, and the rationale for using these different cell types is not explained. In addition, some experimental details are missing from the methods.

There is also a lack of functional studies in patient-derived IPF fibroblasts which means the link between endocytic recycling of collagen and the role of VPS33b and ITGA11 cannot be fully established.

Reviewer #4 (Public Review):

Summary:

Masala N et al showed interesting aberrant calcium microwaves in the hippocampus when synapsin promoter driven GCaMPs were expressed for a long period of time. These aberrant hippocampal Ca2+ micro-waves depend on the viral titre of the GECI. The microwave of Ca2+ was not observed when GECI was expressed only a sparse set of neurons.

Strengths:

These findings are important to wide neuroscience community especially when considering a great number of investigators are using similar approaches. Results look convincing and are consistent across several laboratories.

Weaknesses:

Synapsin promoter labels both excitatory pyramidal neurons and inhibitory neurons. To avoid aberrant Ca2+ microwave, a combination of Flex virus and CaMKII-Cre or Thy-1-GCaMP6s and 6f mice were tested. However, all these approaches limit the number of infected pyramidal neurons. While the comprehensive display of these results is appreciated, one additional important test would be more informative. To distinguish whether the microwave of Ca2+ is sufficiently caused via the expression of GCaMP in interneurons, or just a matter of pyramidal neuron density, testing Flex-GCaMP6 in interneuron specific mouse lines such as PV-Cre and SOM-Cre will provide further clarifications.

Reviewer #3 (Public Review):

Summary:

This paper aims to demonstrate the role of G-quadruplex DNA structures in the establishment of chromosome loops. The authors introduced an array of G4s spanning 275 bp, naturally found within a very well-characterized promoter region of the hTERT promoter, in an ectopic region devoid of G-quadruplex and annotated gene. As a negative control, they used a mutant version of the same sequence in which G4 folding is impaired. Due to the complexity of the region, 3 G4s on the same strand and one on the opposite strand, 12 point mutations were made simultaneously (G to T and C to A). Analysis of the 3D genome organization shows that the WT array establishes more contact within the TAD and throughout the genome than the control array. Additionally, a slight enrichment of H3K4me1 and p300, both enhancer markers, was observed locally near the insertion site. The authors tested whether the expression of genes located either nearby or up to 5 Mb away was up-regulated based on this observation. They found that four genes were up-regulated from 1.5 to 3-fold. An increased interaction between the G4 array compared to the mutant was confirmed by the 3C assay. For in-depth analysis of the long-range changes, they also performed Hi-C experiments and showed a genome-wide increase in interactions of the WT array versus the mutated form.

Strengths:

The experiments were well-executed and the results indicate a statistical difference between the G4 array inserted cell line and the mutated modified cell line.

Weaknesses:

The control non-G4 sequence contains 12 point mutations, making it difficult to draw clear conclusions. These mutations not only alter the formation of G4, but also affect at least three Sp1 binding sites that have been shown to be essential for the function of the hTERT promoter, from which the sequence is derived. The strong intermingling of G4 and Sp1 binding sites makes it impossible to determine whether all the observations made are dependent on G4 or Sp1 binding. As a control, the authors used Locked Nucleic Acid probes to prevent the formation of G4. As for mutations, these probes also interfere with two Sp1 binding sites. Therefore, using this alternative method has the same drawback as point mutations. This major issue should be discussed in the paper. It is also possible that other unidentified transcription factor binding sites are affected in the presented point mutants.

Reviewer #3 (Public Review):

Summary:

The authors study the function of HCN channels in L2/3 pyramidal neurons, employing somatic whole-cell recordings in acute slices of visual cortex in adult mice and a bevy of technically challenging techniques. Their primary claim is a non-uniform HCN distribution across the dendritic arbor with a greater density closer to the soma (roughly opposite of the gradient found in L5 PT-type neurons). The second major claim is that multiple sources of long-range excitatory input (cortical and thalamic) are differentially affected by the HCN distribution. They further describe an interesting interplay of NMDAR and HCN, serotonergic modulation of HCN, and compare HCN-related properties at 1, 2 and 6 weeks of age. Several results are supported by biophysical simulations.

Strengths:

The authors collected data from both male and female mice, at an age (6-10 weeks) that permits comparison with in vivo studies, in sufficient numbers for each condition, and they collected a good number of data points for almost all figure panels. This is all the more positive, considering the demanding nature of multi-electrode recording configurations and pipette-perfusion. The main strength of the study is the question and focus.

Weaknesses:

Unfortunately, in its present form, the main claims are not adequately supported by the experimental evidence: primarily because the evidence is indirect and circumstantial, but also because multiple unusual experimental choices (along with poor presentation of results) undermine the reader's confidence. Additionally, the authors overstate the novelty of certain results and fail to cite important related publications. Some of these weaknesses can be addressed by improved analysis and statistics, resolving inconsistent data across figures, reorganizing/improving figure panels, more complete methods, improved citations, and proofreading. In particular, given the emphasis on EPSPs, the primary data (for example EPSPs, overlaid conditions) should be shown much more.

However, on the experimental side, addressing the reviewer's concerns would require a very substantial additional effort: direct measurement of HCN density at different points in the dendritic arbor and soma; the internal solution chosen here (K-gluconate) is reported to inhibit HCN; bath-applied cesium at the concentrations used blocks multiple potassium channels, i.e. is not selective for HCN (the fact that the more selective blocker ZD7288 was used in a subset of experiments makes the choice of Cs+ as the primary blocker all the more curious); pathway-specific synaptic stimulation, for example via optogenetic activation of specific long-range inputs, to complement / support / verify the layer-specific electrical stimulation.

Reviewer #3 (Public Review):

Summary:

Authors suggest a new biomarker of chronic back pain with the option to predict the result of treatment. The authors found a significant difference in a fractional anisotropy measure in superior longitudinal fasciculus for recovered patients with chronic back pain.

Strengths:<br /> The results were reproduced in three different groups at different studies/sites.

Weaknesses:<br /> - The number of participants is still low.<br /> - An explanation of microstructure changes was not given.<br /> - Some technical drawbacks are presented.

Reviewer #3 (Public Review):

Summary:

The authors aimed to investigate the effectiveness of streptavidin imaging as an alternative to traditional antibody labeling for visualizing proteins within cellular contexts. They sought to address challenges associated with antibody accessibility and inconsistent localization by comparing the performance of streptavidin imaging with a TurboID-HA tandem tag across various protein localization scenarios, including phase-separated regions. They aimed to assess the reliability, signal enhancement, and potential advantages of streptavidin imaging over antibody labeling techniques.

Overall, the study provides a convincing argument for the utility of streptavidin imaging in cellular protein visualization. By demonstrating the effectiveness of streptavidin imaging as an alternative to antibody labeling, the study offers a promising solution to issues of accessibility and localization variability. Furthermore, while streptavidin imaging shows significant advantages in signal enhancement and preservation of protein interactions, the authors must consider potential limitations and variations in its application. Factors such as the fact that tagging may sometimes impact protein function, background noise, non-specific binding, and the potential for off-target effects may impact the reliability and interpretation of results. Thus, careful validation and optimization of streptavidin imaging protocols are crucial to ensure reproducibility and accuracy across different experimental setups.

Strengths:

- Streptavidin imaging utilizes multiple biotinylation sites on both the target protein and adjacent proteins, resulting in a substantial signal boost. This enhancement is particularly beneficial for several applications with diluted antigens, such as expansion microscopy or correlative light and electron microscopy.

- This biotinylation process enables the identification and characterization of interacting proteins, allowing for a comprehensive understanding of protein-protein interactions within cellular contexts.

Weaknesses:

- One of the key advantages of antibodies is that they label native, endogenous proteins, i.e. without introducing any genetic modifications or exogenously expressed proteins. This is a major difference from the approach in this manuscript, and it is surprising that this limitation is not really mentioned, let alone expanded upon, anywhere in the manuscript. Tagging proteins often impacts their function (if not their localization), and this is also not discussed.

- Given that BioID proximity labeling encompasses not only the protein of interest but also its entire interacting partner history, ensuring accurate localization of the protein of interest poses a challenge.

- The title of the publication suggests that this imaging technique is widely applicable. However, the authors did not show the ability to track the localization of several distinct proteins on the same sample, which could be an additional factor demonstrating the outperformance of streptavidin imaging compared with antibody labeling. Similarly, the work focuses only on small 2D samples. It would have been interesting to be able to compare this with 3D samples (e.g. cells encapsulated in an extracellular matrix) or to tissues.

Reviewer #3 (Public Review):

Summary:

Cellulose is a major component of the primary cell wall of growing cells and it is made by cellulose synthases (CESAs) organized into multi-subunit complexes in the plasma membrane. Previous results have resolved the structure of secondary cell wall CESAs, which are only active in a subset of cells. Here, the authors evaluate the structure of CESAs from soybeans (Glycine max, Gm) via cryo-EM and compare these structures to secondary cell wall CESAs. First, they expressed GmCESA1, GmCESA3, or GmCESA6 in insect cells, purified these proteins as both monomers and homotrimers and demonstrated their capacity to incorporate 3H-labelled glucose into the cellulase-sensitive product in a pH and divalent cation (e.g., Mg2+) -dependant fashion (Figure 1). Although CESA1, CESA3, and a CESA6-like isoform are essential for cellulose synthesis in Arabidopsis, in this study, monomers and homotrimers both showed catalytic activity, and there was more variation between individual isoforms than between their oligomerization states (i.e., CESA3 monomers and trimers showed similar activities, which were substantially different from CESA1 monomers or trimers).

They next use cryo-EM to solve the structure of each homotrimer to ~3.0 to 3.3 A (Figure 2). They compare this with PttCESA8 and find important similarities, such as the unidentified density at a positively-charged region near Arg449, Lys452, and Arg453, and differences, such as the position and relatively low resolution (suggesting higher flexibility) of TM7, which presumably creates a large lateral lipid-exposed channel opening, rather than the transmembrane pore in PttCESA8. Like PttCESA8, an oligosaccharide in the translocation channel was co-resolved with the protein structure. Neither the N-terminal domains nor the CSRs (a plant-specific insert into the cytosolic loop between TM2 and TM3) are resolved well.

Several previous models have proposed that the cellulose synthase complexes may be composed of multiple heterotrimers, but since the authors were able to isolate beta-glucan-synthesizing homotrimers, their results challenge this model. Using the purified trimers, the authors investigated how the CESA homotrimers might assemble into higher-order complexes. They detected interactions between each pair of CESA homotrimers via pull-down assays (Figure 3), although these same interactions were also detected among monomers (Supplemental Figure 4). Neither catalytic activity nor these inter-homotrimer interactions required the N-terminal domain (Figure 4). When populations of homotrimers were mixed, they formed larger aggregations in vitro (Figure 4) and displayed increased activity, compared to the predicted additive activity of each enzyme alone (Figure 5). Intriguingly, this synergistic behavior is observed even when one trimer is chemically inactivated before mixing (Supplemental Figure 6), suggesting that the synergistic effects are due to structural interactions.

Strengths:

The main strength of this manuscript is its detailed characterization of the structure of multiple CESAs, which complements previous studies of secondary cell wall CESAs. They provide a comprehensive comparison of these new structures with previously resolved CESA structures and discuss several intriguing similarities and differences. The synergistic activity observed when different homotrimers are mixed is a particularly interesting result. These results provide fundamental in vitro support for a cellulose synthase complex comprised of a hexamer of CESA homotrimers.

Weaknesses:

There are several weaknesses in the manuscript. The authors do not present any data to indicate that GmCESA1, GmCESA3, and GmCESA6 are primary cell wall CESAs (e.g. expression patterns, phylogenetic evidence). Furthermore, their evidence that these proteins make cellulose in vitro is limited to the beta-glucanase-sensitive digestion of the product. Previous reports characterizing CESA structures have used multiple independent methods: sensitivity and resistance of the product to various enzymes, linkage analysis, and importantly, TEM of the product to ensure that it makes genuine cellulose microfibrils, rather than amorphous beta-glucan. Without demonstrating that GmCESA1, GmCESA3, and GmCESA6 are genuinely synthesizing cellulose microfibrils (via TEM) and that they are primary cell wall CESAs (via expression patterns & phylogenetic evidence), it is difficult to place the results into context. Finally, the authors indicate that they were unable to isolate heterotrimers in vitro, but they do not present any evidence of these experiments, which is essential to evaluate their conclusion that these CESAs operate as homotrimers in vitro.

Reviewer #3 (Public Review):

The authors have investigated the effect of noncaloric monosaccharides on angiogenesis in the zebrafish embryo. These compounds are used as substitutes of sugars to sweeten beverages and they are commonly used by diabetic patients. The authors show that noncaloric monosaccharides and glucose similarly induce excessive blood vessel formation due to the increased formation of tip cells by endothelial cells. The authors show that this excessive angiogenesis involved the foxo1a-marcksl1a pathway.

A limitation of the study is that the mechanism of angiogenesis in the retinal circulation and in peripheral vasculature is certainly different.

This result suggests that these noncaloric monosaccharides share common side effects with glucose. Consequently, more caution should be taken with regard to the use of these artificial sweeteners. This work is of interest for better management of diabetes.

Reviewer #3 (Public Review):

This article is the first report to study the effects of T. pallidum on the neural development of an iSPC-derived brain organoid model. The study indicates that T. pallidum inhibits the differentiation of subNPC1B neurons into hindbrain neurons, hence affecting brain organoid neurodevelopment. Additionally, the TCF3 and notch signaling pathways may be involved in the inhibition of the subNPC1B-hindbrain neuron differentiation axis. While the majority of the data in this study support the conclusions, there are still some questions that need to be addressed and data quality needs to be improved. The study provides valuable insights for future investigations into the mechanisms underlying congenital neurodevelopment disability.

Reviewer #3 (Public Review):

Summary:

This study presents a solid framework for the metabolic modeling of microbial species and resources in the rhizosphere environment. It is an ambitious effort to tackle the huge complexity of the rhizosphere and reveal the plant-microbiota interactions therein. Considering previously published data by Berihu et al., going through a series of steps, the framework then finds associations between an apple tree disease state and both microbes and metabolites. The framework is well explained and motivated. I think that further work should be done to validate the method, both using synthetic data, with a known ground truth and following up on key findings experimentally.

Strengths:

- The manuscript is well written with a good balance between detail and readability. The framework steps are well-motivated and explained.

- The authors faithfully acknowledge the limitations of their approach and do not try to "over-sell" their conclusions.

- The presented framework has the potential for significant discovery if the hypotheses generated are followed up with experimental validation.

Weaknesses:

- When presenting a computational framework, best practices include running it on artificial (synthetic) data where the ground truth is known and therefore the precision and accuracy of the method may be assessed. This is not an optional step, the same way that positive/negative controls in lab experiments are not optional. Without this validation step, the manuscript is severely limited. The authors should ask themselves: what have we done to convince the reader that the framework actually works, at least on our minimal synthetic data?

Justification of claims and conclusions:

The claims and conclusions are sufficiently well justified since the limitations of this approach are acknowledged by the authors.

Reviewer #3 (Public Review):

Summary:

Authors performed a genome-wide CRISPR-based screen for synthetic lethal interactions in leukemic cells expressing a mutant form of PPM1D and identified SOD1. Loss of SOD1 or its inhibition with small molecule compounds reduced survival of the cells containing truncated PPM1D. Further analysis revealed that mitochondria are functionally deficient in PPM1D mutant cells resulting in increased levels of ROS. Surprisingly, expression profiling and reverse phase protein arrays revealed that PPM1D mutant cells did not respond appropriately to the increased levels of ROS. The precise molecular mechanism underlying this phenotype remains currently unclear, nevertheless the study convincingly shows that PPM1D mutant cells are vulnerable to oxidative stress.

Strengths:

Experimental procedures used in the study are appropriate and overall the presented data are very convincing. The study identified an important vulnerability of leukemic cells that carry PPM1D mutation and provides a fundamental background for testing SOD1 inhibitors in preclinical research. In the revised version of the manuscript, authors provide several new experiments that support their former conclusions. In particular, they showed that deletion of SOD1 in AML cells improved survival of the transplanted mice and this effect was more prominent when using cells carrying the mutant PPM1D. Further, they included an important control experiment that showed decreased SOD1 activity after treatment with ATN-224 inhibitor.

Weaknesses:

In the opinion of reviewer, there are no obvious weaknesses in this study. In broader view, the findings presented here using in vitro cultures will need to be validated in vivo by future research. Cell lines used in the study were generated by CRSIPR approaches in AML cells that have already been transformed. In addition, genome editing is inheritably connected with a risk of off target effects. It would therefore be great to identify AML samples carrying the PPM1D mutation that has been naturally selected during the transformation process.

Reviewer #3 (Public Review):

The authors comprehensively demonstrated the Cbfβ gene, which is involved in articular cartilage homeostasis, can promote articular cartilage regeneration and repair in osteoarthritis (OA) through regulating Hippo/YAP signaling TGF-β signaling, and canonical Wnt signaling. First, the authors demonstrated the deletion of Cbfβ can induce the OA phenotypes including decreased articular cartilage and osteoblasts, and increased osteoclasts and subchondral bone hyperplasia, and induce the early onset of OA. Additionally, the authors showed that the deficiency of Cbfβ in cartilage can increase canonical Wnt signaling and decrease TGF-β and Hippo signaling. Finally, the authors demonstrated that the overexpression of Cbfβ can inhibit Wnt signaling and enhance Hippo/YAP signaling in knee joints articular cartilage of ACLT-induced OA mice and protect against ACLT-induced OA. The manuscript is overall well-constructed, and the authors provided evidence to support their findings.

In Fig. 7I, it could be better to show the statistical analysis between normal and AAV-mediated Cbfβ ACLT mice groups.

In Fig. 9H-K, in the quantification analysis, the OARSI score in the DMM+AAV-YFP group is higher than in the sham group significantly. However, the SO staining results appear to show no significant difference between the DMM+AAV-luc-YFP group (Fig. 9I) and the sham group (Fig. 9H).

Reviewer #3 (Public Review):

The authors first characterize their mouse model of pancreatic cancer and show that CYRI-B mRNA is detectable in pancreatic lesions and that its amount increases over time. They also show that genetic deletion of CYRI-B accelerates pancreatic ductal adenocarcinoma (PDAC), leading to lower survival of mice. This is accompanied by higher levels of phospho-(i.e. activated)-JNK and -ERK, which are likely two of the factors driving cancer cell proliferation. Using in vivo transplantation, the authors further demonstrate that cancer cells depleted for CYRI-B exhibit decreased numbers of metastases in the mesentery, despite showing similar proliferation as control cells.<br /> Cancer cell migration can be driven by LPA, which binds LPAR1 at the surface of PDAC cells. Investigation of chemotactic migration of cancer cells towards fetal bovine serum as a source of LPA further shows that cancer cells depleted for CYRI-B and expressing GFP as control exhibit strongly reduced chemotactic migration, while cells re-expressing CYRI-B-GFP show normal chemotactic migration. Furthermore, this restored migration is blocked by using the LPAR1/3 inhibitor K116425, showing that CYRI-B is required for the chemotactic migration of PDAC cells in a gradient of serum LPA.

Using live cell imaging, the authors show that CYRI-B-GFP and LPAR1-mCherry localize to macropinocytic cups and to macropinosomes, indicating that LPAR1 can be internalized by PDAC cells through macropinocytosis. This notion is supported by immunofluorescence analyses showing that PDAC cells depleted for CYRI-B have reduced LPAR1-mCherry internalization upon stimulation with LPA, compared to cells rescued by CYRI-B-GFP expression. Collectively, the authors suggest that CYRI-B regulates macropinocytic uptake of LPAR1, thus regulating the chemotactic migration of PDAC cells towards LPA, which supports the metastasis of pancreatic cancer.

This is an interesting manuscript that makes a convincing case for the involvement of CYRI-B as a driver of PDAC. A particular strength is the expert use of different mouse models and derived cancer cell lines. The major conclusions are supported by the data presented. The results could be further strengthened by detecting CYRI-B protein (in addition to mRNA) in cancer lesions and also by staining endogenous CYRI-B and LPAR1 in the macropinocytosis experiments.

Reviewer #3 (Public Review):

The submitted paper by Hoving et al addresses the role of N-cadherin in Schwann cell collective cell migration and its previously unknown relationship with the slit/robo signaling pathway. The main conclusion is that N-cadherin has two distinct functions. One that is dependent on its classical role as a cell-cell junction protein promoting cell clustering and one that promotes cell repulsion and polarity independently of the formation of cell adhesion complexes. The second function is mediated by the Slit/Robo pathway. It is proposed that N-cadherin and Glypican-4 act together to present Slit2/3 at the surface of Schwann cells in order to trigger Robo signaling on neighboring cells.

The data about N-cadherin loss of function and the associated rescue experiments with the various truncated forms of N-cadherin are well substantiated by proper controls for efficiency and specificity. They show that the extracellular domain of Ncadherin is the one required for the repulsive effect. The experiments performed to distinguish the roles in adhesion and repulsion seem clear and conclusive. In addition, the fact the slit signal needs to be provided in a polarized manner for directional migration to occur is also clearly demonstrated in vitro and on slice assays. Overall the model that Ncadherin plays two different roles, a repulsive one via presentation of slit at the cell surface and a cell adhesion one via formation of adherens junctions, is well supported by the data and will be of interest beyond the subfield of the authors.

However, other parts of the manuscript seem weaker. If N-cadherin presentation of the Slit signal is so critical why are repulsion rates still very high in cells without N-cadherin? Same is observed with Glypican4 knockdowns. In both loss of function 50% of cell collisions lead to repulsion (compared to 70% amongst control cells). While significant such drop remains modest. The authors propose a cooperative role of Glypican-4 and N-cadherin at the cell surface as co-binding factors for Slit2/3 but they have not checked whether double knockdown of N-cad and Glypican4 might have a stronger effect. Could Glypican and N-cadherin present Slit at the cell surface independently in a somewhat redundant manner? Can Glypican and Slit interact physically in absence of N-cadherin? They also have not further analyzed the putative colocalization fo Ncad and Glypican at the cell surface.

The data supporting a role for N-cadherin in Slit's trafficking to the cell surface seem also circumstantial. While western blot data seem to indicate no change in Slit protein level after N-cad knockdown, immunostaining for Slit in such condition show a dramatic loss of Slit signal. These two independent data sets are difficult to reconcile and are not designed to address whether Slit reaches the cell surface in control or N-cadherin knockdown conditions.

If Slit signaling is so critical for repulsion why in double sit2/3 knockdown 40% of collisions still lead to repulsion. Also, no analysis of cell collision are provided upon Robo1/2 knockdown for comparison with Slit knockdowns. Altogether, these relatively mild effects of n-cad, slit or glypican knockdown on repulsion seem to indicate that other signals might contribute to contact-inhibition and polarization/repulsion of cells upon physical contact but this is unfortunately not discussed. All statements related to cell polarity stem from the overall cell morphology without being substantiated by actual polarity analysis (using markers such as detection of Rac-GTP or using a proxy such as the golgi-nucleus axis). The authors present the cell cluster generated after Sox2 expression and Sox2 + exposure to recombinant Slit2 as lacking polarity, however in one case cells do not present any flat membrane at their free edge whereas in the other case they do. This suggests a minimal cell polarity with a protrusive-like organization away from the contact. Finally, Robo1/2 siRNA knockdown are used but contrary to the other loss of functions it seems that controls for knockdown efficiency/specificity were not provided.

Therefore, while the study is overall well documented and based on solid data, some weaknesses exist.

The overall topic is clearly of broad interest as N-cadherin is protein essential in various biological settings from development to disease but the range of its biological functions remains to be fully explored. This study clearly adds to the current knowledge and how N-cadherin might act in vivo and in particular how it could mediate crosstalks between various signaling pathways.

Reviewer #3 (Public Review):

B cell receptors are produced through a combination of random V(D)J recombination and somatic hypermutation. Identifying clonal lineages - cells that descend from a common V(D)J rearrangement - is an important part of B cell repertoire analysis. Here, the authors developed a new method to identify clonal lineages from BCR data. This method builds off of prior advances in the field and uses both an adaptive clonal distance threshold and shared somatic hypermutation information to group B cells into clonal lineages.

The major strength of this paper is its thorough quantitative treatment of the subject and integration of multiple improvements into the clonal clustering process. By their simulation results, the method is both highly efficient and accurate.

The only notable weakness we identified is that much of the impact of the method will depend on its superiority to existing approaches, and this is not convincingly demonstrated by Fig. 4. In particular, little detail is given on how the other clonal clustering programs were run, and this can significantly impact their performance. More specifically:

(1) Scoper supports multiple methods for clonal clustering, including both adaptive CDR3 distance thresholds (Nouri and Kleinstein, 2018) and shared V-gene mutations (Nouri and Kleinstein, 2020). It is not clear which method was used for benchmarking. The specific functions and settings used should have been detailed and justified. Spectral clustering with shared V gene mutations would be the most comparable to the authors' method. Similar detail is needed for partis.<br /> (2) It is not clear how the adaptive thresholds and shared mutation analysis in the authors' method differ from prior approaches such as scoper and partis.<br /> (3) The scripts for performing benchmarking analyses, as well as the version numbers of programs tested, are not available.<br /> (4) Similar to above, P. 10 describes single linkage hierarchical clustering with a fixed threshold as a "crude method" that "suffers from inaccuracy as it loses precision in the case of highly-mutated sequences and junctions of short length." As far as we could tell, this statement is not backed up by either citations or analyses in the paper. It should not be difficult for the authors to test this though using their simulations, as this method is also implemented in scoper.

References<br /> Nouri N, Kleinstein SH. 2020. Somatic hypermutation analysis for improved identification of B cell clonal families from next-generation sequencing data. PLOS Comput Biol 16:e1007977. doi:10.1371/journal.pcbi.1007977<br /> Nouri N, Kleinstein SH. 2018. A spectral clustering-based method for identifying clones from high-throughput B cell repertoire sequencing data. Bioinformatics 34:i341-i349. doi:10.1093/bioinformatics/bty235

Reviewer #3 (Public Review):

Mohseni and Elhaik challenge the widespread use of PCA as an analytical and interpretive tool in the study of geometric morphometrics. The standard approach in geometric morphometrics analysis involves Generalised Procrustes Analysis (GPA) followed by Principal Component Analysis (PCA). Recent research challenges PCA outcomes' accuracy, robustness, and reproducibility in morphometrics analysis. In this paper, the authors demonstrate that PCA is unreliable for such studies. Additionally, they test and compare several Machine-Learning methods and present MORPHIX, a Python package of their making that incorporates the tools necessary to perform morphometrics analysis using ML methods.

Mohseni and Elhaik conducted a set of thorough investigations to test PCA's accuracy, robustness, and reproducibility following renewed recent criticism and publications where this method was abused. Using a set of 2 and 3D morphometric benchmark data, the authors performed a traditional analysis using GPA and PCA, followed by a reanalysis of the data using alternative classifiers and rigorous testing of the different outcomes.

In the current paper, the authors evaluated eight ML methods and compared their classification accuracy to traditional PCA. Additionally, common occurrences in the attempted morphological classification of specimens, such as non-representative partial sampling, missing specimens, and missing landmarks, were simulated, and the performance of PCA vs ML methods was evaluated.

The main problem with this manuscript is that it is three papers rolled into one, and the link doesn't work. The title promises a new Python package, but the actual text of the manuscript spends relatively little time on the Python package itself and barely gives any information about the package and what it includes or its usefulness. It is definitely not the focus of the manuscript. The main thrust of the manuscript, which takes up most of the text, is the analysis of the papionin dataset, which shows very convincingly that PCA underperforms in virtually all conditions tested. In addition, the manuscript includes a rather vicious attack against two specific cases of misuse of PCA in paleoanthropological studies, which does not connect with the rest of the manuscript at all.

If the manuscript is a criticism of PCA techniques, this should be reflected in the title. If it is a report of a new Python package, it should focus on the package. Otherwise, there should be two separate manuscripts here.

The criticism of PCA is valid and important. However, pointing out that it is problematic in specific cases and is sometimes misused does not justify labeling tens of thousands of papers as questionable and does not justify vilifying an entire discipline. The authors do not make a convincing enough case that their criticism of the use of PCA in analyzing primate or hominin skulls is relevant to all its myriad uses in morphometrics. The criticism is largely based on statistical power, but it is framed as though it is a criticism of geometric morphometrics in general.

Reviewer #3 (Public Review):

Summary:

Drug resistance is a perennial problem for malaria control and strategies to prevent the acquisition and spread of drug resistance mutations are desperately needed. One strategy is to identify drug resistance mutations that arise in blood-stage parasites, but cannot be readily spread to a new human host. Since malaria parasites must survive and replicate in mosquitoes in order to be transmitted, mutations with elevated mosquito-stage fitness defects will not spread efficiently. Buchanan and coworkers focus on the drug azithromycin and its known role of inhibiting the ribosomes found in parasite apicoplast organelles. Apicoplast organelles are known to have elevated metabolic activity in mosquito stage parasites and azithromycin resistance mutations could interfere with mosquito stage parasite development and parasite transmission.

To address this hypothesis, azithromycin-resistant P. berghei and P. falciparum parasites were generated and analyzed for transmission defects. All lines had mutations in the apicoplast ribosomal protein Rpl4 consistent with the known role of azithromycin inhibiting the 50S ribosomal subunit. Overall, the three lines (3 berghei and one falciparum) had phenotypes that should limit parasite transmission, however, detailed characterization showed that there were surprising differences between the two parasite species and even between the P. berghei lines. The P. berghei lines produced fewer oocysts and sporozoites with aberrant apicoplast morphology compared to wild-type controls. Sporozoites from azithromycin-resistant lines appeared to have motility defects and typically were not able to infect mice (one strain produced infections when 10,000 sporozoites were injected, but not when 1,000 were).

By contrast, the azithromycin-resistant P. falciparum strain did not display any mosquito-stage phenotypes and produced motile sporozoites with intact apicoplast organelles. These sporozoites, however, developed abnormally in a humanized mouse model with reduced liver-stage nuclear division and abnormal apicoplast morphology. These defects combined with a five-fold lower prevalence suggest that azithromycin-resistant P. falciparum parasites experience significant fitness costs during liver stage development (at least those harboring the G76V mutation).

Strengths:

This work was carefully conducted and transparently presented. It provides a comprehensive view of how parasite development is impacted by azithromycin resistance mutations during the mosquito and liver stages in P. berghei and P. falciparum. It adds a new dimension to the growing literature on the transmissibility of drug-resistant parasites, by showing that mutations in the apicoplast genome can impact transmission.

Weaknesses:

Whether these liver-stage defects in P. falciparum are severe enough to completely block subsequent blood-stage infection remains to be seen and would require experiments with humanized mice continuously grafted with human red blood cells - a difficult and expensive model system.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors describe a novel mode of release of small extracellular vesicles. These small EVs are released via the rupture of the membrane of so-called amphiectosomes that resemble "morphologically" Multivesicular Bodies.

These structures have been initially described by the authors as released by colorectal cancer cells (https://doi.org/10.1080/20013078.2019.1596668). In this manuscript, they provide experiments that allow us to generalize this process to other cells. In brief, amphiectosomes are likely released by ectocytosis of amphisomes that are formed by the fusion of multivesicular endosomes with autophagosomes. The authors propose that their model puts forward the hypothesis that LC3 positive vesicles are formed by "curling" of the autophagosomal membrane which then gives rise to an organelle where both CD63 and LC3 positive small EVs co-exist and would be released then by a budding mechanism at the cell surface that appears similar to the budding of microvesicles /ectosomes. Very correctly the authors make the distinction from migrasomes because these structures appear very similar in morphology.

Strengths:

The findings are interesting despite that it is unclear what would be the functional relevance of such a process and even how it could be induced. It points to a novel mode of release of extracellular vesicles.

Weaknesses:

This reviewer has comments and concerns concerning the interpretation of the data and the proposed model. In addition, in my opinion, some of the results in particular micrographs and immunoblots (even shown as supplementary data) are not of quality to support the conclusions.

Reviewer #3 (Public Review):

Summary:

White et al. described laser-induced wound healing of the Drosophila pupal notum. They found that the epithelial monolayer is dynamically induced to form syncytia by cell-cell fusion as an important part of repair. They reveal two processes: cell shrinking and border breakage that occur as part of syncytia formation. Expression of GFP in the cytoplasms of some epithelial cells reveals that cytoplasmic contents mix following injury and the GFP rapidly diffuses between cells. Using live imaging they observe that syncytia expand towards the wound, maintain their positions close to the leading edge, and apparently displace smaller cells. They propose that syncytia redistribute cellular components towards the wound facilitating repair and show that labelled actin becomes concentrated at the leading edge.

Strengths:

The manuscript is interesting and on an important and emerging topic of wound healing in a genetically tractable organism. The manuscript is very well written.

Weaknesses:

There are three major issues that the authors must address:

(1) Is cell-cell fusion sufficient to enhance/facilitate wound healing?

(2) Characterization of "border breakdown"; Is this phenomenon disassembly of apical junctions following membrane fusion?

(3) Are cells really shrinking or is it only the apical domains that "shrink" as the cells join the syncytium?

Reviewer #3 (Public Review):

Summary:

In this manuscript, Weng et al. detect a neuron-specific transcriptome that regulates aging. The authors first profile neuron-specific responses during aging at a time point where a loss in memory function is present. They discover signatures unique to neurons which validate their pipeline and reveal the loss of neuron identity with age. For example, old neurons reduce the expression of genes related to synaptic function and neuropeptide signaling and increase the expression of chromatin regulators, insulin peptides, and glycoproteins. The authors discover the detrimental effect of selected upregulated genes (utx-1, ins-19, and nmgp-1) by knocking them down in the whole body and detecting improvement of short memory functions. They then use their pipeline to test neuronal profiles of long-lived insulin/IGF mutants. They discover that genes related to stress response pathways are upregulated upon longevity (e.g. dod-24, F08H9.4) and that they are required for improved neuron function in long-lived individuals.

Strengths:

Overall, 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. Using neuron-specific transcriptomic analysis in aged animals the authors discover novel regulators of learning and memory, which underlines the importance of cell-specific deep sequencing. The time points 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 showcase on the dod-24 example how powerful this approach is. In long-lived insulin/IGF-1 receptor mutants body-wide dod-24 expression differs from neuron-specific profiles. Importantly, the depletion of dod-24 has an opposing effect on lifespan and learning memory. The dataset will provide a useful resource for the C. elegans and aging community.

Weaknesses:

While this study nicely describes the neuron-specific profiles, the authors do not test the relevance in a tissue-specific way. It remains unclear if modifying the responses only in neurons has implications for either memory or potentially for lifespan. The authors point to this in the text and refer to tissue-specific datasets. However, it is possible that the tissue-specific profile changes with age. The authors should consider mining publicly available cell-specific aging datasets and performing neuron-specific RNAi to test the functional relevance of the neuron-specific response. This would strengthen the importance of cell-specific profiling.

Reviewer #3 (Public Review):

Summary:

The authors have created DHCR24 knockin mice and noted changes in the sperm sterol composition. Concurrently, alterations in the quantity, motility, and function of the sperm in DHCR24 knockin mice were identified.

Strengths:

The manuscript offers an intriguing perspective on how disruptions in sperm sterol composition can lead to sperm abnormalities.

Weaknesses:

From the current data, several issues remain to be clarified, including the fertility test results, which merit a more detailed presentation to ascertain whether differences stem from individual variability or overall changes. The authors suggest an increase in ROS in the sperm of DHCR24 knockin mice, leading to sperm damage, which also requires further confirmation. Moreover, the quality of some data requires verification or improvement, such as the morphological analysis of testicular sections and the OCR experiments.

Reviewer #3 (Public Review):

The author presents a novel theory and computational model suggesting that grid cells do not encode space, but rather encode non-spatial attributes. Place cells in turn encode memories of where those specific attributes occurred. The theory accounts for many experimental results and generates useful predictions for future studies. The model's simplicity and potential explanatory power will interest others in the field, though there are a number of concerns that should first be addressed.

A crucial assumption of the model is that the content of experience must be constant in space. It's difficult to imagine a real-world example that satisfies this assumption. Odors and sounds are used as examples. While they are often more spatially diffuse than an objects on the ground, odors and sounds have sources that are readily detectable. Animals can easily navigate to a food source or to a vocalizing conspecific. This assumption is especially problematic because it predicts that all grid cells should become silent when their preferred non-spatial attribute (e.g. a specific odor) is missing. I'm not aware of any experimental data showing that grid cells become silent. On the contrary, grid cells are known to remain active across all contexts that have been tested, including across sleep/wake states. Unlike place cells, grid cells do not seem to turn off. Since grid cells are active in all contexts, their preferred attribute must also be present in all contexts, and therefore they would not convey any information about the specific content of an experience.

The proposed novelty of this theory is that other models all assume that grid cells encode space. This isn't quite true of models based on continuous attractor networks, the discussion of which is notably absent. More specifically, these models focus on the importance of intrinsic dynamics within the entorhinal cortex in generating the grid pattern. While this firing pattern is aligned to space during navigation and therefore can be used as a representation of that space, the neural dynamics are preserved even during sleep. Similarly, it is because the grid pattern does not strictly encode physical space that grid-like signals are also observed in relation to other two-dimensional continuous variables.

The use of border cells or boundary vector cells as the main (or only) source of spatial information in the hippocampus is not well supported by experimental data. Border cells in the entorhinal cortex are not active in the center of an environment. Boundary-vector cells can fire farther away from the walls but are not found in the entorhinal cortex. They are located in the subiculum, a major output of the hippocampus. While the entorhinal-hippocampal circuit is a loop, the route from boundary-vector cells to place cells is much less clear than from grid cells. Moreover, both border cells and boundary-vector cells (which are conflated in this paper) comprise a small population of neurons compared to grid cells.

Reviewer #3 (Public Review):

In this manuscript, Berger et al. study how internal energy storage influence learning and memory. Since in Drosophila melanogaster, octopamine (OA) is involved in the regulation of energy homeostasis they focus on the roles of OA. To do so they use the tyramine-β-hydroxylase (Tbh) mutant that is lacking the neurotransmitter OA and study short term memory (STM), long-term memory (LTM) and anesthesia-resistant memory (ARM). They show that the duration of starvation affects the magnitude of both short- and long-term memory. In addition, they show that OA has a suppressive effect on learning and memory. In terms of energy storage, they show that internal glycogen storage influences how long sucrose is remembered, and high glycogen suppresses memory. Finally, they show that insulin-like signaling in octopaminergic neurons, which is also related to internal energy storage, suppresses learning and memory.

The revised version of the manuscript is greatly improved, and I thank the authors for taking the comment seriously. This is an important study that extends our knowledge on OA activity in learning and memory and the effects the metabolic state has on learning and memory. The authors nicely use the genetic tools available in flies to try and unravel the complex circuitry of metabolic state level, OA activity and learning and memory. The overall take-home message of the manuscript is clear and supported by the data presented.

Reviewer #3 (Public Review):

The manuscript by Mahapatra and Takahashi addresses the role of presynaptic release site clearance during sustained synaptic activity. The authors characterize the effects of pharmacologically interfering with SV endocytosis (pre-incubation with Dynasore or Pitstop-2) on synaptic short-term plasticity (STP) at two different CNS synapses (calyx of Held synapses and hippocampal SC to CA1 synapses) using patch-clamp recordings in acute slices under experimental conditions designed to closely mimic a physiological situation (37{degree sign}C and 1.3 mM external [Ca2+]). Endocytosis blocker-induced changes in STP and in the recovery from short-term depression (STD) are compared to those seen after pharmacologically inhibiting actin filament assembly (pre-incubation with Latrunculin-B or the selective Cdc42 GTPase inhibitor ML-141). Presynaptic capacitance (Cm) recordings in calyx terminals were used to establish the effects of the pharmacological maneuvers on SV endocytosis.<br /> Latrunculin-B and ML-141 neither affect SV endocytosis (assayed by Cm recordings) nor EPSC recovery following conditioning trains, but strongly enhances STD at calyx synapses. No changes in STP were observed at Latrunculin-B- or ML-141-treated SC to CA1 synapses.

Dynasore and Pitstop-2 slow down endocytosis, limit the total amount of exocytosis in response to long stimuli, enhance STD in response to 100 Hz stimulation, but profoundly accelerate EPSC recovery following conditioning 100 Hz trains at calyx synapses. At SC to CA1 synapses, Dynasore and Pitstop-2 reduce the extend of facilitation and lower relative steady-state EPSCs suggesting a change in the facilitation-depression balance in favor of the latter.

The authors use state-of-the art techniques and their data, which is clearly presented, leads to authors to conclude that endocytosis is universally important for clearance of release sites while the importance of scaffold protein-mediated site clearance is limited to 'fast synapses'.

Unfortunately, and perhaps not completely unexpected in view of the pharmacological tools chosen, there are several observations which remain difficult to understand:

(1) Blocking site clearance affects release sites that have previously been used, i.e. sites at which SV fusion has occurred and which therefore need to be cleared. Calyces use at most 20% of all release sites during a single AP, likely fewer at 1.3 mM external [Ca2+]. Even if all those 20% of release sites become completely unavailable due to a block of release site clearance, the 2nd EPSC in a train should not be reduced by >20% because ~80% of the sites cannot be affected. However, ~50% EPSC reduction was observed (Fig. 2B1, lower right panel) raising the possibility that Dynasore does more than specifically interfering with SVs endocytosis (and possibly Pitstop as well). Non-specific effects are also suggested by the observed two-fold increase in initial EPSC size in SC to CA1 synapses after Dynasore pre-incubation.

(2) More severe depression was observed at calyx synapses after blocking endocytosis which the authors attribute to a presynaptic mechanism affecting pool replenishment. When probing EPSC recovery after conditioning 100 Hz trains, a speed up was observed mediated by an "unknown mechanism" which is "masked in 2 mM [Ca2+]". These two observations, deeper synaptic depression during 100 Hz but faster recovery from depression following 100 Hz, are difficult to align and no attempt was made to find an explanation.

(3) To reconcile previous data reporting a block of Ca2+-dependent recovery (CDR) by Dynasore or Latrunculin (measured at 2 mM external [Ca2+]) with the data presented here (using 1.3 mM external [Ca2+]) reporting no effect or a speed up of recovery from depression, the authors postulate that "CDR may operate only when excessive Ca2+ enters during massive presynaptic activation" (page 10 line 244). While that is possible, such explanation ignores plenty of calyx studies demonstrating fiber stimulation-induced CDR and elucidating molecular pathways mediating fiber stimulation-induced CDR, and it also completely dismisses the strong change in recovery time course after 10 Hz conditioning (single exponential) as compared to 100 Hz conditioning (double exponential with a pronounced fast component).

Strong presynaptic stimuli such as those illustrated in Figs. 1B,C induce massive exocytosis. The illustrated Cm increase of 2 to 2.5 pF represents fusion of 25,000 to 30,000 SVs (assuming a single SV capacitance of 80 aF) corresponding to a 12 to 15% increase in whole terminal membrane surface (assuming a mean terminal capacitance of ~16 pF). Capacitance measurements can only be considered reliable in the absence of marked changes in series and membrane conductance. Documentation of the corresponding conductance traces is therefore advisable for such massive Cm jumps and merely mentioning that the first 450 ms after stimulation were skipped during analysis or referring to previous publications showing conductance traces is insufficient.<br /> All bar graphs in Figures 1 through 6 and Figures S3 through S6 compare three or even four (Fig. 5C) conditions, i.e. one control and at least two treatment data sets. It appears as if repeated t-tests were used to run multiple two-group comparisons (i.e. using the same control data twice for two different comparisons). Either a proper multiple comparison test should be used or a Bonferroni correction or similar multiple-comparison correction needs to be applied.

Finally, the terminology of contrasting "fast-signaling" (calyx synapses) and "slow-plastic" (SC synapses) synapses seems to imply that calyx synapses lack plasticity, as does the wording "conventional bouton-type synapses involved in synaptic plasticity" (page 11, line 251). I assume, the authors primarily refer to the maximum frequencies these two synapse types typically transmit (fast-signaling vs slow-signaling)?

Reviewer #3 (Public Review):

Summary:

This study concerns how macaque visual cortical area MT represents stimuli composed of more than one speed of motion.

Strengths:

The study is valuable because little is known about how the visual pathway segments and preserves information about multiple stimuli. The study presents compelling evidence that (on average) MT neurons represent the average of the two speeds, with a bias that accentuates the faster of the two speeds. An additional strength of the study is the inclusion of perceptual reports from both humans and one monkey participant performing a task in which they judged whether the stimuli involved one vs two different speeds. Ultimately, this study raises intriguing questions about how exactly the response patterns in visual cortical area MT might preserve information about each speed, since such information could potentially be lost in an average response as described here, depending on assumptions about how MT activity is evaluated by other visual areas.

Weaknesses:

My main concern is that the authors are missing an opportunity to make clear that the divisive normalization, while commonly used to describe neural response patterns in visual areas (and which fits the data here), fails on the theoretical front as an explanation for how information about multiple stimuli can be preserved. Thus, there is a bit of a disconnect between the goal of the paper - how does MT represent multiple stimuli? - and the results: mostly averaging responses which, while consistent with divisive normalization, would seem to correspond to the perception of a single intermediate speed. This is in contrast to the psychophysical results which show that subjects can at least distinguish one from two speeds. The paper would be strengthened by grappling with this conundrum in a head-on manner.

Reviewer #3 (Public Review):

Summary:

In order to study the factors and neural dynamics that lead to the suppression of irrelevant information in the brain, the authors trained artificial neural networks in the execution of a task that involved the discrimination of complex stimuli with three main features: color, shape, and width. Specific combinations of color and shape led to a reward, but the temporal structure made color dynamically irrelevant at the beginning of the trial, and then it became relevant once the shape was presented. On the other hand, the width of the stimulus was always irrelevant. Importantly, non-human primates were also trained to execute this task (in a previous study by the authors) and the activity from neural populations from the dorsolateral Prefrontal Cortex (dlPFC) was recorded, allowing to compare the coding of information by the artificial neural network model with what happens in biological neural populations.

The authors changed systematically the amount of noise present in the neural network model, as well as limiting the firing rate of the artificial neurons to simulate the limitations imposed by high metabolic costs in biological neurons. They found that models with medium and low noise, as well as medium and low metabolic cost, developed information encoding patterns that resembled the patterns observed throughout learning in the dlPFC, as follows: early in the learning process, color information was strongly represented during the whole trial, as well as shape and width, whereas the color/shape combination significance (XOR operation) was weakly encoded. Late in learning, color information was initially suppressed (while it was deemed irrelevant) and became more prominent during the shape presentation. Width information coding decreased, and the XOR operation result became more strongly encoded.

Subthreshold activity dynamics were studied by training artificial networks consisting of 2 neurons, with the aim of understanding how dynamically irrelevant information is suppressed and then encoded more strongly at a different time during the trial. Under medium noise and medium metabolic cost, color information is suppressed by the divergence of the activity away from the level that triggers spikes. The authors claim that this subthreshold dynamic explains the suppression of irrelevant information in biological neural networks.

Strengths:

The study leverages the power of computational models to simulate biological networks and do manipulations that are difficult (if not impossible) to perform in vivo. The analyses of the activity of the network model are neat and thorough and provide a clear demonstration of how noise and metabolic costs may affect the information coding in the brain. The mathematical analyses are rigorous and nicely documented.

Weaknesses:

The study does not leverage the fact that they have access to the activity of individual neurons both on a neural network model and in neural recordings. The model/brain comparison results are limited to the decodability of different pieces of information during the execution of the task at different stages of learning. It would have been useful if the authors had shown response profiles of individual neurons, both biological and artificial, to strengthen the claim that the activity patterns are similar. Perhaps showing that the firing rates vary in a similar way in the large models (like they do for the 2-neuron model) would have been informative. For instance, it is possible that suppression is not occurring in the dlPFC, but that the PFC receives input with this information already suppressed. If suppression indeed happens in the PFC, response profiles associated with this process may be observed.

There is no way to say that the 2-neuron models are in any way informative of what happens in brain neurons, or even larger artificial networks since the sources of sensory input, noise, and inhibition will differ between biological and artificial networks. And because the firing patterns are not shown for large networks, it is not clear if some non-coding artificial neurons will become broadly inhibitory but maintain a relatively high firing rate (to mention only one possibility).

Reviewer #3 (Public Review):

Summary

In their manuscript, Vickers and McCormick have demonstrated the potential of leveraging mesoscale two-photon calcium imaging data to unravel complex behavioural motifs in mice. Particularly commendable is their dedication in providing detailed surgical preparations and corresponding design files, a contribution that will greatly benefit the broader neuroscience community as a whole. The quality of the data is high and examples are available to the community. More importantly, the authors have acquired activity-clustered neural ensembles at an unprecedented spatial scale to further correlate with high level behaviour motifs identified by B-SOiD. Such an advancement marks a significant contribution to the field. While the manuscript is comprehensive and the analytical strategy proposed is promising, some technical aspects warrant further clarification. Overall, the authors have presented an invaluable and innovative approach, effectively laying a solid foundation for future research in correlating large scale neural ensembles with behavioural. The implementation of a custom sound insulator for the scanner is a great idea and should be something implemented by others.

This is a methods paper, but there is no large diagram (in the main figures) that shows how all the parts are connected, communicating and triggering between each other. This is described in the methods and now supplemental figure, but a visual representation would greatly benefit the readers looking to implement something similar as a main figure but I guess they can find it in the methods. No stats for the results shown in Figure 6e, it would be useful to know which of these neural densities for all areas show a clear statistical significance across all the behaviors. While I understand that this is a methods paper, it seems like the authors are aware of the literature surrounding large neuronal recordings during mouse behavior. Indeed, in line 178-179 the authors mention how a significant portion of the variance in neural activity can be attributed to changes in "arousal or self-directed movement even during spontaneous behavior." Why then did the authors not make an attempt at a simple linear model that tries to predict the activity of their many thousands of neurons by employing the multitude of regressors at their disposal (pupil, saccades, stimuli, movements, facial changes, etc). These models are straightforward to implement, and indeed it would benefit this work if the model extracts information on par with what it's known from the literature. We also realize such a model could be done in the future.

Reviewer #3 (Public Review):

Summary:

The authors established a new virtual reality place preference task. On the task, rats, which were body-restrained on top of a moveable Styrofoam ball and could move through a circular virtual environment by moving the Styrofoam ball, learned to navigate reliably to a high-reward location over a low-reward location, using allocentric visual cues arranged around the virtual environment.

The authors also showed that functional inhibition by bilateral microinfusion of the GABA-A receptor agonist muscimol, which targeted the dorsal or intermediate hippocampus, disrupted task performance. The impact of functional inhibition targeting the intermediate hippocampus was more pronounced than that of functional inhibition targeting the dorsal hippocampus.

Moreover, the authors demonstrated that the same manipulations did not significantly disrupt rats' performance on a virtual reality task that required them to navigate to a spherical landmark to obtain reward, although there were numerical impairments in the main performance measure and the absence of statistically significant impairments may partly reflect a small sample size (see comments below).

Overall, the study established a new virtual-reality place preference task for rats and established that performance on this task requires the dorsal to intermediate hippocampus. They also established that task performance is more sensitive to the same muscimol infusion (presumably - doses and volumes used were not clearly defined in the manuscript, see comments below) when the infusion was applied to the intermediate hippocampus, compared to the dorsal hippocampus, although this does not offer strong support for the authors claim that dorsal hippocampus is responsible for accurate spatial navigation and intermediate hippocampus for place-value associations (see comments below).

Strengths:

(1) The authors established a new place preference task for body-restrained rats in a virtual environment and, using temporary pharmacological inhibition by intra-cerebral microinfusion of the GABA-A receptor agonist muscimol, showed that task performance requires dorsal to intermediate hippocampus.

(2) These findings extend our knowledge about place learning tasks that require dorsal to intermediate hippocampus and add to previous evidence that, for some place memory tasks, the intermediate hippocampus may be more important than other parts of the hippocampus, including the dorsal hippocampus, for goal-directed navigation based on allocentric place memory.

(3) The hippocampus-dependent task may be useful for future recording studies examining how hippocampal neurons support behavioral performance based on place information.

Weaknesses:<br /> (1) The new findings do not strongly support the authors' suggestion that the dorsal hippocampus is responsible for accurate spatial navigation and the intermediate hippocampus for place-value associations.

The authors base this claim on the differential effects of the dorsal and intermediate hippocampal muscimol infusions on different performance measures. More specifically, dorsal hippocampal muscimol infusion significantly increased perimeter crossings and perimeter crossing deviations, whereas dorsal infusion did not significantly change other measures of task performance, including departure direction and visits to the high-value location. However, these statistical outcomes offer only limited evidence that dorsal hippocampal infusion specifically affected the perimeter crossing, without affecting the other measures. Numerically the pattern of infusion effects is quite similar across these various measures: intermediate hippocampal infusions markedly impaired these performance measures compared to vehicle infusions, and the values of these measures after dorsal hippocampal muscimol infusion were between the values in the intermediate hippocampal muscimol and the vehicle condition (Figures 5-7). Moreover, I am not so sure that the perimeter crossing measures really reflect distinct aspects of navigational performance compared to departure direction and hit rate, and, even if they did, which aspects this would be. For example, in line 316, the authors suggest that 'departure direction and PCD [perimeter crossing deviation] [are] indices of the effectiveness and accuracy of navigation, respectively'. However, what do the authors mean by 'effectiveness' and 'accuracy'? Accuracy typically refers to whether or not the navigation is 'correct', i.e. how much it deviates from the goal location, which would be indexed by all performance measures.

So, overall, I would recommend toning down the claim that the findings suggest that the dorsal hippocampus is responsible for accurate spatial navigation and the intermediate hippocampus for place-value associations.

(2) The claim that the different effects of intermediate and dorsal hippocampal muscimol infusions reflect different functions of intermediate and dorsal hippocampus rests on the assumption that both manipulations inhibit similar volumes of hippocampal tissue to a similar extent, but at different levels along the dorso-ventral axis of the hippocampus. However, this is not a foregone conclusion (e.g., drug spread may differ depending on the infusion site or drug effects may differ due to differential expression of GABA-A receptors in the dorsal and intermediate hippocampus), and the authors do not provide direct evidence for this assumption. Therefore, a possible alternative account of the weaker effects of dorsal compared to intermediate hippocampal muscimol infusions on place-preference performance is that the dorsal infusions affect less hippocampal volume or less markedly inhibit neurons within the affected volume than the intermediate infusions. I would recommend that the authors briefly consider this issue in the discussion. Moreover, from the Methods, it is not clear which infusion volume and muscimol concentration were used for the different infusions (see below, 4.a.), and this must be clarified.

(3) It is good that the authors included a comparison/control study using a spherical beacon-guided navigation task, to examine the specific psychological mechanisms disrupted by the hippocampal manipulations. However, as outlined below (4.b.), the sample size for the comparison study was lower than for the main study, and the data in Figure 8 suggest that the comparison task may be affected by the hippocampal manipulations similarly to the place-preference task, albeit less markedly. This would raise the question as to which mechanisms that are common to the two tasks may be affected by hippocampal functional inhibition, which should be considered in the discussion.

(4) Several important methodological details require clarification:<br /> a. Drug infusions (from line 673):<br /> - '0.3 to 0.5 μl of either phosphate-buffered saline (PBS) or muscimol (MUS) was infused into each hemisphere'; the authors need to clarify when which infusion volume was used and why different infusion volumes were used.<br /> - I could not find the concentration of the muscimol solution that was used. The authors must clarify this and also should include a justification of the doses used, e.g. based on previous studies.<br /> - Please also clarify if the injectors and dummies were flush with the guides or by which distance they protruded from the guides.<br /> b. Sample sizes: The authors should include sample size justifications, e.g. based on considerations of statistical power, previous studies, practical considerations, or a combination of these factors. Importantly, the smaller sample size in the control study using the spherical beacon-guided navigation task (n=5 rats) limits comparability with the main study using the place-preference task (n=8). Numerically, the findings on the control task (Figure 8) look quite similar to the findings on the place-preference task, with intermediate hippocampal muscimol infusions causing the most pronounced impairment and dorsal hippocampal muscimol infusions causing a weaker impairment. These effects may have reached statistical significance if the same sample size had been used in the place-preference study.<br /> c. Statistical analyses: Why were the data of the intermediate and dorsal hippocampal PBS infusion conditions averaged for some of the analyses (Figure 5; Figure 6B and C; Figure 7B and C; Figure 8B) but not for others (Figure 6A and Figure 7A)?

Reviewer #3 (Public Review):

A mechanical model of C. elegans, embedded in a resistive force environment, is used to calculate input torque patterns required to generate output curvature patterns and coordinates, corresponding to a number of different locomotion behaviors in C. elegans.

Strengths:

The use of a mechanical model to study a variety of locomotor sequences and the grounding in empirical data are strengths. The matching of speeds (though requiring adjusted drag coefficients) is a strength.

Weaknesses:

The paper lacks evidence of numerical validation or comparison with the results and tools in the literature. E.g. is it surprising that the uniform torque distribution yields maximal speed? What is the relation between input and output data? How does the input-output relation depend on the parameters of the model? What novel model predictions are made?

In particular, if validated, the breakdown of drag forces and torque distributions during forward locomotion and turning behaviors may be interesting to compare to predictions by other tools, and to empirical measurement. One caveat is that the worm touches itself during such turns, and even crosses over itself in delta turns, and so the estimated drag coefficients and the resultant mechanical forces are likely incorrect.

Reviewer #3 (Public Review):

In Okholm et al., the authors evaluate the functional impact of circHIPK3 in bladder cancer cells. By knocking down circHIPK3 and performing an RNA-seq analysis, the authors found thousands of 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 the STAT3 gene, which also consistently has 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:

Although the number of circRNAs continues to grow, this field lacks many instances of 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. Experiments involving use of time-point knockdown followed by RNA-seq, investigation of miRNA-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 are thorough and convincing.

Weaknesses:

It is unclear why the authors used certain bladder cancer cells versus non-bladder cells in some experiments. The efficacy of certain experiments (specifically rescue experiments) and some control conditions is still questionable. Overall, the presented study adds some further knowledge in describing circHIPK3 function, its capability to regulate some downstream genes, and its interaction and competition for IGF2BP2.

Reviewer #3 (Public Review):

Summary:

The cognitive striatum, also known as the dorsomedial striatum, receives input from brain regions involved in high-level cognition and plays a crucial role in processing cognitive information. However, despite its importance, the extent to which different projection pathways of the striatum contribute to this information processing remains unclear. In this paper, Bruce et al. conducted a study using a range of causal and correlational techniques to investigate how these pathways collectively contribute to interval timing in mice. Their results were consistent with previous research, showing that the direct and indirect striatal pathways perform opposing roles in processing elapsed time. Based on their findings, the authors proposed a revised computational model in which two separate accumulators track evidence for elapsed time in opposing directions. These results have significant implications for understanding the neural mechanisms underlying cognitive impairment in neurological and psychiatric disorders, as disruptions in the balance between direct and indirect pathway activity are commonly observed in such conditions.

Strengths:

The authors employed a well-established approach to study interval timing and employed optogenetic tagging to observe the behavior of specific cell types in the striatum. Additionally, the authors utilized two complementary techniques to assess the impact of manipulating the activity of these pathways on behavior. Finally, the authors utilized their experimental findings to enhance the theoretical comprehension of interval timing using a computational model.

Weaknesses:

The behavioral task used in this study is best suited for investigating elapsed time perception, rather than interval timing. Timing bisection tasks are often employed to study interval timing in humans and animals. The main results from unit recording (opposing slopes of D1/D2 cell firing rate, as shown in Figure 3D) appear to be very sensitive to a couple of outlier cells, and the predictive power of ensemble recording seems to be only slightly above chance levels. In the optogenetic experiment, the laser was kept on for too long (18 seconds) at high power (12 mW). This has been shown to cause adverse effects on population activity (for example, through heating the tissue) that are not necessarily related to their function during the task epochs. Given the systemic delivery of pharmacological interventions, it is difficult to conclude that the effects are specific to the dorsomedial striatum. Future studies should use the local infusion of drugs into the dorsomedial striatum.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors discover that nuclear volume decreases after mitotic exit following cell confinement in a manner that scales with the extent of confinement. This adaptation appears to protect the cells from adverse outcomes of critical confinement such as nuclear blebs and DNA damage. The evidence to support these claims is strong.

The authors also provide a model in which argue that what they call the "apparent nuclear surface area" is modulated by confinement through a mechanism regulated by cPLA2 and myosin II activities. Here there are weaknesses in that the manuscript relies on a single approach, measurements are indirect, and alternative models are not explored. Similarly, additional considerations need to be addressed so that the reader can interpret the data presented - for example whether cell volume is also changing coincident with nuclear volume changes, and whether other aspects of cell physiology such as cytokinesis are altered.

Considerations that could support the manuscript further:

One essential consideration that goes unaddressed is whether the nuclear volume alone is changing under compression (resulting in a higher nuclear to cytoplasmic ratio) or if the cell volume is changing and the nuclear volume is following suit (no change in the N:C ratio). Depending on which of these is the case, the overall model would likely shift. In particular, interpreting the effect of disrupting myosin II activity given its different distribution at the cortex in response to the higher confinement would be influenced by which of these conditions are at play.

A key approach used and interpreted by the investigators is an assessment of the folding of the "inner lamin envelope", which they derive from an image analysis routine of lamin staining that they developed and argue reflects "nuclear envelope tension". I am not convinced of the robustness of this approach or what it mechanistically reveals. It may or may not reflect the contour of the inner nuclear membrane, which (perhaps) is the most relevant to the authors' interpretation of nuclear envelope tension. Given the major contribution of this data to the model, which is based on the "unfolding" of the nuclear envelope, an orthogonal approach (e.g. electron microscopy - which one needs to truly address the high-frequency undulations of the nuclear envelope) is needed to support the larger conclusions.

The authors argue that nuclear tension is lost after mitosis in the confined devices because nuclear volume has decreased. While a smaller nuclear volume might indeed translate to less compressive force from the device on the nucleus, one would imagine that the chromosomes still have to be accommodated and that confining them in a smaller volume could increase the tension. Although arguable, the potential alternative possibilities suggest that actual measurements of nuclear envelope tension are needed to robustly test the model. The authors cite the observation that blebs are less prevalent after mitosis as additional support for this model, but this is expected as nuclear envelope breakdown and reformation will "reset" the nuclear contour while the appearance of blebs at mitotic entry is essential a "memory" of all blebs and ruptures over the entire preceding cell cycle.

Representative images for the pharmacological perturbations other than blebbistatin are notably absent - only the analyzed data are presented in the manuscript or the supplemental material. How these perturbations (e.g. to cPLA2) also affect the cortex is important to interpret the data given the point raised above. Orthogonal approaches would also strengthen the conclusions (for example, the statement that "nuclear adaptation observed during mitosis requires nuclear tension sensing through cPLA2" requires more evidence to be convincing - it is not sufficiently supported by the data presented). Even if this is the case, the authors acknowledge that cPLA2 is likely not the answer to the adaption observed under the lower degrees of confinement. Thus, the mechanisms underlying the adaptive changes to nuclear volume remain enigmatic.

One more consideration that seems to go without comment is that the cells under confinement do not appear to successfully complete cytokinesis (Fig. 5b). At a minimum this seems like a major perturbation to cell physiology and needs to be more fully discussed by the authors as playing a role in the observed changes in nuclear volume.

Reviewer #3 (Public Review):

The authors report on the engineering of an induced Pluripotent Stem Cell (iPSC) line that harbours a single copy of a split mNeonGreen, mNG2(1-10). This cell line is subsequently used to take endogenous protein with a smaller part of mNeonGreen, mNG2(11), enabling complementation of mNG into a fluorescent protein that is then used to visualize the protein. The parental cell is validated and used to construct several iPSC line with endogenously tagged proteins. These are used to visualize and quantify endogenous protein localisation during mitosis.

I see the advantage of tagging endogenous loci with small fragments, but the complementation strategy has disadvantages that deserve some attention. One potential issue is the level of the mNG2(1-10). In addition, this may probably not work for organelle-resident proteins, where the mNG2(11) tag is localised in a membrane enclosed compartment.

Overall the tools and resources reported in this paper will be valuable for the community that aims to study proteins at endogenous levels.

Reviewer #3 (Public Review):

Summary:

Kokinovic et al. presents an interesting paper that addresses an important gap in knowledge about the differences in the development of direct and indirect pathway striatal neurons in the striosome and matrix compartments. The division of the striatum into 4 distinct populations, striosome-dSPNs, striosome-iSPNs, matrix-dSPNs, and matrix-iSPNs is important, but rarely done. This study records all four populations across early development and shows differences in action potential characteristics and intrinsic properties. They also suppress striosome activity during postnatal development and evaluate the characteristics of adult dopaminergic neurons in control and previously striosome-quieted conditions.

Strengths:

The striatal electrophysiology is beautifully and carefully done and shows important developmental differences between neural subtypes.

The idea to test the striatonigral connection is a good idea.

Weaknesses:

The authors didn't actually test the striatonigral connection. The experiments they do instead don't convincingly show that the striosomal or even striatal connection to the dopaminergic neurons is altered after postnatal striosome suppression.

Major concerns:

(1) mIPSCs are measured and are reduced after chemogenetic suppression of striosomal neurons during development. This is an interesting finding, but these mIPSCs could be coming from any inhibitory input onto the SNc neurons. It is unlikely that most of the mIPSCs are coming from the striosomal inputs. The GPe is much more likely to be the source of these mIPSCs than the striatum because the GPe inputs form synapses nearer the soma and have a higher probability of release (Evans et al., 2020). dSPNs inhibit GPe neurons through a non-canonical pathway (Cui et al., 2021; Spix et al., 2021) and striosomes also inhibit the SNr (McGregor et al., 2019). The striatum has the potential to disinhibit SNc neurons through both the SNr or the GPe (Evans, 2022), and modification of the striosome-SNr or striosome-GPe connections during development could be what is causing the mIPSC changes. To claim that the striosome-SNc connection is altered, a direct test of this connection is necessary.

(2) The dopaminergic neurons recorded seem to be randomly selected, but the striosomes do not inhibit all SNc dopamine neurons. They selectively inhibit the ventral tier SNc neurons (Evans et al., 2020). In the present manuscript, it is impossible to know which subpopulation of SNc neurons was recorded, so it is impossible to tell whether the dopaminergic neurons recorded are the ones expected to receive striosomal input.

(3) Very similarly, the striosomes selectively wrap around the "SNr dendrite" of SNc neurons that participate in striosome-dendron bouquets (Crittenden et al., 2016). However, not all SNc neurons have prominent SNr dendrites (Henny et al., 2012). In the morphological images of Supplemental Figure 3, it looks like the recorded cells sometimes have an SNr dendrite and sometimes don't (but it is hard to tell because the medial-lateral rostral-caudal axis is not labeled in the images). The presence or absence of the "SNr dendrite" is a strong determinant of whether an individual dopaminergic neuron receives striosomal inhibition or not (Evans et al., 2020). As above, not knowing whether the neurons recorded have SNr dendrites makes it impossible to know whether they should be receiving striosomal input at all.

(4) It's quite interesting that the dendron-bouquet structure is intact even after striosomal activity suppression, as cannabinoid receptor knockout greatly disrupts the structural integrity of bouquets (Crittenden et al., 2022). However, going along with point 3, the gephyrin puncta analysis only at the somas is very limiting. The striosome-SNc relevant puncta would be primarily on the SNr dendrite. Gephyrin density on the SNr dendrites or in bouquets would be much more informative than density on the soma.

(5) The authors claim that "CNO didn't affect the shape of the DA neuron dendritic tree", but more information about the morphological analysis should be added. It is not clear how the sholl analysis was conducted or whether a full 3D reconstruction was made. This claim seems to be based on only one dendritic measurement (sholl analysis), but many other dendritic or morphological features could be altered.

Crittenden, J.R., Tillberg, P.W., Riad, M.H., Shima, Y., Gerfen, C.R., Curry, J., Housman, D.E., Nelson, S.B., Boyden, E.S., & Graybiel, A.M. (2016) Striosome-dendron bouquets highlight a unique striatonigral circuit targeting dopamine-containing neurons. Proc. Natl. Acad. Sci. U.S.A., 113, 11318-11323.<br /> Crittenden, J.R., Yoshida, T., Venu, S., Mahar, A., & Graybiel, A.M. (2022) Cannabinoid Receptor 1 Is Required for Neurodevelopment of Striosome-Dendron Bouquets. eNeuro, 9, ENEURO.0318-21.2022.<br /> Cui, Q., Du, X., Chang, I.Y.M., Pamukcu, A., Lilascharoen, V., Berceau, B.L., García, D., Hong, D., Chon, U., Narayanan, A., Kim, Y., Lim, B.K., & Chan, C.S. (2021) Striatal Direct Pathway Targets Npas1+ Pallidal Neurons. J Neurosci, 41, 3966-3987.<br /> Evans, R.C. (2022) Dendritic involvement in inhibition and disinhibition of vulnerable dopaminergic neurons in healthy and pathological conditions. Neurobiol Dis, 172, 105815.<br /> Evans, R.C., Twedell, E.L., Zhu, M., Ascencio, J., Zhang, R., & Khaliq, Z.M. (2020) Functional Dissection of Basal Ganglia Inhibitory Inputs onto Substantia Nigra Dopaminergic Neurons. Cell Rep, 32, 108156.<br /> Henny, P., Brown, M.T.C., Northrop, A., Faunes, M., Ungless, M.A., Magill, P.J., & Bolam, J.P. (2012) Structural correlates of heterogeneous in vivo activity of midbrain dopaminergic neurons. Nat. Neurosci., 15, 613-619.<br /> McGregor, M.M., McKinsey, G.L., Girasole, A.E., Bair-Marshall, C.J., Rubenstein, J.L.R., & Nelson, A.B. (2019) Functionally Distinct Connectivity of Developmentally Targeted Striosome Neurons. Cell Rep, 29, 1419-1428.e5.<br /> Spix, T.A., Nanivadekar, S., Toong, N., Kaplow, I.M., Isett, B.R., Goksen, Y., Pfenning, A.R., & Gittis, A.H. (2021) Population-specific neuromodulation prolongs therapeutic benefits of deep brain stimulation. Science, 374, 201-206.

Reviewer #3 (Public Review):

Summary:

The receptor binding domain of SARS-Cov-2 spike protein contains two N-glycans which have been conserved by the variants observed in these last 4 years. Through the use of extensive molecular dynamics, the authors demonstrate that even if glycosylation is conserved, the stabilization role of glycans at N343 differs among the strains. They also investigate the effect of this glycosylation on the binding of RBD towards sialylated gangliosides, as a function of evolution.

Strengths:

The molecular dynamics characterization is well performed and demonstrates differences in the effect of glycosylation as a factor of evolution. The binding of different strains to human gangliosides shows variations of strong interest. Analyzing the structure function of glycans on SARS-Cov-2 surface as a function of evolution is important for the surveillance of novel variants since it can influence their virulence.

Weaknesses:

The article is difficult to read, with no sufficient efforts of clarification for non-glycobiology audiences. The presentation of previous knowledge about RBD glycosylation and its effect on structure is very difficult to follow and should be reorganized. The choice of the nature of the biantennary glycan at N343 is not rationalized. A major weakness is the absence of data supporting the proposed binding site for ganglioside.

Reviewer #3 (Public Review):

Summary:

This work aims to demonstrate how recent advances in thermal stability assays can be utilised to screen chemical libraries and determine the compound mechanism of action. Focusing on 96 compounds with known mechanisms of action, they use the PISA assay to measure changes in protein stability upon treatment with a high dose (10uM) in live K562 cells and whole cell lysates from K562 or HCT116. They intend this work to showcase a robust workflow that can serve as a roadmap for future studies.

Strengths:

The major strength of this study is the combination of live and whole cell lysates experiments. This allows the authors to compare the results from these two approaches to identify novel ligand-induced changes in thermal stability with greater confidence. More usefully, this also enables the authors to separate the primary and secondary effects of the compounds within the live cell assay.

The study also benefits from the number of compounds tested within the same framework, which allows the authors to make direct comparisons between compounds.

These two strengths are combined when they compare CHEK1 inhibitors and suggest that AZD-7762 likely induces secondary destabilisation of CRKL through off-target engagement with tyrosine kinases.

Weaknesses:

One of the stated benefits of PISA compared to the TPP in the original publication (Gaetani et al 2019) was that the reduced number of samples required allows more replicate experiments to be performed. Despite this, the authors of this study performed only duplicate experiments. They acknowledge this precludes the use of frequentist statistical tests to identify significant changes in protein stability. Instead, they apply an 'empirically derived framework' in which they apply two thresholds to the fold change vs DMSO: absolute z-score (calculated from all compounds for a protein) > 3.5 and absolute log2 fold-change > 0.2. They state that the fold-change threshold was necessary to exclude non-specific interactors. While the thresholds appear relatively stringent, this approach will likely reduce the robustness of their findings in comparison to an experimental design incorporating more replicates. Firstly, the magnitude of the effect size should not be taken as a proxy for the importance of the effect. They acknowledge this and demonstrate it using their data for PIK3CB and p38α inhibitors (Figures 2B-C). They have thus likely missed many small, but biologically relevant changes in thermal stability due to the fold-change threshold. Secondly, this approach relies upon the fold-changes between DMSO and compound for each protein being comparable, despite them being drawn from samples spread across 16 TMT multiplexes. Each multiplex necessitates a separate MS run and the quantification of a distinct set of peptides, from which the protein-level abundances are estimated. Thus, it is unlikely the fold changes for unaffected proteins are drawn from the same distribution, which is an unstated assumption of their thresholding approach. The authors could alleviate the second concern by demonstrating that there is very little or no batch effect across the TMT multiplexes. However, the first concern would remain. The limitations of their approach could have been avoided with more replicates and the use of an appropriate statistical test. It would be helpful if the authors could clarify if any of the missed targets passed the z-score threshold but fell below the fold-change threshold.

The authors use a single, high, concentration of 10uM for all compounds. Given that many of the compounds likely have low nM IC50s, this concentration will often be multiple orders of magnitude above the one at which they inhibit their target. This makes it difficult to assess the relevance of the off-target effects identified to clinical applications of the compounds or biological experiments. The authors acknowledge this and use ranges of concentrations for follow-up studies (e.g. Figure 2E-F). Nonetheless, this weakness is present for the vast bulk of the data presented.

The authors claim that combining cell-based and lysate-based assays increases coverage (Figure 3F) is not supported by their data. The '% targets' presented in Figure 3F have a different denominator for each bar. As it stands, all 49 targets quantified in both assays which have a significant change in thermal stability may be significant in the cell-based assay. If so, the apparent increase in % targets when combining reflects only the subsetting of the data. To alleviate this lack of clarity, the authors could update Figure 3F so that all three bars present the % targets figure for just the 60 compounds present in both assays.

Aims achieved, impact and utility:

The authors have achieved their main aim of presenting a workflow that serves to demonstrate the potential value of this approach. However, by using a single high dose of each compound and failing to adequately replicate their experiments and instead applying heuristic thresholds, they have limited the impact of their findings. Their results will be a useful resource for researchers wishing to explore potential off-target interactions and/or mechanisms of action for these 96 compounds, but are expected to be superseded by more robust datasets in the near future. The most valuable aspect of the study is the demonstration that combining live cell and whole cell lysate PISA assays across multiple related compounds can help to elucidate the mechanisms of action.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Long et al. demonstrated that the deletion of SUL1, which encodes a sulfate transporter localized on the plasma membrane, extends the replicative lifespan in S. cerevisiae. The authors further investigated the mechanism underlying this lifespan extension. They found that, unlike sul1∆ mutants, other mutants that have been shown to have a deficiency in sulfate transport cannot extend lifespan, from which they concluded that it is unlikely that SUL1 deletion extends lifespan by impairing sulfate intake. The authors then performed a series of characterizations on sul1∆ mutants and found that consistent with previous studies, PKA activity is downregulated when SUL1 is deleted. The authors demonstrated that SUL1 deletion promotes the nuclear localization of Msn2, as well as autophagy, which are known downstream signals of the PKA pathway. In addition, the authors show that MSN2 and ATG8 are indispensable for the lifespan extension in sul1∆ cells. Altogether, this manuscript suggests that SUL1 deletion extends lifespan by affecting PKA activity.

Strengths:

This study reported an interesting phenotype that the deletion of SUL1, but not SUL2, promotes lifespan extension in budding yeast. The authors performed some characterizations on sul1∆ mutants and epistatic studies to demonstrate that this lifespan extension requires MSN2 and ATG8, which further support the importance of the PKA pathway in regulating lifespan.

Weaknesses:

However, one of the major findings in this paper that SUL1 deletion extends lifespan independently of its role in sulfate uptake was merely based on lifespan measurements on sul2∆, SUL1E427Q, and met3∆ mutants, which cannot exclude the possibility that yeast lifespan is affected by sulfate intake. In addition, the strength of evidence for whether SUL1 deletion extends lifespan through affecting PKA activity is incomplete. It has been shown that Sul1 and Sul2 have redundant functions in both sulfate transport and PKA activation (Kankipati et al. 2015). However, in this manuscript, as shown by the authors, the deletion of SUL2 does not extend the lifespan compared with sul1∆ mutants. Without a further characterization on why deletion of SUL1, but not SUL2, extends lifespan, it is likely that SUL1 deletion extends lifespan independently of either sulfate transport or PKA activation.

Reviewer #3 (Public Review):

Summary

The authors of this work aim to address the challenge of accurately and efficiently identifying protein binding sites from sequences. They recognize that the limitations of current methods, including reliance on multiple sequence alignments or experimental protein structure, and the under-explored geometry of the structure, which limit the performance and genome-scale applications. The authors have developed a multi-task network, GPSite, that predicts binding residues for a range of biologically relevant molecules, including DNA, RNA, peptides, proteins, ATP, HEM, and metal ions, using sequence embeddings from protein language models and ESMFold-predicted structures. The reported results showed to be superior to current sequence-based and structure-based methods in terms of accuracy and efficiency.

Strengths<br /> (1) The GPSite model's ability to predict binding sites for a wide variety of molecules, including DNA, RNA, peptides, and various metal ions.<br /> (2) Based on the presented results, GPSite outperforms state-of-the-art methods in several benchmark datasets in terms of accuracy and efficiency.<br /> (3) GPSite adopts predicted structure instead of native structures as input, enabling the model to be applied to a wider range of scenarios where native structures are rare.<br /> (4) The low computational cost of GPSite is beneficial, which enables rapid genome-scale binding residue annotations, indicating the model's potential for large-scale downstream applications and discoveries.

Weaknesses

There are no major weaknesses after the revision.

Reviewer #3 (Public Review):

Summary:

How the microbial composition of the human body is influenced by and influences disease progression is an important topic. For people with COVID-19, symptomatic progression and deterioration can be difficult to predict. This manuscript attempts to associate the nasal and fecal microbiomes of COVID-19 patients with the severity of disease symptoms, with the goal of identifying microbial markers that can predict disease outcomes. However, the value of this work is held back by unclear methods and data presentation.

Strengths:

Analysis of microbiomes from two distinct anatomical locations and across three distinct patient groups is a substantial undertaking. How these microbiomes influence and are influenced by COVID-19 disease progression is an important question. In particular, the putative biomarker identified here could be of clinical value with additional research.

Weaknesses:

The methods and statistics used for several figures and comparisons are unclear or used in non-standard ways. For instance: the description of the Bray-Curtis test for Figure 1 is inaccurate and conflicts between the text and figure legend; the method used to compare the relative abundance of genera in Figure 2 is not clear; and it is not stated how the "total amount" of detected bacteria is inferred from the data presented in Figures 2C and 2D.

The description of results for Figure 1 is overstated or unclear for both the alpha diversity among disease groups and the overlap for nasal samples.

The most abundant phyla from nasal samples cumulatively account for less than 1% of abundance and it is unclear why this would be expected or how it compares to other work. Relatedly, the potential biological relevance of the very small proportional changes among phyla in the nasal samples is also not clear.

There is no real discussion of how the identified biomarkers might work in practice. While some microbes are detected in one condition but not others, it is unclear whether these organisms are expected to already exist below the detection threshold and then increase in abundance along with disease severity, or if they are picked up from the environment. For instance, would the presence of these 'severe' - associated microbes in patients with mild or moderate disease justify additional treatment to prevent disease progression?

The authors use the term "nasopharyngeal-faecal axis", but there is no substantial discussion of how these two microbiomes interact to influence disease progression, or how they are jointly affected to yield useful biomarkers. With one exception, correlation values between nasal and fecal microbes range from negligible to modest. It is unclear, then, how much parallel influence disease has on these microbiomes.

Silent weapons for quiet wars<br /> Operations Research Technical Manual<br /> TW-SW7905.1

Welcome Aboard

This publication marks the 25th anniversary of the Third World War, called the "Quiet War", being conducted using subjective biological warfare, fought with "silent weapons".<br /> This book contains an introductory description of this war, its strategies, and its weaponry.<br /> May 1979 #74-1120

Security

It is patently impossible to discuss social engineering or the automation of a society, i.e., the engineering of social automation systems (silent weapons) on a national or worldwide scale without implying extensive objectives of social control and destruction of human life, i.e., slavery and genocide.<br /> This manual is in itself an analog declaration of intent. Such a writing must be secured from public scrutiny. Otherwise, it might be recognized as a technically formal declaration of domestic war. Furthermore, whenever any person or group of persons in a position of great power and without full knowledge and consent of the public, uses such knowledge and methodologies for economic conquest - it must be understood that a state of domestic warfare exists between said person or group of persons and the public.<br /> The solution of today's problems requires an approach which is ruthlessly candid, with no agonizing over religious, moral or cultural values.<br /> You have qualified for this project because of your ability to look at human society with cold objectivity, and yet analyze and discuss your observations and conclusions with others of similar intellectual capacity without the loss of discretion or humility. Such virtues are exercised in your own best interest. Do not deviate from them.

https://ia802300.us.archive.org/10/items/silent-weapons-for-quiet-wars_202110/Silent%20Weapons%20for%20Quiet%20Wars.pdf

Reviewer #3 (Public Review):

This study explores sensory prediction errors in the sensory cortex. It focuses on the question of how these signals are shaped by non-hierarchical interactions, specifically multimodal signals arising from same-level cortical areas. The authors used 2-photon imaging of mouse auditory cortex in head-fixed mice that were presented with sounds and/or visual stimuli while moving on a ball. First, responses to pure tones, visual stimuli, and movement onset were characterized. Then, the authors made the running speed of the mouse predictive of sound intensity and/or visual flow. Mismatches were created through the interruption of sound and/or visual flow for 1 second while the animal moved, disrupting the expected sensory signal given the speed of movement. As a control, the same sensory stimuli triggered by the animal's movement were presented to the animal decoupled from its movement. The authors suggest that auditory responses to the unpredicted silence reflect mismatch responses. That these mismatch responses were enhanced when the visual flow was congruently interrupted, indicates the cross-modal influence of prediction error signals.

This study's strengths are the relevance of the question and the design of the experiment. The authors are experts in the techniques used. The analysis explores neither the full power of the experimental design nor the population activity recorded with 2-photon, leaving open the question of to what extent what the authors call mismatch responses are not sensory responses to sound interruption. The auditory system is sensitive to transitions and indeed responses to the interruption of the sound are similar in quality, if not quantity, in the predictive and the control situation.

Reviewer #3 (Public Review):

Summary:

In this article, Hermannova et al catalog the changes in ribosome association with mRNAs when the eukaryotic translation initiation factor 3 is disrupted by knocking down subunits of the multisubunit protein. They find that RNAs relying on TOP motifs for translation, such as ribosomal protein RNAs, and RNAs encoding proteins that modify other proteins in the ER or components of the lysosome are upregulated. In contrast, proteins encoding components of MAP kinase cascades are downregulated when subunits of eIF3 are knocked down.

Strengths:

The authors use ribosome profiling of well-characterized mutants lacking subunits of eIF3 and assess the changes in translation that take place. They supplement the ribosome association studies with western blotting to determine protein level changes of affected transcripts. They analyze what is being encoded by the transcripts undergoing translation changes, which is important for understanding more broadly how translation initiation factor levels affect cancer cell translatomes.

Weaknesses:

(1) The data are presented as a catalog of effects, and the paper would be strengthened if there were a clear model tying the various effects together or linking individual subunit knockdown to cancerous phenotypes. It is unclear what the hypothesis is for cells having more MAPK activity with less of the MAPK proteins being translated, so the main findings of the paper become observational without context.

(2) The conclusions drawn are presented as very generalized other than in the last paragraph, but the experiments were only done in Hela cells. Since conclusions are being made about how translation changes affect MAP kinase signaling and there is mention in the abstract that dysregulation of these subunits is observed in cancer, at least one other cell line would need to be analyzed to provide evidence that the effects of subunit knockdown aren't cell-line specific.

(3) It is also unclear how replicates were performed and how many replicates were performed for several experiments. Biological replicates are mentioned, but what the authors did for biological replicates isn't defined and the description of the collection of cells for polysome/ribosome footprint/RNA seq samples makes it unclear whether the "biological replicates" are samples from separate transfections (true biological replicates) or different aliquots or wells from a single transfection (technical replicates) being run over a separate gradient. If using technical replicates, the data comparing the effects of knocking down D vs E vs H subunits are substantially weakened because subunit-specific differences could be the result of non-specific events that occurred in a transfection. It's also notable that while the pooled siRNAs will increase the potency of knockdown, it is possible that one or more of the siRNAs could have off-target effects, and analyzing individual siRNAs would be better for ensuring effects are specific.

(4) Many of the changes in protein levels reported by Western are subtle. Data from all western blots making claims of quantitative differences should really be quantified relative to nontreated over-loading control or total protein quantified from the gel, and presented with a degree of error from biological replicates to make conclusions about differences in protein levels between samples.

Reviewer #3 (Public Review):

This study investigates subtelomeric repetitive sequences in the budding yeast Saccharomyces cerevisiae, known as Y' and X-elements. Taking advantage of yeast strain SY12 that contains only 3 chromosomes and six telomeres (normal yeast strains contain 32 telomeres) the authors are able to generate a strain completely devoid of Y'- and X-elements.

Strengths:

They demonstrate that the SY12 delta XY strain displays normal growth, with stable telomeres of normal length that were transcriptionally silenced, a key finding with wide implications for telomere biology. Inactivation of telomerase in the SY12 and SY12 delta XY strains frequently resulted in survivors that had circularized all three chromosomes, hence bypassing the need for telomeres altogether. They show that survivors with fused chromosomes and so-called atypical survivors arise independently of the central recombination protein Rad52. The SY12 and SY12 delta XY yeast strains can become a useful tool for future studies of telomere biology. The conclusions of this manuscript are well supported by the data and are valuable for researchers studying telomeres.

Weaknesses:

A weakness of the manuscript is the analysis of telomere transcriptional silencing. They state: "The results demonstrated a significant increase in the expression of the MPH3 and HSP32 upon Sir2 deletion, indicating that telomere silencing remains effective in the absence of X and Y'-elements". However, for the SY12 strain, their analyses indicate that the difference between the WT and sir2 strains is nonsignificant. In addition, a striking observation is that the SY12 strain (with only three chromosomes) express much less of both MPH3 and HSP32 than the parental strain BY4742 (16 chromosomes), both in the presence and absence of Sir2.

Reviewer #3 (Public Review):

Summary:

This valuable study by Semenova and colleagues describes a large cross-sectional cohort of 115 individuals on ART. Participants contributed a single blood sample which underwent IPDA, and 25-color flow with various markers (pre and post-stimulation). The authors then used clustering, decision tree analyses, and machine learning to look for correlations between these immunophenotypic markers and several measures of HIV reservoir volume. They identified two distinct clusters that can be somewhat differentiated based on total HIV DNA level, intact HIV DNA level, and multiple T cell cellular markers of activation and exhaustion.

The conclusions of the paper are supported by the data but the relationships between independent and dependent variables in the models are correlative with no mechanistic work to determine causality. It is unclear in most cases whether confounding variables could explain these correlations. If there is causality, then the data is not sufficient to infer directionality (ie does the immune environment impact the HIV reservoir or vice versa or both?). In addition, even with sophisticated and appropriate machine learning approaches, the models are not terribly predictive or highly correlated. For these reasons, the study is very much hypothesis-generating and will not impact cure strategies or HIV reservoir measurement strategies in the short term.

Strengths:

The study cohort is large and diverse in terms of key input variables such as age, gender, and duration of ART. Selection of immune assays is appropriate. The authors used a wide array of bioinformatic approaches to examine correlations in the data. The paper was generally well-written and appropriately referenced.

Weaknesses:

(1) The major limitation of this work is that it is highly exploratory and not hypothesis-driven. While some interesting correlations are identified, these are clearly hypothesis-generating based on the observational study design.

(2) The study's cross-sectional nature limits the ability to make mechanistic inferences about reservoir persistence. For instance, it would be very interesting to know whether the reservoir cluster is a feature of an individual throughout ART, or whether this outcome is dynamic over time.

(3) A fundamental issue is that I am concerned that binarizing the 3 reservoir metrics in a 50/50 fashion is for statistical convenience. First, by converting a continuous outcome into a simple binary outcome, the authors lose significant amounts of quantitative information. Second, the low and high reservoir outcomes are not actually demonstrated to be clinically meaningful: I presume that both contain many (?all) data points above levels where rebound would be expected soon after interruption of ART. Reservoir levels would also have no apparent outcome on the selection of cure approaches. Overall, dividing at the median seems biologically arbitrary to me.

(4) The two reservoir clusters are of potential interest as high total and intact with low % intact are discriminated somewhat by immune activation and exhaustion. This was the most interesting finding to me, but it is difficult to know whether this clustering is due to age, time on ART, other co-morbidity, ART adherence, or other possible unmeasured confounding variables.

(5) At the individual level, there is substantial overlap between clusters according to total, intact, and % intact between the clusters. Therefore, the claim in the discussion that these 2 cluster phenotypes may require different therapeutic approaches seems rather speculative. That said, the discussion is very thoughtful about how these 2 clusters may develop with consideration of the initial insult of untreated infection and / or differences in immune recovery.

(6) The authors state that the machine learning algorithms allow for reasonable prediction of reservoir volume. It is subjective, but to me, 70% accuracy is very low. This is not a disappointing finding per se. The authors did their best with the available data. It is informative that the machine learning algorithms cannot reliably discriminate reservoir volume despite substantial amounts of input data. This implies that either key explanatory variables were not included in the models (such as viral genotype, host immune phenotype, and comorbidities) or that the outcome for testing the models is not meaningful (which may be possible with an arbitrary 50/50 split in the data relative to median HIV DNA volumes: see above).

(7) The decision tree is innovative and a useful addition, but does not provide enough discriminatory information to imply causality, mechanism, or directionality in terms of whether the immune phenotype is impacting the reservoir or vice versa or both. Tree accuracy of 80% is marginal for a decision tool.

(8) Figure 2: this is not a weakness of the analysis but I have a question about interpretation. If total HIV DNA is more predictive of immune phenotype than intact HIV DNA, does this potentially implicate a prior high burden of viral replication (high viral load &/or more prolonged time off ART) rather than ongoing reservoir stimulation as a contributor to immune phenotype? A similar thought could be applied to the fact that clustering could only be detected when applied to total HIV DNA-associated features. Many investigators do not consider defective HIV DNA to be "part of the reservoir" so it is interesting to speculate why these defective viruses appear to have more correlation with immunophenotype than intact viruses.

(9) Overall, the authors need to do an even more careful job of emphasizing that these are all just correlations. For instance, HIV DNA cannot be proven to have a causal effect on the immunophenotype of the host with this study design. Similarly, immunophenotype may be affecting HIV DNA or the correlations between the two variables could be entirely due to a separate confounding variable.

(10) In general, in the intro, when the authors refer to the immune system, they do not consistently differentiate whether they are referring to the anti-HIV immune response, the reservoir itself, or both. More specifically, the sentence in the introduction listing various causes of immune activation should have citations. (To my knowledge, there is no study to date that definitively links proviral expression from reservoir cells in vivo to immune activation as it is next to impossible to remove the confounding possible imprint of previous HIV replication.) Similarly, it is worth mentioning that the depletion of intact proviruses is quite slow such that provial expression can only be stimulating the immune system at a low level. Similarly, the statement "Viral protein expression during therapy likely maintains antigen-specific cells of the adaptive immune system" seems hard to dissociate from the persistence of immune cells that were reactive to viremia.

(11) Given the many limitations of the study design and the inability of the models to discriminate reservoir volume and phenotype, the limitations section of the discussion seems rather brief.