Reviewer #3 (Public Review):

Summary: The manuscript by Ryan and colleagues uses a well-established object recognition task to examine memory retrieval and forgetting. They show that memory retrieval requires activation of the acquisition engram in the dentate gyrus and failure to do so leads to forgetting. Using a variety of clever behavioural methods, the authors show that memories can be maintained and retrieval slowed when animals are reared in environmental enrichment and that normally retrieved memories can be forgotten if exposed to the environment in which the expected objects are no longer presented. Using a series of neural methods, the authors also show that activation or inhibition of the acquisition engram is key to memory expression and that forgetting is due to Rac1.

Strengths:<br /> This is an exemplary examination of different conditions that affect successful retrieval vs forgetting of object memory. Furthermore, the computational modelling that captures in a formal way how certain parameters may influence memory provides an important and testable approach to understanding forgetting.<br /> The use of the Rescorla-Wagner model in the context of object recognition and the idea of relevance being captured in negative prediction error are novel (but see below).<br /> The use of gain and loss of function approaches are a considerable strength and the dissociable effects on behaviour eliminate the possibility of extraneous variables such as light artifacts as potential explanations for the effects.

Weaknesses:<br /> Knowing what process (object retrieval vs familiarity) governed the behavioural effect in the present investigation would have been of even greater significance.

The impact of the paper is somewhat limited by the use of only one sex.

While relevance is an interesting concept that has been operationalized in the paper, it is unclear how distinct it is from extinction. Specifically, in the case where the animals are exposed to the context in the absence of the object, the paper currently expresses this as a process of relevance - the objects are no longer relevant in that context. Another way to think about this is in terms of extinction - the association between the context and the objects is reduced results in a disrupted ability of the context to activate the object engram.

Reviewer #3 (Public Review):

Summary:<br /> Nguyen et al show data indicating that the vomeronasal organ (VNO) and ventromedial hypothalamus (VMH) are part of a circuit that elicits defensive responses induced by predator odors. They also show that using fresh or old predator saliva may be a method to change the perceived imminence of predation. The authors also identify a family of VNO receptors that are activated by cat saliva. Next, the authors show how different components of this defensive circuit are activated by saliva, as measured by fos expression. Though interesting, the findings are not all integrated into a single narrative, and some of the results are only replications of earlier findings using modern methods. Overall, these findings provide incremental advance.

Strengths:<br /> 1 Predator saliva is a stimulus of high ethological relevance<br /> 2 The authors performed a careful quantification of fos induction across the anterior-posterior axis in Figure 6.

Weaknesses:<br /> 1 It is unclear if fresh and old saliva indeed alter the perceived imminence predation, as claimed by the authors. Prior work indicates that lower imminence induces anxiety-related actions, such as re-organization of meal patterns and avoidance of open spaces, while slightly higher imminence produces freezing. Here, the authors show that fresh and old predator saliva only provoke different amounts of freezing, rather than changing the topography of defensive behaviors, as explained above. Another prediction of predatory imminence theory would be that lower imminence induced by old saliva should produce stronger cortical activation, while fresh saliva would activate the amygdala, if these stimuli indeed correspond to significantly different levels of predation imminence.

2 It is known that predator odors activate and require AOB, VNO, and VMH, thus replications of these findings are not novel, decreasing the impact of this work.

3 There is a lack of standard circuit dissection methods, such as characterizing the behavioral effects of increasing and decreasing the neural activity of relevant cell bodies and axonal projections, significantly decreasing the mechanistic insights generated by this work.

4 The correlation shown in Figure 5c may be spurious. It appears that the correlation is primarily driven by a single point (the green square point near the bottom left corner). All correlations should be calculated using Spearman correlation, which is non-parametric and less likely to show a large correlation due to a small number of outliers. Regardless of the correlation method used, there are too few points in Figure 5c to establish a reliable correlation. Please add more points to 5c.

5 Some of the findings are disconnected from the story. For example, the authors show that V2R-A4-expressing cells are activated by predator odors. Are these cells more likely to be connected to the rest of the predatory defense circuit than other VNO cells?

6 Were there other behavioral differences induced by fresh compared to old saliva? Do they provoke differences in stretch-attend risk evaluation postures, number of approaches, the average distance to odor stimulus, the velocity of movements towards and away from the odor stimulus, etc?

Reviewer #3 (Public Review):

Summary: In this manuscript, Mure et al. describe interactions between diet, microbiome, and host development using Drosophila as a model. By characterizing microbial communities in food sources and animals, the authors showed that microbial community dynamics in the food source is critical for host development.

Strengths: This is a very interesting study where authors managed to tackle a difficult question in an elegant manner. How the interactions between different microbial species within the microbiome shape host physiology is an area of great interest but equally challenging due to the complexity of intercellular interactions in complex, host-associated microbial communities. By using a simplified model and interrogating not only microbe-microbe and host-microbe interactions, but also the role played by diet, authors were able to identify significant interactions during fly development.

Weaknesses: All weaknesses observed in the original manuscript have been corrected in the current version.

Exercise 19.5.1 The voltage will be given to us correct?

Exercise 19.3.7 What would the answer look like if both electrodes are consumed?

Exercise 19.2.1b Does it matter which side these are displayed? Mine tend to be on the opposite sides

Exercise 19.23 Similar to our last Quiz Question

Equation put on index card

Write equation on index card, will be on exam

19.3.4 Cathode is reducing and an anode is oxidating

Quiz Question, will be on exam

• for: visualizations - sea level rise at 3 Deg C

• comment

  • Look to canal cities like Venice or Amsterdam for inspiration because if it cities are salvagable, parts of them will become canal cities.

Reviewer #3 (Public Review):

In this article, Faisal et. al., use a combinatorial approach to look at the mechanisms of HIV-capsid inhibition by the highly potent drug Lenacepavir (LEN). The authors conclude that LEN induces capsid opening, but hyper-stabilizes the remaining capsid lattice during the early stages, and adversely affects the assembly of capsids during late stages of HIV-1 infection. Additionally, they suggest that hyper-stabilization effects of LEN on the capsid-lattice are induced by a low occupancy of this highly potent drug, while less potent inhibitors like PF74 need high occupancy on the lattice to induce similar effects. Taken together their findings shine a light on the importance of the capsid binding pocket targeted by multiple inhibitors including LEN, PF74, BI-2, and host-factor CPSF6 on overall capsid assembly, its stability in cells, and its role in HIV-1 infection.

Strengths:<br /> 1. Combinatorial approach using single-molecule imaging, cryoET and cellular assays show the adverse effects of LEN on HIV-1 capsid assembly, capsid disassembly, and block to virus infectivity.<br /> 2. Several novel insights are obtained in this paper, including the cryoET-data showing 2-layers of capsid formation in the presence of LEN. CPSF6-peptide binding to capsids, and their effect on stability.

Weakness:<br /> 1. Interpretation of the capsid stability data is focused on single virus traces rather than population analysis, which might paint a different picture of the conclusions.<br /> 2. The description and interpretation of the data in the results sections and the conclusions are inconsistent, and somewhat confusingly presented for the general non-expert audience.

Reviewer #3 (Public Review):

To identify direct targets of BMP signal in Nematostella, the authors performed ChIP-seq using an antibody against phosphorylated SMAD1/5 (pSMAD1/5) at late gastrula and late planula stages. In accordance with the highly dynamic BMP activity detected using immunofluorescence, pSMAD1/5 binding profiles change drastically as the larvae develop, with only a fraction of target genes shared between these two time points. The authors then followed up with RNA-seq in control versus BMP2/4 KD embryos and identified significant expression changes in many transcription factors and signaling molecules, including the Gbx-Hox genes, which are known to regulate endoderm patterning. These results, in conjunction with a thorough validation using in situ hybridization, strongly support the authors' claim that the BMP signal in Nematostella directly controls a small set of second-tier targets which in turn execute the morphogenic functions.

Next, the authors explored the conservation of BMP downstream targets by intersecting their candidate list with two published datasets from Drosophila (2-3hpf) and Xenopus (NF20 stage). Results from such an analysis should be taken with a grain of salt, as the developmental time points and biological context examined here are not necessarily comparable. Furthermore, whole genome duplication in vertebrates means multiple copies of transcription factors and signaling molecules belonging to the same family exist in Xenopus, making a homology-based comparison difficult. A handful of shared targets were identified between different species, although no statics were provided to support the significance of such an observation.

The authors then focused on Zswim4-6, one of the identified BMP targets with a high pSMAD1/5 enrichment level, and dissected its regulatory properties on BMP activity. Using complimentary knockdown and overexpression experiments, the authors rigorously demonstrated that Zswim4-6 is crucial to maintaining the proper pSMAD1/5 gradient at the late gastrula stage. By ectopically overexpressing a GFP tagged form of Zswim4-6, the authors performed low input ChIP-qPCR and confirmed that Zswim4-6 selectively binds to a regulatory region of a BMP-repressed gene, suggesting it may function as a co-repressor for certain BMP targets.<br /> Lastly, the authors examined the effect of Zswim5, a bilaterian homolog of Zswim4-6, during zebrafish D-V axis establishment. Overexpression of Zswim5 leads to a dampened pSMAD1/5 gradient and dorsalization of the fish larvae, hinting at the possibility that Zswim5 may function as a BMP modulator in zebrafish as well.

Overall, despite certain caveats, the experimental evidence supports the claims from the authors that Zswim4-6 is directly activated by and reciprocally modulates the BMP activity in Nematostella. The work presented here opens exciting possibilities to further dissect the gene regulatory networks downstream of the cnidarian BMP signaling pathway and expands our knowledge on the evolution of a bilaterally symmetric body plan.

Reviewer #3 (Public Review):

The study is interesting and the results are informative in how well people can report colors of two superimposed dot clouds. It reveals that there are trade-offs between reporting two colors. However, I have a few basic but major concerns with the present study and its conclusions about people's abilities to continuously track color values and the rate at which attention may be allocated across the two streams which I am outlining below.

1) The first concern regards the task that was used to measure continuous tracking of feature values, which in my view is ambiguous in whether it truly assesses active tracking of features or rather short-term memory of the last-seen colors. Specifically, participants were viewing two colored dot clouds that then turned gray, and were asked to report each of the colors they saw using continuous report. The test usually occurred after 6-8s (in Exp. 1 &2), so while not completely predictable, participants could easily perform the task without tracking both feature streams continuously and simply perform the color report based on the very last colors they saw. In other words, it does not seem necessary to know which color belonged to which stream, or what color it was before, to perform the task successfully. Thus, it is unclear to what extent this task is actually measuring active tracking, the same way tracking of spatial locations in multiple-object tracking tasks has been studied, which is the literature that the authors are trying to draw parallels to. In multiple-object tracking tasks, targets and nontarget objects look identical and so to keep track of which of the moving objects are targets, participants need to attend to them actively and selectively. (Similarly, the original feature-tracking study by Blaser et al., at least in their main experiment, people were asked to track an object superimposed on a second object which required continuous and selective tracking of that object).

2) The main claim that tracking two colors relies on a shared and strictly limited resource is primarily based on the relation between the two responses people give, such that the first response about one color tends to be higher accuracy than for the second response of the other color across participants. In my view, this is a relatively weak version of looking at trade-offs in resources, and it would have been more compelling to show such trade-offs at a single-trial level, or assess them with well-established methods that have been developed to look at attentional bottlenecks such as attention-operating characteristics that allow quantifying the cost of adding an additional task in a precise and much more direct manner.

3) Finally, the data of the last experiment is taken as evidence that feature-based selection oscillates at 1Hz between the two streams. This is based on response errors changing across time points with respect to an exogenous cue that is thought to "reset" attentional allocation to one stream. Only one of three data sets (which uses relatively sparse temporal sampling) shows a significant interaction between cue and time, and given that there was no a priori prediction of when such interaction should occur, this result begs for a replication to ensure that this is not a false positive result. Furthermore, based on the analyses done in the paper, it may very well be the case that the presumed "switching rate" is entirely non-oscillatory based on a recent very important paper by Geoffrey Brookshire (2022, Nature Human Behavior) that demonstrates that frequency analysis are not just sensitive to periodic but also aperiodic temporal structures. The paper also has a series of suggested analyses that could be used here to further test the current conclusions.

Reviewer #3 (Public Review):

This paper studies the role of the core PCP pathway on tissue morphogenesis of the Drosophila pupil wing. The authors used three different core PCP mutants to compare the cell dynamics with the wild type and conclude that core PCP does not guide the global patterns of cell dynamics during pupal wing morphogenesis. They use the previously published "triangle method" to extract modes of deformation (total shear, cell elongation, cell rearrangements) and find that they are the same (to within error) in the core PCP mutants. Moreover, the global shape of the wing at the end of the process is nearly the same, too.

Using laser ablation and a rheological model, the paper also investigates the effect of the core PCP pathway on the short-time mechanical properties of the tissue. The authors find that the short-time mechanical response is different in core PCP mutants. This is surprising, as most researchers in the field assume that the short-time recoil velocity is a proxy for tissue mechanics, and therefore also predictive of global tissue deformations. So the observation that the short-time recoils are different, while the global response is the same, is important for the field to understand.

A challenge with the paper as written is that it does not clearly explain how to reconcile these two observations, stating in the discussion that "the proportionality factor [which relates short-time recoil to tissue mechanics] can depend on the genotype and can change in time". It is possible that the data and model in the paper could be used to make a more convincing and clear statement.

The paper is conceptually interesting, methodologically sound, and likely impactful to the broad area of tissue mechanics and mechanobiology.

Reviewer #3 (Public Review):

In this study, the authors present the first comprehensive transcriptome map of the human locus coeruleus using two independent but complementary approaches, spatial transcriptomics and single-nucleus RNA sequencing. Several canonical features of locus coeruleus neurons that have been described in rodents were conserved, but potentially important species differences were also identified. This work lays the foundation for future descriptive and experimental approaches to understanding the contribution of the locus coeruleus to healthy brain function and disease.

This study has many strengths. It is the first reported comprehensive map of the human LC transcriptome and uses two independent but complementary approaches (spatial transcriptomics and snRNA-seq). Some of the key findings confirmed what has been described in the rodent LC, as well as some intriguing potential genes and modules identified that may be unique to humans and have the potential to explain LC-related disease states. The main limitations of the study were acknowledged by the authors and include the spatial resolution probably not being at the single cell level and the relatively small number of samples (and questionable quality) for the snRNA-seq data. Overall, the strengths greatly outweigh the limitations. This dataset will be a valuable resource for the neuroscience community, both in terms of methodology development and results that will no doubt enable important comparisons and follow-up studies.

Reviewer #3 (Public Review):

In this manuscript, the authors define the developmental trajectory resulting in a diverse mTEC compartment. Using a variety of approaches, including a novel CCL21-fate mapping model, data is presented to argue that embryonic CCL21-expressing thymocyte attracting mTECs naturally convert to into self-antigen displaying mTEC subsets, including Aire+ mTECs and thymic tuft cells. Perhaps somewhat surprisingly, a large fraction of cTECs were also marked for having expressed CCL21, suggesting that there exists some conversion of mTEC (progenitors) into cTEC, a developmentally interesting observation that could be followed up later. Overall, the experimental setup, writing, and conclusions, are all outstanding.

Reviewer #3 (Public Review):

Summary: Despite being preventable and treatable, cervical cancer remains the second most common cause of cancer death globally. This cancer, and associated deaths, occur overwhelmingly in low- and middle-income countries (LMIC), reflecting a lack of access to vaccination, screening and treatment services. Cervical screening is the second pillar in the WHO strategy to eliminate cervical cancer as a public health problem and will be critical in delivering early gains in cervical cancer prevention as the impact of vaccination will not be realized for several decades. However, screening strategies implemented in high income countries are not feasible or affordable in LMICs. This ambitious multi-center study aims to address these issues by developing and systematically evaluating a novel approach to cervical screening. The approach, based on primary screening with self-collected specimens for HPV testing, is focused on optimizing triage of people in whom HPV is detected, so that sensitivity for the detection of pre-cancer and cancer is maximized while treatment of people without pre-cancer or cancer is minimized.

Strengths:

The triage proposed for this study builds on the authors' previously published work in designing the ScreenFire test to appropriately group the 13 detected genotypes into four channels and to develop automated visual evaluation (AVE) of images of the cervix, taken by health workers.

The move from mobile telephone devices to a dedicated device to acquire and evaluate images, overcomes challenges previously encountered whereby updates of mobile phone models required retraining of the AVE algorithm.

The separation of the study into two phases, an efficacy phase in which screen positive people will be triaged and treated according to local standard of care and the performance of AVE will be evaluated against biopsy outcomes will be followed by the second phase in which the effectiveness, cost-effectiveness, feasibility and acceptability will be evaluated.

The setting in a range of low resource settings which are geographically well spread and reflective of where the global cancer burden is highest.

Weaknesses:

Potential ascertainment bias due to the lack of specified biopsy (such as small four quadrant biopsies or small biopsies across the transformation zone) when aceto-white areas are not identified. This has the potential lead to lead to an over-estimate of sensitivity of the triage approach, particularly in the setting of VIA as compared to colposcopy. While the authors specify endocervical sampling in this setting, using curette or brush (for cytology), this may not be as sensitive unless clinicians are experienced in endocervical curette procedures.

Reviewer #3 (Public Review):

Summary:<br /> The authors focus on the role of formin-like protein 2 in the mouse oocyte, which could play an important role in actin filament dynamics. The cytoskeleton is known to influence a number of cellular processes from transcription to cytokinesis. The results show that downregulation of FMNL2 affects spindle migration with resulting abnormalities in cytokinesis in oocyte meiosis I.

Weaknesses:<br /> The overall description of methods and figures is overall dismissively poor. The description of the sample types and number of replicate experiments is impossible to interpret throughout, and the quantitative analysis methods are not adequately described. The number of data points presented is unconvincing and unlikely to support the conclusions. On the basis of the data presented, the conclusions appear to be preliminary, overstated, and therefore unconvincing.

Reviewer #3 (Public Review):

In this improved version of the manuscript, Chang et al set out to find direct interactions with the Eph-B2 receptor, as our knowledge of its function/regulation is still incomplete. Using proteomic analysis of Hela cells expressing EPHB2, they identified MYCBP2 a potential binder, which they then confirm using extensive biochemical analyses, an interaction that seems to be negatively affected by binding of ephrin-B2 (but not B1). Furthermore, they find that FBXO45, a known MYCBP2 interaction, strongly facilitates its binding to EPHB2. Intriguingly, these interactions depend on the extracellular domains of EPHB2, suggesting the involvement of additional proteins as MYCBP2 is thought to be a cytoplasmic protein. Finally, they find that, in contrast to what could be expected given the known function of MYCBP2 as a ubiquitin E3 ligase, it actually positively regulates EPHB2 protein stability, and function.

The strength of this manuscript is the extensive biochemical analysis of the EPHB2/MYCBP2/FBXO43 interactions. The vast majority of the conclusions are supported by the data.

The attempt to extend the study to an in vivo animal using the worm is important, however the additive insight is, unfortunately, minimal.

Reviewer #3 (Public Review):

Summary:

In this manuscript, Krause and colleagues identify miR-182 as diabetes-associated microRNA: miR-182 is increased in bariatric surgery patients with versus without T2D; miR-182 was the only microRNA associated with three metabolic traits; miR-182 levels were associated with increased body weight in mice under different dietary manipulations; overexpression in Hep-G2 led to a decrease in LRP6; and overexpression in HFD fed mice led to increased insulin and liver TG. The manuscript provides a potentially useful resource for microRNA expression in human livers, though the functional importance of miR-182 remains unclear.

Strengths:

The use of human tissues and good sample sizes is strong.

Weaknesses:

The study is primarily correlative; the in vivo overexpression is non-physiological; and the mechanisms by which miR-182 exerts its effects are not rigorously tested.

Reviewer #3 (Public Review):

In this work, Kita et al., aim to understand the activation mechanisms of the kinesin-3 motors KLP-6 and UNC-104 from C. elegans. As many other motor proteins involved in intracellular transport processes, KLP-6 and UNC-104 motors suppress their ATPase activities in the absence of cargo molecules. Relieving the autoinhibition is thus a crucial step that initiates directional transport of intracellular cargo. To investigate the activation mechanisms, the authors make use of mass photometry to determine the oligomeric states of the full length KLP-6 and the truncated UNC-104(1-653) motors at sub-micromolar concentrations. While full length KLP-6 remains monomeric, the truncated UNC-104(1-653) displays a sub-population of dimeric motors that is much more pronounced at high concentrations, suggesting a monomer-to-dimer conversion. The authors push this equilibrium towards dimeric UNC-104(1-653) motors solely by introducing a point mutation into the coiled-coil domain and ultimately unleash a robust processivity of the UNC-104 dimer. The authors find that the same mechanistic concept does not apply to the KLP-6 kinesin-3 motor, suggesting an alternative activation mechanism of the KLP-6 that remains to be resolved. The present study encourages further dissection of the kinesin-3 motors with the goal of uncovering the main factors needed to overcome the 'self-inflicted' deactivation.

Reviewer #3 (Public Review):

In this study, Kierdorf and colleagues investigated the function of hemocytes in oxidative stress response and found that non-canonical DNA damage response (DDR) is critical for controlling JNK activity and the expression of cytokine unpaired3. Hemocyte-mediated expression of upd3 and JNK determines the susceptibility to oxidative stress and systemic energy metabolism required for animal survival, suggesting a new role for hemocytes in the direct mediation of stress response and animal survival.

In the revised manuscript, the authors provide additional evidence to support the role of DNA damage-modulated cytokine release by hemocytes during oxidative stress responses and strengthen the connection between DNA damage and the regulation of upd3 release from hemocytes. The authors have also included new analyses to emphasize the significance of hemocytes in the regulation of energy during oxidative stress. Following the reviewers' suggestions, the authors made improvements to the manuscript and the graphical abstract to better display their findings. Overall, the revised manuscript makes it easier to understand the main points, flows better, and is supported by convincing data and analysis throughout.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript by Coberski et al describes a combined experimental and computational study aimed to shed light on the catalytic mechanism in a methyltransferase that transfers a methyl group from S-adenosylmethionine (SAM) to a substrate adenosine to form N6-methyladenosine (m6A).

Strengths:<br /> The authors determine crystal structures in complex with so-called bi-substrate analogs that can bridge across the SAM and adenosine binding sites and mimic a transition state or intermediate of the methyl-transfer reaction. The crystal structures suggest dynamical motions of the substrate(s) that are examined further using classical MD simulations. The authors then use QM/MM calculations to study the methyl-transfer process. Together with biochemical assays of ligand/substrate binding and enzyme turnover, the authors use this information to suggest what the key steps are in the catalytic cycle. The manuscript is in most places easy to read.

Weaknesses:<br /> My main suggestion for the authors is that they show better how their conclusions are supported by the data. This includes how the electron density maps for example support the key interactions and water molecules in the active site and a better error analysis of the computational analyses.

Reviewer #3 (Public Review):

Summary:<br /> The authors were trying to show that a novel neuronal metallothionein of poorly defined function, GIF/MT3, is actually heavily persulfidated in both the Zn-bound and apo (metal-free) forms of the molecule as purified from a heterologous or native host. Evidence in support of this conclusion is compelling, with both spectroscopic and mass spectrometry evidence strongly consistent with this general conclusion. The authors would appear to have achieved their aims.

Strengths:<br /> The analytical data are compelling in support of the author's primary conclusions are strong. The authors also provide some modeling evidence that strongly supports the contention that MT3 (and other MTs) can readily accommodate sulfane sulfur on each of the 20 cysteines in the Zn-bound structure, with little perturbation of the structure. This is not the case with Cys trisulfides, which suggests that the persulfide-metallated state is clearly positioned at lower energy relative to the immediately adjacent thiolate- or trisulfidated metal coordination complexes.

Weaknesses:<br /> The biological significance of the findings is not entirely clear. On the one hand, the analytical data are clearly solid (albeit using a protein derived from a bacterial over-expression experiment), and yes, it's true that sulfane S can protect Cys from overoxidation, but everything shown in the summary figure (Fig. 8D) can be done with Zn release from a thiol by ROS, and subsequent reduction by the Trx/TR system. In addition, it's long been known that Zn itself can protect Cys from oxidation. I view this as a minor weakness that will motivate follow-up studies. Fig. 1 was incomplete in its discussion and only suggests that a few S atoms may be covalently bound to MT3 as isolated. This is in contrast to the sulfate S "release" experiment, which I find quite compelling.

Impact:<br /> The impact will be high since the finding is potentially disruptive to the metals in the biology field in general and the MT field for sure. The sulfane sulfur counting experiment (the HPE-IAM electrophile trapping experiment) may well be widely adopted by the field. Those of us in the metals field always knew that this was a possibility, and it will interesting to see the extent to which metal-binding thiolates broadly incorporate sulfate sulfur into their first coordination shells.

Reviewer #3 (Public Review):

Summary:<br /> This manuscript describes some biochemical experiments on the crucial virulence factor EsxA (ESAT-6) of Mycobacterium tuberculosis. EsxA is secreted via the ESX-1 secretion system. Although this system is recognized to be crucial for virulence the actual mechanisms employed by the ESX-1 substrates are still mostly unknown. The EsxA substrate is attracting the most attention as the central player in virulence, especially phagosomal membrane disruption. EsxA is secreted as a dimer together with EsxB. The authors show that EsxA is also able to form homodimers and even tetramers, albeit at very low pH (below 5). Furthermore, the addition of a nanobody that specifically binds EsxA blocks intracellular survival, as well as if the nanobody is produced in the cytosol of the infected macrophages.

Strengths:<br /> -Decent biochemical characterization of EsxA and identification of a new and interesting tool to study the function of EsxA (nanobody).

-The manuscript is well-written.

Weaknesses:<br /> The findings are not critically evaluated using extra experiments or controls.

For instance, tetrameric EsxA in itself is interesting and could reveal how EsxA works. But one would say that this is a starting point to make small point mutations that specifically affect tetramer formation and then evaluate what the effect is on phagosomal membrane lysis. Also one would like to see experiments to indicate whether these structures can be produced under in vitro conditions, especially because it seems that this mainly happens when the pH is lower than 5, which is not normally happening in phagosomes that are loaded with M. tuberculosis.

Also, the fact that the addition of the nanobody, either directly to the bacteria or produced in the cytosol of macrophages is interesting, but again it is the starting point for further experimentation. As a control, one would like to see the effect on an Esx-1 secretion mutant. Furthermore, does cytosolic production or direct addition of the nanobody affect phagosomal escape? What happens if an EsxA mutant is produced that does not bind the nanobody?

Finally, it is a bit strange that the authors use a non-native version of esxA that has not only an additional His-tag but also an additional 12 amino acids, which makes the protein in total almost 20% bigger. Of course, these additions do not have to alter the characteristics, but they might. On the other hand, they easily discard the natural acetylation of EsxA by mycobacteria itself (proven for M. marinum) as not relevant for the function because it might not happen in (the close homologue) M. tuberculosis.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

2. The article identified a potential pathogenesis of AIS.

Weaknesses:

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

Reviewer #3 (Public Review):

Summary:

In this study, Warfvinge and colleagues use CITE-seq to interrogate how CML stem cells change between diagnosis and after one year of TKI therapy. This provides important insight into why some CML patients are "optimal responders" to TKI therapy while others experience treatment failure. CITE-seq in CML patients revealed several important findings. First, substantial cellular heterogeneity was observed at diagnosis, suggesting that this is a hallmark of CML. Further, patients who experienced treatment failure demonstrated increased numbers of primitive cells at diagnosis compared to optimal responders. This finding was validated in a bulk gene expression dataset from 59 CML patients, in which it was shown that the proportion of primitive cells versus lineage-primed cells correlates to treatment outcome. Even more importantly, because CITE-seq quantifies cell surface protein in addition to gene expression data, the authors were able to identify that BCR/ABL+ and BCR/ABL- CML stem cells express distinct cell surface markers (CD26+/CD35- and CD26-/CD35+, respectively). In optimal responders, BCR/ABL- CD26-/CD35+ CML stem cells were predominant, while the opposite was true in patients with treatment failure. Together, these findings represent a critical step forward for the CML field and may allow more informed development of CML therapies, as well as the ability to predict patient outcomes prior to treatment.

Strengths:

This is an important, beautifully written, well-referenced study that represents a fundamental advance in the CML field. The data are clean and compelling, demonstrating convincingly that optimal responders and patients with treatment failure display significant differences in the proportion of primitive cells at diagnosis, and the ratio of BCR-ABL+ versus negative LSCs. The finding that BCR/ABL+ versus negative LSCs display distinct surface markers is also key and will allow for a more detailed interrogation of these cell populations at a molecular level.

Weaknesses:

CITE-seq was performed in only 9 CML patient samples and 2 healthy donors. Additional samples would greatly strengthen the very interesting and notable findings.

Reviewer #3 (Public Review):

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

The authors have addressed my previous comments.

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

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

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

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

Reviewer #3 (Public Review):

The authors introduce two new concepts for antimicrobial resistance borrowed from pharmacology, "variant vulnerability" (how susceptible a particular resistance gene variant is across a class of drugs) and "drug applicability" (how useful a particular drug is against multiple allelic variants). They group both terms under an umbrella term "drugability". They demonstrate these features for an important class of antibiotics, the beta-lactams, and allelic variants of TEM-1 beta-lactamase. In the revised version, they investigate a second drug class that targets dihydrofolate reductase in Plasmodium (the causative agent of malaria).

The strength of the result is in its conceptual advance and that the concepts seem to work for beta-lactam resistance and DHFR inhibitors in a protozoan. However, I do not necessarily see the advance of lumping both terms under "drugability", as this adds an extra layer of complicaton in my opinion.

I think that the utility of the terms will be more comprehensively demonstrated by using examples across a breadth of drug classes classes and/or resistance genes. For instance, another good bacterial model with published data might have been trimethoprim resistance, which arises through point mutations in the folA gene (although, clinical resistance tends to be instead conferred by a suite of horizontally acquired dihydrofolate reductase genes, which are not so closely related as the TEM variants explored here).

The impact of the work on the field depends on a more comprehensive demonstration of the applicability of these new concepts to other drugs. This would be demonstrated in future work.

Reviewer #3 (Public Review):

Summary:<br /> The authors have undertaken a study to rigorously characterize the possible role of eIF2A in regulating translation in yeast. The authors test for the role of eIF2A in the absence or presence of cellular stress and conclude that eIF2A does not play any significant role in regulating translation initiation in yeast.

Strengths:<br /> The authors have used rigorous experimental approaches, including genome-wide ribosome profiling analysis in the absence or presence of stress, to show that eIF2A does not function in translation initiation on most mRNAs in yeast. Interestingly, the authors do identify a small number of mRNAs that possess some eIF2A dependency, so they constructed reporters to rigorously test them. One mRNA, HKR1, appears to possess a degree of eIF2A-dependent translation regulation.

Weaknesses:<br /> While no role of eIF2A in translation initiation is apparent, the authors do not determine what function eIF2A does play in yeast. Whether it plays a role in regulating translation in a different stress response is not determined.

Reviewer #3 (Public Review):

The manuscript from Mandal et al. aims to show that the actin cytoskeleton is the key mechanosensitive element in cytotoxic T lymphocytes, enabling them to discriminate between target cells of different cortical stiffness. They further examine whether WASP activation is sensitive to substrate stiffness, and thus modulates actin polymerization and early T cell signaling in a mechanosensitive manner. Overall, the mechanosensitivity of CTLs has attracted a lot of attention in the last few years and this study explores new and interesting facets. The manuscript asks an important question regarding the mechanisms underlying the stiffness dependent response observed in T cells. The authors have used a variety of techniques ranging from mouse models and in vivo studies, cell biological manipulations and biophysical measurements which is commendable. Their work suggests that the actin cytoskeleton regulated by WASP plays a key role in mechanosensitivity - which is an intriguing finding.

While this manuscript has wide-ranging experiments and interesting results, a number of points need to be carefully addressed to support the central claims.

The first major issue is that the irreversible actin inhibitor myca can have a number of non-specific effects on CTL activation. It is not clear that the effects observed are due to the change in stiffness alone. Since Myca depolymerizes actin, the B16 target cells would have altered MHC mobility or impaired receptor-ligand engagement - which might affect actin foci formation and signaling. There is also no gain of function experiment, wherein the stiffness of the target cell is enhanced. Moreover, there are two populations in both the control and myca-treated Young's modulus histograms for B16 cells. Are these sub-populations fundamentally different in their cytoskeletal organization? This can also confound or introduce variability in results on stiffness-dependence of CTL function, given the second sub-population of Myca-treated cells overlaps with the first sub-population of control cells. The authors need to provide a justification for these.

Secondly, the WASP knockout still shows mechanosensitivity but at reduced force levels (Fig. 3B). Similarly, other measures (Fig. 3) still show increases with stiffness. Thus, it is not clear whether WASP is necessary for mechanosensing but simply for maintaining force levels and (expectedly) lower actin levels and foci in the WASP knockout. In fact, Fig 3 implies WASP is required for signaling and not for mechanosensing, undermining the main claim of the paper. At the very least, ANOVA or factor analysis (stiffness x WASP) needs to be done to demonstrate the requirement of WASP for CTL mechanosensitivity.

Third, there are some concerns regarding the traction force microscopy. The authors do not present key details in the manuscript about the methods used. Secondly, the traction values are entirely too high compared to reported values in the literature for CTLs. A back-of-the-envelope calculation of the total force yields ~30 nN for wild-type cells) on 10 kPa gels, which is about an order of magnitude higher than reported values (Tamzalit et al. 2020, Hui et al. 2017, Bashour et al. 2014, Pathni et al. 2022). The authors should clearly demonstrate and justify that their measured values are reasonable and accurate. The lack of representative movies and displacement maps used for the traction force measurements make it hard to evaluate the results. Typical bead displacements for CTLs on softer gels are on the order of 1 micron (Mustapha et al. 2022), which should decrease to 0.1 micron or less on 50 kPa gels. These would make the tractions hard to estimate accurately. The authors should evaluate and show the displacements underneath the cell and outside the cell boundaries to give estimates of the noise floor for tractions. Finally, there is no discussion of how the tractions were calculated from the displacements - was Fourier Transform or Finite element method used? What is the noise level of the measurements and how were the traction estimates regularized?

Fourth, many of the plots in the manuscripts are not accompanied by representative images to show how these aspects (distribution of actin and signaling markers for example) change qualitatively under different conditions (e.g. stiffness). Details of analysis and quantification need to be provided for a clearer understanding of the results and interpretations. All figures and captions should include information about the number of cells and experiments. Along these lines, there is very little detail in the methods, statistical power, calculations are not mentioned, there is little description of the pmel-1 knockout mouse, all of which make it hard to evaluate the soundness of the results.

Finally, the study as presented, doesn't conclusively show that WASP is required for mechanosensitive CTL function. The results presented show that WASP is required for early and longer-term signaling events and cytolytic activity, and that knocking out WASP reduces early TCR signaling, actin foci formation in response to substrate stiffness. To make the claim of WASP-mediated regulation of CTL mechanosensitivity stronger, it would be helpful to see how WASP knockout affects CTL killing in response to softened and (possibly) stiffened B16 targets.

Reviewer #3 (Public Review):

The authors investigate the role of commensal microbes and molecules in the antigen presentation pathway in the development and phenotype of CD8 T cells specific for the Qa-1b-restricted peptide FL9 (QFL). The studies track both endogenous QFL-specific T cells and utilize a recently generated TCR transgenic model. The authors confirm that QFL-specific T cells in the spleen and small intestine intraepithelial lymphocyte (IEL) pool show an antigen-experienced phenotype as well as unique phenotypic and innate-like functional traits, especially among CD8+ T cells expressing Va3.2+ TCRs. They find that deficiency in the TAP transporter leads to almost complete loss of QFL-specific T cells but that loss of either Qa1 or the ERAAP aminopeptidase does not impact QFL+ T cell numbers but does cause them to maintain a more conventional, naïve-like phenotype. In germ-free (GF) mice, the QFL-specific T cells are present at similar numbers and with a similar phenotype to SPF animals, but in older animals (>18w) there is a notable loss of IEL QFL-specific cells. This drop can be avoided by neonatal colonization of GF mice with the commensal microbe Pediococcus pentosaceus but not a different commensal, Lactobacillus johnsonii, and the authors show that P. pentosaceus encodes a peptide that weakly stimulates QFL-specific T cells, while the homologous peptide from L. johnsonii does not stimulate such cells.

This study provides new insights into the way in which the differentiation, phenotype, and function of CD8+ T cells specific for Qa-1b/FL9 is regulated by peptide processing and Qa1 expression, and by interactions with the microbiota. The approaches are well designed, the data compelling, and the interpretation, for the most part, appropriate.

The response to several of my concerns involved reference to a different manuscript from the authors (which has not been through peer review), and for point #3, it would have been useful to provide experimental evidence (e.g., competitive inhibition assays) to justify their hypothesis that P4 serves as a TCR contact while P6 may be a Qa-1b contact residue. Nevertheless, the authors have made considerable efforts to clarify their approaches and interpretation, which strengthens the manuscript.

Reviewer #3 (Public Review):

In this work, the authors conduct transcriptional profiling experiments with Mtb under various different stress conditions (oxidative, nitrosative, low pH, starvation, and SDS). The Mtb transcriptional responses to these stress conditions are not particularly new, having been reported extensively in the literature over the past ~20 years in various forms. A common theme from the current work is that L-cysteine synthesis genes are seemingly up-regulated by many stresses. Thus, the authors focused on deleting two of the three L-cysteine synthesis genes (cysM and cysK2) in Mtb to better understand the roles of these genes in Mtb physiology.

The cysM and cysK2 mutants display fitness defects in various media (Sautons media, starvation, oxidative and nitrosative stress) noted by CFU reductions. Transcriptional profiling studies with the cysM and cysK2 mutants revealed that divergent gene signatures are generated in each of these strains under oxidative stress, suggesting that cysM and cysK2 have non-redundant roles in Mtb's oxidative stress response which likely reflects the different substrates used by these enzymes, CysO-L-cysteine and O-phospho-L-serine, respectively. Note that these studies lack genetic complementation and are thus not rigorously controlled for the engineered deletion mutations.

The authors quantify the levels of sulfur-containing metabolites (methionine, ergothioneine, mycothiol, mycothionine) produced by the mutants following exposure to oxidative stress. Both the cysM or cysK2 mutants produce more methionine, ergothioneine, and mycothionine relative to WT under oxidative stress. Both mutants produce less mycothiol relative to WT under the same condition. These studies lack genetic complementation and thus, do not rigorously control for the engineered mutations.

Next, the mutants were evaluated in infection models to reveal fitness defects associated with oxidative and nitrosative stress in the cysM or cysK2 mutants. In LPS/IFNg activated peritoneal macrophages, the cysM or cysK2 mutants display marked fitness defects which can be rescued with exogenous cysteine added to the cell culture media. Peritoneal macrophages lacking the NADPH oxidase (Phox) or IFNg fail to produce fitness phenotypes in the cysM or cysK2 mutants suggesting that oxidative stress is responsible for the phenotypes. Similarly, chemical inhibition of iNOS partly abrogated the fitness defect of the cysM or cysK2 mutants. Similar studies were conducted in mice lacking IFNg and Phox establishing that cysM or cysK2 mutants have fitness defects in vivo that are dependent on oxidative and nitrosative stress.

Lastly, the authors use small molecule compounds to inhibit cysteine synthases. It is demonstrated that the compounds display inhibition of Mtb growth in 7H9 ADC media. No evidence is provided to demonstrate that these compounds are specifically inhibiting the cysteine synthases via "on-target inhibition" in the whole Mtb cells. Additionally, it is wrongly stated in the discussion that "combinations of L-cys synthase inhibitors with front-line TB drugs like INH, significantly reduced the bacterial load inside the host". This statement suggests that the INH + cysteine synthase inhibitor combinations reduce Mtb loads within a host in an infection assay. No data is presented to support this statement.

https://www.maxtonandcompany.com/alfion

Maxton & Company also carries Notsu cards.

Reviewer #3 (Public Review):

Summary:<br /> Here, Osnes et al examine the population dynamics of Neisseria gonorrhoeae. They develop new methodologies to deal with the issue of recombination, as well as using ancestral state reconstruction approaches to quantify the number of import and export transmission events occurring in different regions in the world. Overall, they provide a framework for understanding intercontinental transmission that could be applied to other microbial pathogens.

Strengths:<br /> A major strength of this study is the incredibly large number of genomes analysed, which span a wide temporal range with significant geographical diversity. The use of ancestral state reconstruction to quantitatively determine the number of import and export events of N. gonorrhoeae in densely sampled Norway and Victoria, Australia, is an interesting application of this well-known method and could be applied to other bacterial species that have been well-sampled.

Weaknesses:<br /> The methods development to deal with the issue of recombination in their dataset to ensure that the recombination signal does not affect their dating estimates and effective population size analysis is thorough but has likely not been able to remove all bias. Additionally, the authors discuss the utility of using the identified transmission lineages in this study to better type N. gonorrhoeae as there are issues with traditional typing, such as MLST, due to the highly recombinogenic nature of this species. However, no method seems to be provided to enable future researchers to easily assign their genomes to the transmission lineages identified in this study.

Reviewer #3 (Public Review):

The authors develop a variational autoencoder (VAE), termed d-VAE (or distill VAE) that aims to tease apart the behaviorally relevant and irrelevant sections of each neuron's firing rate. The input to the VAE is the population activity for a given time step, and the output is the inferred behaviorally relevant section of the population activity at that time step. The residual is referred to as behaviorally irrelevant: total neural activity = behaviorally relevant + behaviorally irrelevant (x = x_r + x_i). The mapping from the raw neural signals (x) to the bottlenecked latent in the autoencoder (called z, z=f(x)) and back to the inferred behaviorally relevant single-neuron activities (x_r = g(z)) is applied per time step (does not incorporate any info from past/future time steps) and, critically, it is nonlinear (f and g are nonlinear feedforward neural networks). The key technical novelty that encourages x_r to encode behaviorally relevant information is a term added to the loss, which penalizes bad linear behavior decoding from the latent z. Otherwise the method is very similar to a prior method called pi-VAE, which should be explained more thoroughly in the manuscript to clearly highlight the technical novelty.

The authors apply their method to 3 non-human primate datasets to infer behaviorally relevant signals and contrast them with the raw neural signals and the residual behaviorally irrelevant signals. As a key performance metric, they compute the accuracy of decoding behavior from the inferred behaviorally relevant signals (x_r) using a linear Kalman filter (KF) or alternatively using a nonlinear feed forward neural network (ANN). They highlight 3 main conclusions in the abstract: first, that single neurons from which behavior is very poorly decodable do encode considerable behavior information in a nonlinear manner, which the ANN can decode. Second, they conclude from various analyses that behavior is occupying a higher dimensional neural space than previously thought. Third, they find that linear KF decoding and nonlinear ANN decoding perform similarly when provided with the inferred behaviorally relevant signals (x_r), from which they conclude that a linear readout must be performed in motor cortex.

The paper is well-written in many places and has high-quality graphics. The questions that it aims to address are also of considerable interest in neuroscience. However, unfortunately, several main conclusions, including but not limited to all 3 conclusions that are highlighted in the abstract, are not fully supported by the results due to confounds, some of which are fundamental to the method. Several statements in the text also seem inaccurate due to use of imprecise language. Moreover, the authors fail to compare with some more relevant existing methods that are specifically designed for extracting behaviorally relevant signals. In addition, for some of the methods they compare with, they do not use an appropriate setup for the benchmark methods, rendering the validation of the proposed method unconvincing. Finally, in many places imprecise language that is not accompanied with an operational definition (e.g., smaller R2 [of what], similar [per what metric]) makes results hard to follow, unless most of the text is read very carefully. Some key details of the methods are also not explained anywhere.

Reviewer #3 (Public Review):

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

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

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

Reviewer #3 (Public Review):

Summary:<br /> Non-enzymatic replication of RNA or a similar polymer is likely to be important for the origin of life. The authors present a model of how a functional catalytic sequence could emerge from a mixture of sequences undergoing non-enzymatic replication.

Strengths:<br /> Interesting model describing details of the proposed replication mechanism.

Weaknesses:<br /> A discussion of the virtual circular genome idea proposed in [33] is included in the discussion section together with the problem of sequence scrambling faced by this mechanism that was raised in [34]. However, the authors state that sequence scrambling is a special case of the classical error catastrophe. This should be reworded, because these phenomena are completely different. The error catastrophe occurs due to single-point mutational errors in a model that assumes that a complete template is being copied in one cycle. Sequence scrambling arises in models that assume cycles of melting and reannealing, in which case only part of a template is copied in one cycle. Scrambling is due to the many alternative ways in which pairs of sequences can reanneal. Many of these alternatives are incorrect and this leads to the disappearance of the original sequence. This problem exists even in the limit where there is zero mutational error rate. Therefore, it cannot be called a special case of the error catastrophe problem.

The authors seem to believe that their model avoids the scrambling problem. If this is the case, a clear explanation should be added about why this problem is avoided. Two possible points are mentioned.<br /> (i) Replication is bidirectional in this model. This seems like a small detail to me. I don't think it makes any difference to whether scrambling occurs.<br /> (ii) The functional activity is located in a short sequence region. I can imagine that if the length of a strand that is synthesized in a single cycle is long enough to cover the complete functional region, then sometimes the complete functional sequence can be copied in one cycle. Is this what is being argued? If so, it depends a lot on rates of primer extension and lengths of melting cycles etc, and some comment on this should be made.

Reviewer #3 (Public Review):

In their study the authors analyze the localization of multiple organelles and subcellular structure of blood stage malaria parasites with unprecedented detail. They use a 3D super-resolution imaging technique that has gained popularity in the protozoan field, ultrastructure expansion microscopy. Building on markers and labels established in the field they generate an appealing collection of images for all stages of the intraerythrocytic developmental stages of asexual blood stage parasites with some focus on nuclear division and cell segmentation stages.

The authors have made a very good effort to address all the comments raised by the reviewers providing more clarity to the manuscript and appropriate interpretations of their results. Particularly the sharing of their image data in the Dryad repository adds significant value to their work.

Reviewer #3 (Public Review):

Summary:<br /> In this work, the authors propose a novel method for analyzing spiking neuron network models with delays. By modeling the delay as an additional axonal component to relay spikes, the infinite-dimensional system of the delayed network is transformed into a system of finite dimensions. This allows the calculation of the entire spectrum of Lyapunov exponents which provide information on the dimensionality of attractor and noise entropy of network responses. The authors demonstrate that chaos intensifies at the onset of oscillations as synaptic delay increases. This is surprising since network oscillation has been thought to indicate regular firing activity. The authors find similar results in different types of networks and in networks driven by oscillatory inputs, suggesting that the boosting of chaos by oscillation can be a general feature of spiking networks.

Strengths:<br /> This work builds on the authors' past work on characterizing chaos in spiking networks and extends to include synaptic delays. The transformation of a delayed network into a network of two-compartment neurons, modeling the spike generation and transmission, is novel and interesting. This allows for an analytical expression of the single spike Jacobian of the network dynamics, which can be used to calculate the full spectrum of Lyapunov exponents.

The analysis is rigorous and the parameter study is comprehensive.

Weaknesses:<br /> Because the delayed interaction is spike-triggered, effectively it only requires N variables to count the remaining time since the last spike from each neuron. The axon component only implements the delay time to transmit a spike with no interaction with other neurons. It seems that the axon component can be simply modeled as a variable counting the time since the last spike and does not need to be modeled as a QIF model. Is there any advantage of modeling the axon component as a QIF model? The supplemental figure S2 considers the case of "dynamic delay", where delay time can depend on network activity, but the Lyapunov exponents seem to be largely independent of the reset parameter.

In most of the results, the network mean firing rate is kept at a fixed value while the delay time parameter varies. What would be the results if only the delay parameter changes? It would be helpful if the authors could provide some reasoning as to why it is a better comparison with the network rate kept as a constant.

The majority of the neurons have a CV below 1 (Fig 2d and Fig S3c). This indicates that many neurons are in the mean-driven regime. This is different from balanced networks where CVs are around 1. It would be helpful for the authors to comment on this discrepancy.

Reviewer #3 (Public Review):

This study utilizes saccade metrics to explore, what the authors term the "past and future" of working memory. The study features an original design: in each trial, two pairs of stimuli are presented, first a vertical pair and then a horizontal one. Between these two pairs comes the cue that points the participant to one target of the first pair and another of the second pair. The task is to compare the two cued targets. The design is novel and original but it can be split into two known tasks - the first is a classic working memory task (a post-cue informs participants which of two memorized items is the target), which the authors have used before; and the second is a classic spatial attention task (a pre-cue signal that attention should be oriented left or right), which was used by numerous other studies in the past. The combination of these two tasks in one design is novel and important, as it enables the examination of the dynamics and overlapping processes of these tasks, and this has a lot of merit. However, each task separately is not new. There are quite a few studies on working memory and microsaccades and many on spatial attention and microsaccades. I am concerned that the interpretation of "past vs. future" could mislead readers to think that this is a new field of research, when in fact it is the (nice) extension of an existing one. Since there are so many studies that examined pre-cues and post-cues relative to microsaccades, I expected the interpretation here to rely more heavily on the existing knowledge base in this field. I believe this would have provided a better context of these findings, which are not only on "past" vs. "future" but also on "working memory" vs. "spatial attention".

Reviewer #3 (Public Review):

Summary:<br /> The authors used cTBS TMS, magnetic resonance spectroscopy (MRS), and functional magnetic resonance imaging (fMRI) as the main methods of investigation. Their data show that cTBS modulates GABA concentration and task-dependent BOLD in the ATL, whereby greater GABA increase following ATL cTBS showed greater reductions in BOLD changes in ATL. This effect was also reflected in the performance of the behavioural task response times, which did not subsume to practice effects after AL cTBS as opposed to the associated control site and control task. This is in line with their first hypothesis. The data further indicates that regional GABA concentrations in the ATL play a crucial role in semantic memory because individuals with higher (but not excessive) GABA concentrations in the ATLs performed better on the semantic task. This is in line with their second prediction. Finally, the authors conducted additional analyses to explore the mechanistic link between ATL inhibitory GABAergic action and semantic task performance. They show that this link is best captured by an inverted U-shaped function as a result of a quadratic linear regression model. Fitting this model to their data indicates that increasing GABA levels led to better task performance as long as they were not excessively low or excessively high. This was first tested as a relationship between GABA levels in the ATL and semantic task performance; then the same analyses were performed on the pre and post-cTBS TMS stimulation data, showing the same pattern. These results are in line with the conclusions of the authors.

Strengths:<br /> I thoroughly enjoyed reading the manuscript and appreciate its contribution to the field of the role of the ATL in semantic processing, especially given the efforts to overcome the immense challenges of investigating ATL function by neuroscientific methods such as MRS, fMRI & TMS. The main strengths are summarised as follows:

• The work is methodologically rigorous and dwells on complex and complementary multimethod approaches implemented to inform about ATL function in semantic memory as reflected in changes in regional GABA concentrations. Although the authors previously demonstrated a negative relationship between increased GABA levels and BOLD signal changes during semantic processing, the unique contribution of this work lies within evidence on the effects of cTBS TMS over the ATL given by direct observations of GABA concentration changes and further exploring inter-individual variability in ATL neuroplasticity and consequent semantic task performance.

• Another major asset of the present study is implementing a quadratic regression model to provide insights into the non-linear relationship between inhibitory GABAergic activity within the ATLs and semantic cognition, which improves with increasing GABA levels but only as long as GABA levels are not extremely high or low. Based on this finding, the authors further pinpoint the role of inter-individual differences in GABA levels and cTBS TMS responsiveness, which is a novel explanation not previously considered (according to my best knowledge) in research investigating the effect of TMS on ATLs.

• There are also many examples of good research practice throughout the manuscript, such as the explicitly stated exploratory analyses, calculation of TMS electric fields, using ATL optimised dual echo fRMI, links to open source resources, and a part of data replicates a previous study by Jung et. al (2017).

Weaknesses:<br /> • Research on the role of neurotransmitters in semantic memory is still very rare and therefore the manuscript would benefit from more context on how GABA contributes to individual differences in cognition/behaviour and more justification on why the focus is on semantic memory. A recommendation to the authors is to highlight and explain in more depth the particular gaps in evidence in this regard.

• The focus across the experiments is on the left ATL; how do the authors justify this decision? Highlighting the justification for this methodological decision will be important, especially given that a substantial body of evidence suggests that the ATL should be involved in semantics bilaterally (e.g. Hoffman & Lambon Ralph, 2018; Lambon Ralph et al., 2009; Rice et al., 2017; Rice, Hoffman, et al., 2015; Rice, Ralph, et al., 2015; Visser et al., 2010).

• When describing the results, (Pg. 11; lines 233-243), the authors first show that the higher the BOLD signal intensity in ATL as a response to the semantic task, the lower the GABA concentration. Then, they state that individuals with higher GABA concentrations in the ATL perform the semantic task better. Although it becomes clearer with the exploratory analysis described later, at this point, the results seem rather contradictory and make the reader question the following: if increased GABA leads to less task-induced ATL activation, why at this point increased GABA also leads to facilitating and not inhibiting semantic task performance? It would be beneficial to acknowledge this contradiction and explain how the following analyses will address this discrepancy.

• There is an inconsistency in reporting behavioural outcomes from the performance on the semantic task. While experiment 1 (cTBS modulates regional GANA concentrations and task-related BOLD signal changes in the ATL) reports the effects of cTBS TMS on response times, experiment 2 (Regional GABA concentrations in the ATL play a crucial role in semantic memory) and experiment 3 (The inverted U-shaped function of ATL GABA concentration in semantic processing) report results on accuracy. For full transparency, the manuscript would benefit from reporting all results (either in the main text or supplementary materials) and providing further explanations on why only one or the other outcome is sensitive to the experimental manipulations across the three experiments.

Overall, the most notable impact of this work is the contribution to a better understanding of individual differences in semantic behaviour and the potential to guide therapeutic interventions to restore semantic abilities in neurological populations. While I appreciate that this is certainly the case, I would be curious to read more about how this could be achieved.

Reviewer #3 (Public Review):

Summary:<br /> The study investigates the longitudinal changes in hearing threshold, speech recognition behavior, and speech neural responses in 2 years, and how these changes correlate with each other. A slight change in the hearing threshold is observed in 2 years (1.2 dB on average) but the speech recognition performance remains stable. The main conclusion is that there is no significant correlation between longitudinal changes in neural and behavioral measures.

Strengths:<br /> The sample size (N>100) is remarkable, especially for longitudinal studies.

Weaknesses:<br /> The participants are only tracked for 2 years and relatively weak longitudinal changes are observed, limiting how the data may shed light on the relationships between basic auditory function, speech recognition behavior, and speech neural responses.

Suggestions<br /> First, it's not surprising that a 1.2 dB change in hearing threshold does not affect speech recognition, especially for the dichotic listening task and when speech is always presented 50 dB above the hearing threshold. For the same listener, if the speech level is adjusted for 1.2 dB or much more, the performance will not be influenced during the dichotic listening task. Therefore, it is important to mention in the abstract that "sensory acuity" is measured using the hearing threshold and the change in hearing threshold is only 1.2 dB.

Second, the lack of correlation between age-related changes in "neuronal filtering" and behavior may not suggest that they follow independent development trajectories. The index for "neuronal filtering" does not seem to be stable and the correlation between the two tests is only R = 0.21. This low correlation probably indicates low test-retest reliability, instead of a dramatic change in the brain between the two tests. In other words, if the "neuronal filtering" index only very weakly correlates with itself between the two tests, it is not surprising that it does not correlate with other measures in a different test. If the "neuronal filtering" index is measured on two consecutive days and the index remains highly stable, I'm more convinced that it is a reliable measure that just changes a lot within 2 years, and the change is dissociated with the changes in behavior.

The authors attempted to solve the problem in the section entitled "Neural filtering reliably supports listening performance independent of age and hearing status", but I didn't follow the logic. As far as I could tell, the section pooled together the measurements from two tests and did not address the test-retest stability issue.

Third, the behavioral measure that is not correlated with "neuronal filtering" is the response speed. I wonder if the participants are asked to respond as soon as possible (not mentioned in the method). If not, the response speed may strongly reflect general cognitive function or a personal style, which is not correlated with the changes in auditory functions. This can also explain why the hearing threshold affects speech recognition accuracy but not the response speed (lines 263-264).

Reviewer #3 (Public Review):

Summary:<br /> This manuscript shows that mutations that disable the gene encoding the PTTH gene cause an increase in female receptivity (they mate more quickly), a phenotype that can be reversed by feeding these mutants the molting hormone, 20-hydoxyecdysone (20E). The use of an inducible system reveals that inhibition or activation of PTTH neurons during the larval stages increases and decreases female receptivity, respectively, suggesting that PTTH is required during the larval stages to affect the receptivity of the (adult) female fly. Showing that these neurons express the sex-determining gene dsx leads the authors to show that interfering with 20E actions in pC1 neurons, which are dsx-positive neurons known to regulate female receptivity, reduces female receptivity and increases the arborization pattern of pC1 neurons. The work concludes by showing that targeted knockdown of EcRA in pC1 neurons causes 527 genes to be differentially expressed in the brains of female flies, of which 123 passed a false discovery rate cutoff of 0.01; interestingly, the gene showing the greatest down-regulation was the gene encoding dopamine beta-monooxygenase.

Stengths<br /> This is an interesting piece of work, which may shed light on the basis for the observation noted previously that flies lacking PTTH neurons show reproductive defects ("... females show reduced fecundity"; McBrayer, 2007; DOI 10.1016/j.devcel.2007.11.003).

Weaknesses:<br /> There are some results whose interpretation seem ambiguous and findings whose causal relationship is implied but not demonstrated.<br /> 1- At some level, the findings reported here are not at all surprising. Since 20E regulates the profound changes that occur in the central nervous system (CNS) during metamorphosis, it is not surprising that PTTH would play a role in this process. Although animals lacking PTTH (rather paradoxically) live to adulthood, they do show greatly extended larval instars and a corresponding great delay in the 20E rise that signals the start of metamorphosis. For this reason, concluding that PTTH plays a SPECIFIC role in regulating female receptivity seems a little misleading, since the metamorphic remodeling of the entire CNS is likely altered in PTTH mutants. Since these mutants produce overall normal (albeit larger--due to their prolonged larval stages) adults, these alterations are likely to be subtle. Courtship has been reported as one defect expressed by animals lacking PTTH neurons, but this behavior may stand out because reduced fertility and increased male-male courtship (McBrayer, 2007) would be noticeable defects to researchers handling these flies. By contrast, detecting defects in other behaviors (e.g., optomotor responses, learning and memory, sleep, etc) would require closer examination. For this reason, I would ask the authors to temper their statement that PTTH is SPECIFICALLY involved in regulating female receptivity.<br /> 2- The link between PTTH and the role of pC1 neurons in regulating female receptivity is not clear. Again, since 20E controls the metamorphic changes that occur in the CNS, it is not surprising that 20E would regulate the arborization of pC1 neurons. And since these neurons have been implicated in female receptivity, it would therefore be expected that altering 20E signaling in pC1 neurons would affect this phenotype. However, this does not mean that the defects in female receptivity expressed by PTTH mutants are due to defects in pC1 arborization. For this, the authors would at least have to show that PTTH mutants show the changes in pC1 arborization shown in Fig. 6. And even then the most that could be said is that the changes observed in these neurons "may contribute" to the observed behavioral changes. Indeed, the changes observed in female receptivity may be caused by PTTH/20E actions on different neurons.<br /> 3- Some of the results need commenting on, or refining, or revising:<br /> a- For some assays PTTH behaves sometimes like a recessive gene and at other times like a semi-dominant, and yet at others like a dominant gene. For instance, in Fig. 1D-G, PTTH[-]/+ flies behave like wildtype (D), express an intermediate phenotype (E-F), or behave like the mutant (G). This may all be correct but merits some comment.<br /> b- Some of the conclusions are overstated. i) Although Fig. 2E-G does show that silencing the PTTH neurons during the larval stages affects copulation rate (E) the strength of the conclusion is tempered by the behavior of one of the controls (tub-GAL80[ts]/+, UAS-Kir2.1/+) in panels F and G, where it behaves essentially the same as the experimental group (and quite differently from the PTTH-GAL4/+ control; blue line).(Incidentally, the corresponding copulation latency should also be shown for these data.). ii) For Fig. 5I-K, the conclusion stated is that "Knock-down of EcR-A during pupal stage significantly decreased the copulation rate." Although strictly correct, the problem is that panel J is the only one for which the behavior of the control lacking the RNAi is not the same as that of the experimental group. Thus, it could just be that when the experiment was done at the pupal stage is the only situation when the controls were both different from the experimental. Again, the results shown in J are strictly speaking correct but the statement is too definitive given the behavior of one of the controls in panels I and K. Note also that panel F shows that the UAS-RNAi control causes a massive decrease in female fertility, yet no mention is made of this fact.

Reviewer #3 (Public Review):

Summary:<br /> In humans, short photoperiods are associated with hypersomnolence. The mechanisms underlying these effects are, however, unknown. Chen et al. use the fly Drosophila to determine the mechanisms regulating sleep under short photoperiods. They find that mutations in the circadian photoreceptor cryptochrome (cry) increase sleep specifically under short photoperiods (e.g. 4h light : 20 h dark). They go on to show that cry is required in GABAergic neurons. Further, they suggest that the relevant subset of GABAergic neurons are the well-studied small ventral lateral neurons that they suggest inhibit the arousal-promoting large ventral neurons via GABA signalling.

Strengths:<br /> Genetic analysis to show that cryptochrome (but not other core clock genes) mediates the increase in sleep in short photoperiods, and circuit analysis to localise cry function to GABAergic neurons.

Weaknesses:<br /> The authors' conclusion that the sLNvs are GABAergic is not well supported by the data. Better immunostaining experiments and perhaps more specific genetic driver lines would help with this point (details below).

1. The sLNvs are well known as a key component of the circadian network. The finding that they are GABAergic would if true, be of great interest to the community. However, the data presented in support of this conclusion are not convincing. Much of the confocal images are of insufficient resolution to evaluate the paper's claims. The Anti-GABA immunostaining in Fig 4 and 5 seem to have a high background, and the GRASP experiments in Fig 4 supplement 1 low signal.

Transcriptomic datasets are available for the components of the circadian network (e.g. PMID 33438579, and PMID 19966839). It would be of interest to determine if transcripts for GAD or other GABA synthesis/transport components were detected in sLNvs. Further, there are also more specific driver lines for GAD, and the lLNvs, sLNVs that could be used.

2. The authors' model posits that in short photoperiods, cry functions to suppress GABA secretion from sLNvs thereby disinhibiting the lNVs. In Fig 4I they find that activating the lLNvs (and other peptidergic cells) by c929>NaChBac in a cryb background reduces sleep compared to activating lLNVs in a wild-type background. It's not clear how this follows from the model. A similar trend is observable in Fig 4H with TRP-mediated activation of lNVs, although it is not clear from the figure if the difference b/w cryb vs wild-type background is significant.

Reviewer #3 (Public Review):

In the manuscript by Klug and colleagues, the investigators use a rabies virus-based methodology to explore potential differences in connectivity from cortical inputs to the dorsal striatum. They report that the connectivity from cortical inputs onto D1 and D2 MSNs differs in terms of their projections onto the opposing cell type, and use these data to infer that there are differences in cross-talk between cortical cells that project to D1 vs. D2 MSNs. Overall, this manuscript adds to the overall body of work indicating that there are differential functions of different striatal pathways which likely arise at least in part by differences in connectivity that have been difficult to resolve due to difficulty in isolating pathways within striatal connectivity and several interesting and provocative observations were reported. Several different methodologies are used, with partially convergent results, to support their main points.

However, I have significant technical concerns about the manuscript as presented that make it difficult for me to interpret the results of the experiments. My comments are below.

Major:<br /> There is generally a large caveat to the rabies studies performed here, which is that both TVA and the ChR2-expressing rabies virus have the same fluorophore. It is thus essentially impossible to determine how many starter cells there are, what the efficiency of tracing is, and which part of the striatum is being sampled in any given experiment. This is a major caveat given the spatial topography of the cortico-striatal projections. Furthermore, the authors make a point in the introduction about previous studies not having explored absolute numbers of inputs, yet this is not at all controlled in this study. It could be that their rabies virus simply replicates better in D1-MSNs than D2-MSNs. No quantifications are done, and these possibilities do not appear to have been considered. Without a greater standardization of the rabies experiments across conditions, it is difficult to interpret the results.

The authors claim using a few current clamp optical stimulation experiments that the cortical cells are healthy, but this result was far from comprehensive. For example, membrane resistance, capacitance, general excitability curves, etc are not reported. In Figure S2, some of the conditions look quite different (e.g., S2B, input D2-record D2, the method used yields quite different results that the authors write off as not different). Furthermore, these experiments do not consider the likely sickness and death that occurs in starter cells, as has been reported elsewhere. The health of cells in the circuit is overall a substantial concern that alone could invalidate a large portion, if not all, of the behavioral results. This is a major confound given those neurons are thought to play critical roles in the behaviors being studied. This is a major reason why first-generation rabies viruses have not been used in combination with behavior, but this significant caveat does not appear to have been considered, and controls e.g., uninfected animals, infected with AAV helpers, etc, were not included.

The overall purity (e.g., EnvA pseudotyping efficiency) of the RABV prep is not shown. If there was a virus that was not well EnvA-pseudotyped and thus could directly infect cortical (or other) inputs, it would degrade specificity.

While most of the study focuses on the cortical inputs, in slice recordings, inputs from the thalamus are not considered, yet likely contribute to the observed results. Related to this, in in vivo optogenetic experiments, technically, if the thalamic or other inputs to the dorsal striatum project to the cortex, their method will not only target cortical neurons but also terminals of other excitatory inputs. If this cannot be ruled it, stating that the authors are able to selectively activate the cortical inputs to one or the other population should be toned down.

The statements about specificity of connectivity are not well-founded. It may be that in the specific case where they are assessing outside of the area of injections, their conclusions may hold (e.g., excitatory inputs onto D2s have more inputs onto D1s than vice versa). However, how this relates to the actual site of injection is not clear. At face value, if such a connectivity exists, it would suggest that D1-MSNs receive substantially more overall excitatory inputs than D2s. It is thus possible that this observation would not hold over other spatial intervals. This was not explored and thus the conclusions are over-generalized. e.g., the distance from the area of red cells in the striatum to recordings was not quantified, what constituted a high level of cortical labeling was not quantified, etc. Without more rigorous quantification of what was being done, it is difficult to interpret the results.

The results in figure 3 are not well controlled. The authors show contrasting effects of optogenetic stimulation of D1-MSNs and D2-MSNs in the DMS and DLS, results which are largely consistent with the canon of basal ganglia function. However, when stimulating cortical inputs, stimulating the inputs from D1-MSNs gives the expected results (increased locomotion) while stimulating putative inputs to D2-MSNs had no effect. This is not the same as showing a decrease in locomotion - showing no effect here is not possible to interpret.

In light of their circuit model, the result showing that inputs to D2-MSNs drive ICSS is confusing. How can the authors account for the fact that these cells are not locomotor-activating, stimulation of their putative downstream cells (D2-MSNs) does not drive ICSS, yet the cortical inputs drive ICSS? Is the idea that these inputs somehow also drive D1s? If this is the case, how do D2s get activated, if all of the cortical inputs tested net activate D1s and not D2s? Same with the results in figure 4 - the inputs and putative downstream cells do not have the same effects. Given the potential caveats of differences in viral efficiency, spatial location of injections, and cellular toxicity, I cannot interpret these experiments.

Reviewer #3 (Public Review):

Summary: Well-illustrated new material is documented for Acanthomeridion, a formerly incompletely known Cambrian arthropod. The formerly known facial sutures are shown to be associated with ventral plates that the authors very reasonably homologise with the free cheeks of trilobites. A slight update of a phylogenetic dataset developed by Du et al, then refined slightly by Chen et al, then by Schmidt et al, and again here, permits another attempt to optimise the number of origins of dorsal ecdysial sutures in trilobites and their relatives.

Strengths: Documentation of an ontogenetic series makes a sound case that the proposed diagnostic characters of a second species of Acanthomeridion are variations within a single species. New microtomographic data shed some light on appendage morphology that was not formerly known. The new data on ventral plates and their association with the ecdysial sutures are valuable in underpinning homologies with trilobites.

Weaknesses: The main conclusion remains clouded in ambiguity because of a poorly resolved Bayesian consensus and is consistent with work led by the lead author in 2019 (thus compromising the novelty of the findings). The Bayesian trees being majority rules consensus trees, optimising characters onto them (Figure 7b, d) is problematic. Optimising on a consensus tree can produce spurious optimisations that inflate tree length or distort other metrics of fit. Line 264 refers to at least three independent origins of cephalic sutures in artiopodans but the fully resolved Figure 7c requires only two origins. We can't say how many origins are required by Figures 7b and 7d.

The question of how many times dorsal ecdysial sutures evolved in Artiopoda was addressed by Hou et al (2017), who first documented the facial sutures of Acanthomeridion and optimised them onto a phylogeny to infer multiple origins, as well as in a paper led by the lead author in Cladistics in 2019. Du et al. (2019) presented a phylogeny based on an earlier version of the current dataset wherein they discussed how many times sutures evolved or were lost based on their presence in Zhiwenia/Protosutura, Acanthomeridion, and Trilobita. To their credit, the authors acknowledge this (lines 62-65). The answer here is slightly different (because some topologies unite Acanthomeridion and trilobites).

The following points are not meant to be "Weaknesses" but rather are refinements:

I recommend changing the title of the paper from "cephalic sutures" to "dorsal ecdysial sutures" to be more precise about the character that is being tracked evolutionarily. Lots of arthropods have cephalic sutures (e.g., the ventral marginal suture of xiphosurans; the Y-shaped dorsomedian ecdysial line in insects). The text might also be updated to change other instances of "cephalic sutures" to a more precise wording.

The authors have provided (but not explicitly identified) support values for nodes in their Bayesian trees but not in their parsimony ones. Please do the jackknife or bootstrap for the parsimony analyses and make it clear that the Bayesian values are posterior probabilities.

In line 65 or somewhere else, it might be noted that a single origin of the dorsal facial sutures in trilobites has itself been called into question. Jell (2003) proposed that separate lineages of Eutrilobita evolved their facial sutures independently from separate sister groups within Olenellina.

I have provided minor typographic or terminological corrections to the authors in a list of recommendations that may not be publicly available.

Reviewer #3 (Public Review):

Summary:<br /> 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:<br /> Based on these data, the authors propose a mechanism in which LRMP specifically binds to HCN4 via its isotype-specific N-terminal sequence and thus prevents the cAMP transduction mechanism by acting at the interface between the elbow Clinker, the S4S5-linker, the HCND.

Weaknesses:<br /> Although the work is interesting, there are some discrepancies between data that need to be addressed.

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

2. 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).

This problem is spread throughout the manuscript, and the measured mean effects are indeed always at the limit of statistical significance. Why so? Is this a problem with the analysis, or with the recordings?

There are several other problems with Figure 1 and in all figures of the manuscript: the Y scale is very narrow while the mean values are marked with large square boxes. Moreover, the exemplary activation curve of Figure 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.

On this ground, it is difficult to judge the conclusions and it would also greatly help if exemplary current traces would be also shown.

3. "....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, remains to be proven.

Moreover, I suggest analysing the putative role of P545 and T547 in light of the available HCN4 structures. In particular, T547 (elbow) points towards the underlying shoulder of the adjacent subunit and, therefore, 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 also favours this hypothesis. In this light, it will be also interesting to see whether a single T547F mutation is sufficient to prevent the LRMP effect.

Reviewer #3 (Public Review):

In this study, O'Brien et al. address the need for scalable and cost-effective approaches to finding lead compounds for the treatment of the growing number of Mendelian diseases. They used state-of-the-art phenotypic screening based on an established high-dimensional phenotypic analysis pipeline in the nematode C. elegans.

First, a panel of 25 C. elegans models was created by generating CRISPR/Cas9 knock-out lines for conserved human disease genes. These mutant strains underwent behavioral analysis using the group's published methodology. Clustering analysis revealed common features for genes likely operating in similar genetic pathways or biological functions. The study also presents results from a more focused examination of ciliopathy disease models.

Subsequently, the study focuses on the NALCN channel gene family, comparing the phenotypes of mutants of nca-1, unc-77, and unc-80. This initial characterization identifies three behavioral parameters that exhibit significant differences from the wild type and could serve as indicators for pharmacological modulation.

As a proof-of-concept, O'Brien et al. present a drug repurposing screen using an FDA-approved compound library, identifying two compounds capable of rescuing the behavioral phenotype in a model with UNC80 deficiency. The relatively short time and low cost associated with creating and phenotyping these strains suggest that high-throughput worm tracking could serve as a scalable approach for drug repurposing, addressing the multitude of Mendelian diseases. Interestingly, by measuring a wide range of behavioural parameters, this strategy also simultaneously reveals deleterious side effects of tested drugs that may confound the analysis.

Considering the wealth of data generated in this study regarding important human disease genes, it is regrettable that the data is not actually made accessible. This diminishes the study's utility. It would have a far greater impact if an accessible and user-friendly online interface were established to facilitate data querying and feature extraction for specific mutants. This would empower researchers to compare their findings with the extensive dataset created here. Otherwise, one is left with a very limited set of exploitable data.

Another technical limitation of the study is the use of single alleles. Large deletion alleles were generated by CRISPR/Cas9 gene editing. At first glance, this seems like a good idea because it limits the risk that background mutations, present in chemically-generated alleles, will affect behavioral parameters. However, these large deletions can also remove non-coding RNAs or other regulatory genetic elements, as found, for example, in introns. Therefore, it would be prudent to validate the behavioral effects by testing additional loss-of-function alleles produced through early stop codons or targeted deletion of key functional domains.

This an interesting point - shopify can argue they do it to avoid double counting, but that’s not really what scope 3 is designed for

Reviewer #3 (Public Review):

Summary:

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

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public Review):

Summary:<br /> Radial muscle growth involves an increase in overall muscle cross-sectional area. For decades this process has been described as the splitting of myofibrils to produce more myofibrils during the growth process. However, a closer look at the original papers shows that the evidence underlying this description was incomplete. In this paper, the authors have developed a novel method using fluorescence microscopy to directly measure myofibril size and number. Using a mouse model of mechanical loading and a human model of resistance exercise they discovered that myofibrillogenesis is playing a key role in the radial growth of muscle fibers.

Strengths:<br /> 1. Well-written and clear description of hypothesis, background, and experiments.<br /> 2. Compelling series of experiments.<br /> 3. Different approaches to test the hypothesis.<br /> 4. Rigorous study design.<br /> 5. Clear interpretation of results.<br /> 6. Novel findings that will be beneficial to the muscle biology field.<br /> 7. Innovative microscopy methods that should be widely available for use in other muscle biology labs.

Weaknesses:<br /> Supplemental Figure 1 is not very clear.

Reviewer #3 (Public Review):

Summary:<br /> By conducting QM/MM free energy simulations, the authors aimed to characterize the mechanism and transition state for the phosphoryl transfer in adenylate kinase. The qualitative reliability of the QM/MM results has been supported by several interesting experimental kinetic studies. However, the interpretation of the QM/MM results is not well supported by the current calculations.

Strengths:<br /> The QM/MM free energy simulations have been carefully conducted. The accuracy of the semi-empirical QM/MM results was further supported by DFT/MM calculations, as well as qualitatively by several experimental studies.

Weaknesses:<br /> 1. One key issue is the definition of the transition state ensemble. The authors appear to define this by simply considering structures that lie within a given free energy range from the barrier. However, this is not the rigorous definition of transition state ensemble, which should be defined in terms of committor distribution. This is not simply an issue of semantics, since only a rigorous definition allows a fair comparison between different cases - such as the transition state in an enzyme vs in solution, or with and without the metal ion. For a chemical reaction in a complex environment, it is also possible that many other variables (in addition to the breaking and forming P-O bonds) should be considered when one measures the diversity in the conformational ensemble.

2. While the experimental observation that the activation entropy differs significantly with and without the Ca2+ ion is interesting, it is difficult to connect this result with the "wide" transition state ensemble observed in the QM/MM simulations so far. Even without considering the definition of the transition state ensemble mentioned above, it is unlikely that a broader range of P-O distances would explain the substantial difference in the activation entropy measured in the experiment. Since the difference is sufficiently large, it should be possible to compute the value by repeating the free energy simulations at different temperatures, which would lead to a much more direct evaluation of the QM/MM model/result and the interpretation.

Reviewer #3 (Public Review):

Summary:<br /> Jojoa-Cruz et al provide a new structure of At-OSCA3.1. The structure of OSCA 3.1 is similar to previous OSCA cryo-em structures of both OSCA3.1 and other homologues validating the new structure. Using the novel structure of OSCA3.1 as a guide they created several point mutations to investigate two different mechanosensitive modalities: poking and stretching. To investigate the ability of OSCA channels to gate in response to poking they created point mutations in OSCA1.2 to reduce sensitivity to poking based on the differences between the OSCA1.2 and 3.1 structures. Their results suggest that two separate regions are responsible for gating in response to poking and stretching.

Strengths:<br /> Through a detailed structure-based analysis, the authors identified structural differences between OSCA3.1 and OSCA1.2. These subtle structural changes identify regions in the amphipathic helix and near the pore that are essential for the gating of OSCA1.2 in response to poking and stretching. The use of point mutations to understand how these regions are involved in mechanosensation clearly shows the role of these residues in mechanosensation.

Weaknesses:<br /> In general, the point mutations selected all show significant alterations to the inherent mechanosensitive regions. This often suggests that any mutation would disrupt the function of the region, additional mutations that are similar in function to the WT channel would support the claims in the manuscript. Mutations in the amphipathic helix at W75 and L80 show reduced gating in response to poking stimuli. The gating observed occurs at poking depths similar to cellular rupture, the similarity in depths suggests that these mutations could be a complete loss of function. For example, a mutation to L80I or L80Q would show that the addition of the negative charge is responsible for this disruption not just a change in the steric space of the residue in an essential region.

Reviewer #3 (Public Review):

Several labs in the 1970s published fundamental work revealing that almost all eukaryotes organize their DNA into repeating units called nucleosomes, which form the chromatin fiber. Decades of elegant biochemical and structural work indicated a primarily octameric organization of the nucleosome with 2 copies of each histone H2A, H2B, H3 and H4, wrapping 147bp of DNA in a left handed toroid, to which linker histone would bind.

This was true for most species studied (except, yeast lack linker histone) and was recapitulated in stunning detail by in vitro reconstitutions by salt dialysis or chaperone-mediated assembly of nucleosomes. Thus, these landmark studies set the stage for an exploding number of papers on the topic of chromatin in the past 45 years.

An emerging counterpoint to the prevailing idea of static particles is that nucleosomes are much more dynamic and can undergo spontaneous transformation. Such dynamics could arise from intrinsic instability due to DNA structural deformation, specific histone variants or their mutations, post-translational histone modifications which weaken the main contacts, protein partners, and predominantly, from active processes like ATP-dependent chromatin remodeling, transcription, repair and replication.

This paper is important because it tests this idea whole-scale, applying novel cryo-EM tomography tools to examine the state of chromatin in yeast lysates or cryo-sections. The experimental work is meticulously performed, with vast amount of data collected. The main findings are interpreted by the authors to suggest that majority of yeast nucleosomes lack a stable octameric conformation. The findings are not surprising in that alternative conformations of nucleosomes might exist in vivo, but rather in the sheer scale of such particles reported, relative to the traditional form expected from decades of biochemical, biophysical and structural data. Thus, it is likely that this work will be perceived as controversial. Nonetheless, we believe these kinds of tools represent an important advance for in situ analysis of chromatin. We also think the field should have the opportunity to carefully evaluate the data and assess whether the claims are supported, or consider what additional experiments could be done to further test the conceptual claims made. It is our hope that such work will spark thought-provoking debate in a collegial fashion, and lead to the development of exciting new tools which can interrogate native chromatin shape in vivo. Most importantly, it will be critical to assess biological implications associated with more dynamic - or static forms- of nucleosomes, the associated chromatin fiber, and its three-dimensional organization, for nuclear or mitotic function.

Reviewer #3 (Public Review):

Several labs in the 1970s published fundamental work revealing that almost all eukaryotes organize their DNA into repeating units called nucleosomes, which form the chromatin fiber. Decades of elegant biochemical and structural work indicated a primarily octameric organization of the nucleosome with 2 copies of each histone H2A, H2B, H3 and H4, wrapping 147bp of DNA in a left handed toroid, to which linker histone would bind.

This was true for most species studied (except, yeast lack linker histone) and was recapitulated in stunning detail by in vitro reconstitutions by salt dialysis or chaperone-mediated assembly of nucleosomes. Thus, these landmark studies set the stage for an exploding number of papers on the topic of chromatin in the past 45 years.

An emerging counterpoint to the prevailing idea of static particles is that nucleosomes are much more dynamic and can undergo spontaneous transformation. Such dynamics could arise from intrinsic instability due to DNA structural deformation, specific histone variants or their mutations, post-translational histone modifications which weaken the main contacts, protein partners, and predominantly, from active processes like ATP-dependent chromatin remodeling, transcription, repair and replication.

This paper is important because it tests this idea whole-scale, applying novel cryo-EM tomography tools to examine the state of chromatin in yeast lysates or cryo-sections. The experimental work is meticulously performed, with vast amount of data collected. The main findings are interpreted by the authors to suggest that majority of yeast nucleosomes lack a stable octameric conformation. The findings are not surprising in that alternative conformations of nucleosomes might exist in vivo, but rather in the sheer scale of such particles reported, relative to the traditional form expected from decades of biochemical, biophysical and structural data. Thus, it is likely that this work will be perceived as controversial. Nonetheless, we believe these kinds of tools represent an important advance for in situ analysis of chromatin. We also think the field should have the opportunity to carefully evaluate the data and assess whether the claims are supported, or consider what additional experiments could be done to further test the conceptual claims made. It is our hope that such work will spark thought-provoking debate in a collegial fashion, and lead to the development of exciting new tools which can interrogate native chromatin shape in vivo. Most importantly, it will be critical to assess biological implications associated with more dynamic - or static forms- of nucleosomes, the associated chromatin fiber, and its three-dimensional organization, for nuclear or mitotic function.

Reviewer #3 (Public Review):

Synopsis:<br /> The lack of visualizing the dynamic nature of biomolecules is a major weakness of crystallography or electron microscopy to study structure-function relationship of proteins. Such a challenge can be exemplified by the case of prestin, which shares high structural similarity to SLC26A9 anion transporter but is not an ion transporter. In this study, Lin et al aimed to use hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to investigate the mobility of prestin and its response to anions. The authors exploited the nature of anion-dependent folding of this type of transporter to systematically analyze the mobility of transmembrane helices of both transporters by HDX. The authors found that the anion-binding helices engage in the stabilization of the anion-binding site. When stripped from Cl-, the site exposes to the transporter's extracellular side. More importantly, the authors narrowed down TM3 and TM10 with experimental data supporting the notion of R399's unique role in prestin's function. The results thus provide a working model of how the charged residue works in conjunction with the cooperativity of helix unfolding at the anion-binding site to drive the electromotive force of prestin.

Strengths:<br /> The use of HDX-MS to probe the dynamic nature of prestin is a major strength of this study, which provides experimental evidence revealing the global and local differences in the folding events between prestin and SLC26A9. The mass experimental data led to the identification of TM3 and TM10 as the primary contributors to the folding changes, as well as a calculation of ΔΔG of ~2.4 kcal/mol, within the thermodynamic range of the dipole between the two helices. The latter also suggests the role of R399 as previously speculated in cryo-EM structures.

This study went further to dissect the cooperativity during the folding and unfolding events on TM3, in which the authors observed a helix fraying at the anion-binding site and cooperative unfolding at the distal lipid-facing helices. This provides strong evidence of why prestin can undergo fast electromechanical rearrangement.

Weakness:<br /> The authors tried to investigate the allostery by probing the intermediate folding/unfolding states by using sulfate or salicylate in the absence of chloride. Sulfate-bound proteins appear in an apo state earlier than normal chloride binding, and salicylate treatment led to a stable TMD state with slower HDX. It is unclear from the data (Fig 4) how the allostery works without titrating chloride ions into the reaction. The sulfate or salicylate experiments seem to show two extreme folding events outside the normal chloride conditions.

TM3 and TM10 contribute to the anion-binding site together, and the authors beautifully showed the cooperativity of TM3. Does TM10 show the same cooperativity in prestin and SLC26A9? In addition, it is unclear whether the folding model at the anion-binding helices (Fig. 5B) remains the same when expressing prestin on live cells, such as thermodynamic data derived from electrophysiology studies.

The authors observed increased stability upon chloride binding at the subunit interface in the cytosol for both prestin and SLC26A9 (Fig 1). How does this similarity in the cytosolic region contribute to the differential mechanisms as seen in the TMD in both transporters? It is unclear in this version of the manuscript.

Reviewer #3 (Public Review):

Synopsis:<br /> The lack of visualizing the dynamic nature of biomolecules is a major weakness of crystallography or electron microscopy to study structure-function relationship of proteins. Such a challenge can be exemplified by the case of prestin, which shares high structural similarity to SLC26A9 anion transporter but is not an ion transporter. In this study, Lin et al aimed to use hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to investigate the mobility of prestin and its response to anions. The authors exploited the nature of anion-dependent folding of this type of transporter to systematically analyze the mobility of transmembrane helices of both transporters by HDX. The authors found that the anion-binding helices engage in the stabilization of the anion-binding site. When stripped from Cl-, the site exposes to the transporter's extracellular side. More importantly, the authors narrowed down TM3 and TM10 with experimental data supporting the notion of R399's unique role in prestin's function. The results thus provide a working model of how the charged residue works in conjunction with the cooperativity of helix unfolding at the anion-binding site to drive the electromotive force of prestin.

Strengths:<br /> The use of HDX-MS to probe the dynamic nature of prestin is a major strength of this study, which provides experimental evidence revealing the global and local differences in the folding events between prestin and SLC26A9. The mass experimental data led to the identification of TM3 and TM10 as the primary contributors to the folding changes, as well as a calculation of ΔΔG of ~2.4 kcal/mol, within the thermodynamic range of the dipole between the two helices. The latter also suggests the role of R399 as previously speculated in cryo-EM structures.

This study went further to dissect the cooperativity during the folding and unfolding events on TM3, in which the authors observed a helix fraying at the anion-binding site and cooperative unfolding at the distal lipid-facing helices. This provides strong evidence of why prestin can undergo fast electromechanical rearrangement.

Weakness:<br /> The authors tried to investigate the allostery by probing the intermediate folding/unfolding states by using sulfate or salicylate in the absence of chloride. Sulfate-bound proteins appear in an apo state earlier than normal chloride binding, and salicylate treatment led to a stable TMD state with slower HDX. It is unclear from the data (Fig 4) how the allostery works without titrating chloride ions into the reaction. The sulfate or salicylate experiments seem to show two extreme folding events outside the normal chloride conditions.

TM3 and TM10 contribute to the anion-binding site together, and the authors beautifully showed the cooperativity of TM3. Does TM10 show the same cooperativity in prestin and SLC26A9? In addition, it is unclear whether the folding model at the anion-binding helices (Fig. 5B) remains the same when expressing prestin on live cells, such as thermodynamic data derived from electrophysiology studies.

The authors observed increased stability upon chloride binding at the subunit interface in the cytosol for both prestin and SLC26A9 (Fig 1). How does this similarity in the cytosolic region contribute to the differential mechanisms as seen in the TMD in both transporters? It is unclear in this version of the manuscript.

Reviewer #3 (Public Review):

SUMMARY:<br /> The manuscript by Bian et al. promotes the idea that creatine is a new neurotransmitter. The authors conduct an impressive combination of mass spectrometry (Fig. 1), genetics (Figs. 2, 3, 6), biochemistry (Figs. 2, 3, 8), immunostaining (Fig. 4), electrophysiology (Figs. 5, 6, 7), and EM (Fig. 8) in order to offer support for the hypothesis that creatine is a CNS neurotransmitter.

STRENGTHS:<br /> There are many strengths to this study.<br /> • The combinatorial approach is a strength. There is no shortage of data in this study.<br /> • The careful consideration of specific criteria that creatine would need to meet in order to be considered a neurotransmitter is a strength.<br /> • The comparison studies that the authors have done in parallel with classical neurotransmitters are helpful.<br /> • Demonstration that creatine has inhibitory effects is another strength.<br /> • The new genetic mutations for Slc6a8 and AGAT are strengths and potentially incredibly helpful for downstream work.

WEAKNESSES:<br /> • Some data are indirect. Even though Slc6a8 and AGAT are helpful sentinels for the presence of creatine, they are not creatine themselves. Therefore, the conclusions that are drawn should be circumspect.<br /> • Regarding Slc6a8, it seems to work only as a reuptake transporter - not as a transporter into SVs. Therefore, we do not know what the transporter is.<br /> • Puzzlingly, Slc6a8 and AGAT are in different cells, setting up the complicated model that creatine is created in one cell type and then processed as a neurotransmitter in another.<br /> • No candidate receptor for creatine has been identified postsynaptically.<br /> • Because no candidate receptor has been identified, is it possible that creatine is exerting its effects indirectly through other inhibitory receptors (e.g., GABAergic Rs)?<br /> • More broadly, what are the other possibilities for roles of creatine that would explain these observations other than it being a neurotransmitter? Could it simply be a modifier that exists in the SVs (lots of molecules exist in SVs)?<br /> • The biochemical studies are helpful in terms of comparing relevant molecules (e.g., Figs. 8 and S1), but the images of the westerns are all so fuzzy that there are questions about processing and the accuracy of the quantification.

APPRAISAL OF WHETHER THE AUTHORS ACHIEVED THEIR AIMS AND WHETHER THE RESULTS SUPPORT THE CONCLUSIONS:<br /> There are several criteria that define a neurotransmitter. The authors nicely delineated many criteria in their discussion, but it is worth it for readers to do the same with their own understanding of the data.

By this reviewer's understanding (and the Purves' textbook definition) a neurotransmitter: 1) must be present within the presynaptic neuron and stored in vesicles; 2) must be released by depolarization of the presynaptic terminal; 3) must require Ca2+ influx upon depolarization prior to release; 4) must bind specific receptors present on the postsynaptic cell; 5) exogenous transmitter can mimic presynaptic release; 6) there exists a mechanism of removal of the neurotransmitter from the synaptic cleft.

For a paper to claim that the work has identified a new neurotransmitter, several of these criteria would be met - and the paper would acknowledge in the discussion which ones have not been met. For this particular paper, this reviewer finds that condition 1 is clearly met.

Conditions 2 and 3 seem to be met by electrophysiology, but there are caveats here. High KCl stimulation is a blunt instrument that will depolarize absolutely everything in the prep all at once and could result in any number of non-specific biological reactions as a result of K+ rushing into all neurons in the prep. Moreover, the results in 0 Ca2+ are puzzling. For creatine (and for the other neurotransmitters), why is there such a massive uptick in release, even when the extracellular saline is devoid of calcium?

Condition 4 is not discussed in detail at all. In the discussion, the authors elide the criterion of receptors specified by Purves by inferring that the existence of postsynaptic responses implies the existence of receptors. True, but does it specifically imply the existence of creatinergic receptors? This reviewer does not think that is necessarily the case. The authors should be appropriately circumspect and consider other modes of inhibition that are induced by activation or potentiation of other receptors (e.g., GABAergic or glycinergic).

Condition 5 may be met, because the authors applied exogenous creatine and observed inhibition (Fig. 7). However, this is tough to know without understanding the effects of endogenous release of creatine. if they were to test if the absence of creatine caused excess excitation (at putative creatinergic synapses), then that would be supportive of the same.

For condition 6, the authors made a great effort with Slc6a8. This is a very tough criterion to understand for many synapses and neurotransmitters.

DISCUSSION OF THE LIKELY IMPACT OF THE WORK:<br /> In terms of fundamental neuroscience, the story would be impactful if proven correct. There are certainly more neurotransmitters out there than currently identified.

The impact as framed by the authors in the abstract and introduction for intellectual disability is uncertain (forming a "new basis for ID pathogenesis") and it seems quite speculative beyond the data in this paper.

Reviewer #3 (Public Review):

SUMMARY:

The manuscript by Bian et al. promotes the idea that creatine is a new neurotransmitter. The authors conduct an impressive combination of mass spectrometry (Fig. 1), genetics (Figs. 2, 3, 6), biochemistry (Figs. 2, 3, 8), immunostaining (Fig. 4), electrophysiology (Figs. 5, 6, 7), and EM (Fig. 8) in order to offer support for the hypothesis that creatine is a CNS neurotransmitter.

STRENGTHS:

There are many strengths to this study.

• The combinatorial approach is a strength. There is no shortage of data in this study.<br /> • The careful consideration of specific criteria that creatine would need to meet in order to be considered a neurotransmitter is a strength.<br /> • The comparison studies that the authors have done in parallel with classical neurotransmitters is helpful.<br /> • Demonstration that creatine has inhibitory effects is another strength.<br /> • The new genetic mutations for Slc6a8 and AGAT are strengths and potentially incredibly helpful for downstream work.

WEAKNESSES:

• Some data are indirect. Even though Slc6a8 and AGAT are helpful sentinels for the presence of creatine, they are not creatine themselves. Of note, these molecules themselves are not essential for making the case that creatine is a neurotransmitter.<br /> • Regarding Slc6a8, it seems to work only as a reuptake transporter - not as a transporter into SVs. Therefore, we do not know what the transporter into the TVs is.<br /> • Puzzlingly, Slc6a8 and AGAT are in different cells, setting up the complicated model that creatine is created in one cell type and then processed as a neurotransmitter in another. This matter will likely need to be resolved in future studies.<br /> • No candidate receptor for creatine has been identified postsynaptically. This will likely need to be resolved in future studies.<br /> • Because no candidate receptor has been identified, it is important to fully consider other possibilities for roles of creatine that would explain these observations other than it being a neurotransmitter? There is some attention to this in the Discussion.

There are several criteria that define a neurotransmitter. The authors nicely delineated many criteria in their discussion, but it is worth it for readers to do the same with their own understanding of the data.

By this reviewer's understanding (and combining some textbook definitions together) a neurotransmitter: 1) must be present within the presynaptic neuron and stored in vesicles; 2) must be released by depolarization of the presynaptic terminal; 3) must require Ca2+ influx upon depolarization prior to release; 4) must bind specific receptors present on the postsynaptic cell; 5) exogenous transmitter can mimic presynaptic release; 6) there exists a mechanism of removal of the neurotransmitter from the synaptic cleft.

For a paper to claim that the published work has identified a new neurotransmitter, several of these criteria would be met - and the paper would acknowledge in the discussion which ones have not been met. For this particular paper, this reviewer finds that condition 1 is clearly met.

Conditions 2 and 3 seem to be met by electrophysiology, but there are caveats here. High KCl stimulation is a blunt instrument that will depolarize absolutely everything in the prep all at once and could result in any number of non-specific biological reactions as a result of K+ rushing into all neurons in the prep. Moreover, the results in 0 Ca2+ are puzzling. For creatine (and for the other neurotransmitters), why is there such a massive uptick in release, even when the extracellular saline is devoid of calcium?

Condition 4 is not discussed in detail at all. In the discussion, the authors elide the criterion of receptors specified by Purves by inferring that the existence of postsynaptic responses implies the existence of receptors. True, but does it specifically imply the existence of creatinergic receptors? This reviewer does not think that is necessarily the case. The authors should be appropriately circumspect and consider other modes of inhibition that are induced by activation or potentiation of other receptors (e.g., GABAergic or glycinergic).

Condition 5 may be met, because authors applied exogenous creatine and observed inhibition. However, this is tough to know without understanding the effects of endogenous release of creatine. if they were to test if the absence of creatine caused excess excitation (at putative creatinergic synapses), then that would be supportive of the same. Nicely, Ghirardini et al., 2023 study cited by the reviewers does provide support for this exact notion in pyramidal neurons.

For condition 6, the authors made a great effort with Slc6a8. This is a very tough criterion to understand or prove for many synapses and neurotransmitters.

In terms of fundamental neuroscience, the story should be impactful. There are certainly more neurotransmitters out there than currently identified and by textbook criteria, creatine seems to be one of them taking all of the data in this study and others into account.

Reviewer #3 (Public Review):

Summary:

The authors investigated that learning processes relied on distinct reward or punishment outcomes in probabilistic instrumental learning tasks were involved in functional interactions of two different cortico-cortical gamma-band modulations, suggesting that learning signals like reward or punishment prediction errors can be processed by two dominated interactions, such as areas lOFC-vmPFC and areas aINS-dlPFC, and later on integrated together in support of switching conditions between reward and punishment learning. By performing the well-known analyses of mutual information, interaction information, and transfer entropy, the conclusion was accomplished by identifying directional task information flow between redundancy-dominated and synergy-dominated interactions. Also, this integral concept provided a unifying view to explain how functional distributed reward and/or punishment information were segregated and integrated across cortical areas.

Strengths:

The dataset used in this manuscript may come from previously published works (Gueguen et al., 2021) or from the same grant project due to the methods. Previous works have shown strong evidence about why gamma-band activities and those 4 areas are important. For further analyses, the current manuscript moved the ideas forward to examine how reward/punishment information transfer between recorded areas corresponding to the task conditions. The standard measurements such mutual information, interaction information, and transfer entropy showed time-series activities in the millisecond level and allowed us to learn the directional information flow during a certain window. In addition, the diagram in Figure 6 summarized the results and proposed an integral concept with functional heterogeneities in cortical areas. These findings in this manuscript will support the ideas from human fMRI studies and add a new insight to electrophysiological studies with the non-human primates.

Weaknesses:

After reading through the manuscript, the term "non-selective" in the abstract confused me and I did not actually know what it meant and how it fits the conclusion. If I learned the methods correctly, the 4 areas were studied in this manuscript because of their selective responses to the RPE and PPE signals (Figure 2). The redundancy- and synergy-dominated subsystems indicated that two areas shared similar and complementary information, respectively, due to the negative and positive value of interaction information (Page 6). For me, it doesn't mean they are "non-selective", especially in redundancy-dominated subsystem. I may miss something about how you calculate the mutual information or interaction information. Could you elaborate this and explain what the "non-selective" means?

The directional information flows identified in this manuscript were evidenced by the recording contacts of iEEG with levels of concurrent neural activities to the task conditions. However, are the conclusions well supported by the anatomical connections? Is it possible that the information was transferred to the target via another area? These questions may remain to be elucidated by using other approaches or animal models. It would be great to point this out here for further investigation.

Reviewer #3 (Public Review):

The mechanism controlling plant gravity sensing has fascinated researchers for centuries. It has been clear for at least the past decade that starch-filled plastids (termed statoliths) in specialised gravity-sensing columella cells sense changes in root orientation, triggering an asymmetric auxin gradient that alters root growth direction. Nevertheless, exactly how statolith movement triggers PIN auxin efflux carrier activation and auxin gradient formation has remained unclear until very recently. A series of new papers (in Science and Cell) and this manuscript report how LAZY proteins (also referred to as NEGATIVE GRAVITROPIC 50 RESPONSE OF ROOTS; NGR) play a pivotal role in regulating root gravitropism. In terms of their overall significance, their collective findings provide seminal insights into the very earliest steps for how plant roots sense gravity which are arguably the most important papers about root gravitropism in the past decade.

In the current manuscript, Kulich et al initially report (through creating a functional NGR1-GFP reporter) that "NGR1-GFP displayed a highly specific columella expression, which was most prominent at the PM and the statolith periphery." Is NGR1-GFP expressed in shoot tissues? If yes, is it in starch sheath (the gravity-sensing equivalent of root columella cells)? The authors also note "NGR1-GFP signal from the PM was not evenly distributed, but rather polarized to the lower side of the columella cells in the vicinity of the sedimented statoliths (Fig. 1A)." and (when overexpressing NGR-GFP) "chloroplasts in the vicinity of the PM strongly correlated with NGR1 accumulating at the PM nearby, similar to the scenario in columella" suggesting that NGR1 does not require additional tissue-specific factors (i.e. trafficking proteins or lipids) to assist in its intracellular movement from plastid to PM.

Next, the authors study the spatiotemporal dynamics of NGR1-GFP re-localisation with other early gravitropic signals and/or components Calcium, auxin, and PIN3. The temporal data presented in Figure 1 illustrates how the GCaMP calcium reporter (in panel E) revealed "the first signaling event in the root gravitropic bending is the statolith removal from the top membrane, rather than its arrival at the bottom" It appeared that the auxin DII-VENUS reporter was also changing rapidly (panel G) - was this detectable BEFORE statolith re-sedimentation?<br /> Please can the authors explain their NPA result in Fig 1E? Why would treatment with the auxin transport inhibitor NPA block Ca signalling (unless the latter was dependent on the former)?<br /> They go on to note "This initial auxin asymmetry is mediated by PIN-dependent auxin transport, despite visible polarization of PIN3 can be detected only later" which suggests that PIN activity was being modified prior to PIN polarisation.

In contrast to other proteins involved in gravity response like RLDs and PINs, NGR1 localization and gravity-induced polarization does not undergo BFA-sensitive endocytic recycling by ARF-GEF GNOM. This makes sense given NGR1 is initially targeted to plastids, THEN the PM. Does NGR1 contain a cleavable plastid targeting signal? The authors go on to elegantly demonstrate that NGR1 PM targeting relies on palmitoylation through imaging and mutagenesis-based transgenic ngr rescue assays.

Finally, the authors demonstrate that gravitropic-induced auxin gradient formation is initially dependent on PIN3 auxin efflux activation (prior to PIN3 re-localisation). This early PIN3 activation process is dependent on NGR1 re-targeting D6PK (a PIN3 activating kinase). This elegant molecular mechanism integrates all the regulatory components described in the paper into a comprehensive root gravity sensing model.

Reviewer #3 (Public Review):

Summary:<br /> The manuscript from Tariq and Maurici et al. presents important biochemical and biophysical data linking protein phosphorylation to phase separation behavior in the repressive arm of the Neurospora circadian clock. This is an important topic that contributes to what is likely a conceptual shift in the field. While I find the connection to the in vivo physiology of the clock to be still unclear, this can be a topic handled in future studies.

Strengths: The ability to prepare purified versions of unphosphorylated FRQ and P-FRQ phosphorylated by CK-1 is a major advance that allowed the authors to characterize the role of phosphorylation in structural changes in FRQ and its impact on phase separation in vitro.

Weaknesses: The major question that remains unanswered from my perspective is whether phase separation plays a key role in the feedback loop that sustains oscillation (for example by creating a nonlinear dependence on overall FRQ phosphorylation) or whether it has a distinct physiological role that is not required for sustained oscillation.

Reviewer #3 (Public Review):

The study presents strong evidence for allosteric activation of plant receptor kinases, which enhances our understanding of the non-catalytic mechanisms employed by this large family of receptors.

Plant receptor kinases (RKs) play a critical role in transducing extracellular signals. The activation of RKs involves homo- or heterodimerization of the RKs, and it is believed that mutual phosphorylation of their intracellular kinase domains initiates downstream signaling. However, this model faces a challenge in cases where the kinase domain exhibits pseudokinase characteristics. In their recent study, Mühlenbeck et al. reveal the non-catalytic activation mechanisms of the EFR-BAK1 complex in plant receptor kinase signaling. Specifically, they aimed to determine that the EFR kinase domain activates BAK1 not through its kinase activity, but rather by utilizing a "conformational toggle" mechanism to enter an active-like state, enabling allosteric trans-activation of BAK1. The study sought to elucidate the structural elements and mutations of EFR that affect this conformational switch, as well as explore the implications for immune signaling in plants. To investigate the activation mechanisms of the EFR-BAK1 complex, the research team employed a combination of mutational analysis, structural studies, and hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis. For instance, through HDX-MS analysis, Mühlenbeck et al. discovered that the EFR (Y836F) mutation impairs the accessibility of the active-like conformation. On the other hand, they identified the EFR (F761H) mutation as a potent intragenic suppressor capable of stabilizing the active-like conformation, highlighting the pivotal role of allosteric regulation in BAK1 kinase activation. The data obtained from this methodology strengthens their major conclusion. Moreover, the researchers propose that the allosteric activation mechanism may extend beyond the EFR-BAK1 complex, as it may also be partially conserved in the Arabidopsis LRR-RK XIIa kinases. This suggests a broader role for non-catalytic mechanisms in plant RK signaling.

The allosteric activation mechanism was demonstrated for receptor tyrosine kinases (RTKs) many years ago. A similar mechanism has been suggested for the activation of plant RKs, but experimental evidence for this conclusion is lacking. Data in this study represent a significant advancement in our understanding of non-catalytic mechanisms in plant RK signaling. By shedding light on the allosteric regulation of BAK1, the study provides a new paradigm for future research in this area.

• for: interesting fact - many Albertans are from America

• interesting fact

  • 3 million people settled the Canadian Praries between 1880 and 1913
  • 30% of them were fromNebrask and Montana

Reviewer #3 (Public Review):

This is an interesting manuscript that builds off of this group's previous work focused on the interface between Hsf1, heat shock protein (HSP) mRNA production, and 3D genome topology. Here the group subjects the yeast Saccharomyces cerevisiae to either heat stress (HS) or ethanol stress (ES) and examines Hsf1 and Pol II chromatin binding, Histone occupancy, Hsf1 condensates, HSP gene coalescence (by 3C and live cell imaging), and HSP mRNA expression (by RT-qPCR and live cell imaging). The manuscript is well written, and the experiments seem well done, and generally rigorous, with orthogonal approaches performed to support conclusions. The main findings are that both HS and ES result in Hsf1/Pol II-dependent intergenic interactions, along with the formation of Hsf1 condensates. Yet, while HS results in rapid and strong induction of HSP gene expression and Hsf1 condensate resolution, ES results in slow and weak induction of HSP gene expression without Hsf1 condensate resolution. Thus, the conclusion is somewhat phenomenological - that the same transcription factor can drive distinct transcription, topologic, and phase-separation behavior in response to different types of stress. While identifying a mechanistic basis for these results would be a tough task perhaps beyond the scope of this study, it would nevertheless be helpful to place these results in context with a series of other studies demonstrating across various organisms showing Hsf1 driving distinct activities dependent on the context of activation. Perhaps even more importantly, this work left out PMID: 32015439 which is particularly relevant considering that it shows that it is human HSF1 condensate resolution rather than simple condensate formation that is associated with HSF1 transcriptional activity - which is similar to the findings here with this particular dose of HS resulting in resolution and high transcriptional activity versus ES resulting in resolution failure and lower activity. It is also worth noting that the stresses themselves are quite different - ethanol can be used as a carbon source and so beyond inducing proteotoxic stress, the yeast are presumably adapting to this distinct metabolic state. Basically, it is not clear whether these differences are due to the dose of stress, versus we are looking at an early timepoint as ES initiates a genome-wide chromatin restructuring and gene expression reprogramming that goes beyond a response to proteotoxic stress. This reviewer is not suggesting a barrage of new experiments, but perhaps discussion points to contextualize results.

Reviewer #3 (Public Review):

The study presents a systematic analysis of how a range of dystroglycan mutations alter CCK/CB1 axonal targeting and inhibition in hippocampal CA1 and impact seizure susceptibility. The study follows up on prior literature identifying a role for dystroglycan in CCK/CB1 synapse formation. The careful assay includes comparison of 5 distinct dystroglycan mutation types known to be associated with varying degrees of muscular dystrophy phenotypes: a forebrain specific Dag1 knockout in excitatory neurons at 10.5, a forebrain specific knockout of the glycosyltransferase enzyme in excitatory neurons, mice with deletion of the intracellular domain of beta-Dag1 and 2 lines with missense mutations with milder phenotypes. They show that forebrain glutamatergic deletion of Dag1 or glycosyltransferase alters cortical lamination while lamination is preserved in mice with deletion of the intracellular domain or missense mutation. The study extends prior works by identifying that forebrain deletion of Dag1 or glycosyltransferase in excitatory neurons impairs CCK/CB1 and not PV axonal targeting and CB1 basket formation around CA1 pyramidal cells. Mice with deletion of the intracellular domain or missense mutation show<br /> limited reductions in CCK/CB1 fibers in CA1. Carbachol enhancement of CA1 IPSCs was reduced both in forebrain knockouts. Interestingly, carbachol enhancement of CA1 IPSCs was reduced when the intracellular domain of beta-Dag1was deleted, but not I the missense mutations, suggesting a role of the intracellular domain in synapse maintenance. All lines except the missense mutations , showed increased susceptibility to chemically induced behavioral seizures. Together, the study, is carefully designed, well controlled and systematic. The results advance prior findings of the role for dystroglycans in CCK/CB1 innervations of PCs by demonstrating effects of more selective cellular deletions and site specific mutations in extracellular and intracellular domains.

Prior concerns regarding CCK/CB1 cell numbers and potential changes in basal synaptic inhibition are addressed in the revision.

Reviewer #3 (Public Review):

This manuscript investigates how a seemingly random choice of odourant receptor (OR) gene expression is organised into sterotypic zones of OR expression along the olfactory epithelium. Using a varietty of functional genomics methods, the authors find that along the differentiation axis (progenitor to mature olfactory sensory neuron, OSN) multiple ORs are initally transcribed and from among these, only one OR is selected for expression. The rest are suppressed through chromatin silencing. In addition to this, the authors report a dorso-ventral gradient in OR expression at the immature stage - dorsally expressed ORs are also expressed ventrally and these then get silenced. The expression of the ventrally expressed ORs, on the other hand, are restricted to the ventral region. They suggest a role for the transcription factor NF1 in this dorsoventral process.

This is a valuable study. The data are compelling and generally well presented.

Reviewer #3 (Public Review):

The manuscript by Lin et al. reveals a novel positive regulatory loop between ZEB2 and ACSL4, which promotes lipid droplets storage to meet the energy needs of breast cancer metastasis.

Reviewer #3 (Public Review):

This study on drug repurposing presents the identification of potent activators of the Hippo pathway. The authors successfully screen a drug library and identify two CLK kinase inhibitors as YAP activators, with SM04690 targeting specifically CLK2. They further investigate the molecular basis of SM04690-induced YAP activity and identify splicing events in AMOTL2 as strongly affected by CLK2 inhibition. Exon skipping within AMOTL2 decreases the interactions with membrane bound proteins and is sufficient to induce YAP target gene expression. Importantly, inhibitor concentrations that are sufficient to change YAP target gene expression show differential alternative splicing of AMOTL2. Overall the study is well designed, the conclusions are supported by sufficient data and represent an exciting connection between alternative splicing and the HIPPO pathway.

Reviewer #3 (Public Review):

Summary:<br /> The study by Alfatah et al. presented a role for YBR238C in mediating lifespan through improved mitochondrial function in a TOR1-dependent metabolic pathway. The authors used a dataset comparison approach to identify genes positively modulating yeast chronological (CLS) and Replicative (RLS) lifespan when deleted, and their expression is reduced under Rapamycin treatment condition. This approach revealed an unknown, mitochondria-localized yeast gene YBR238C, and through mechanistic studies, they identified its paralogous gene RMD9 regulating lifespan in an antagonistic effect.

Strengths:<br /> Findings have valuable implications for understanding the YBR238C-mediated, mitochondrial-dependent yeast lifespan regulation, and the interplay between two paralogous genes in the regulation of mitochondrial function represents an inserting case for gene evolution.

Weaknesses:<br /> Overall, the implication/findings of this study are restricted only to the yeast model since these two genes do not have any homology in higher eukaryotes. The primary methods must be carefully designed by considering two different metabolic states: respiration-associated with CLS and fermentation-associated with RLS in a single comparative approach. Yeast CLS and RLS are two completely different processes. It is already known that most gene-regulating CLS is not associated with RLS or vice versa. The method section is poorly written and missing important information. The experimental approaches are poorly designed, and variability across the datasets (e.g., media condition "YPD," "SC" etc.) and their experimental conditions are not well described/considered; thus, presented data are not conclusive, which decreases the overall rigor of the study.

Reviewer #3 (Public Review):

Summary:<br /> In this manuscript, the authors use inhibitors and mimetics of juvenile hormone (JH) to demonstrate that JH has a key role in late embryonic development in Thermobia, specifically in gut and eye development but also resorption of the extraembryonic fluid and hatching. They then exogenously apply JH early in development (when it is not normally present) to examine the biological effects of JH at these stages. This causes a plethora of defects including developmental arrest, deposition of chitin, limb development, and enhanced muscle differentiation. The authors interpret these early effects on development as JH being important for the shift from morphogenetic growth to differentiation - a role that they speculate may have facilitated the evolution of metamorphosis (hemi- and holo-metaboly). This paper will be of interest to insect evo-devo researchers, particularly those with interests in the evolution of metamorphosis.

Strengths:<br /> The experiments are generally conducted very well with appropriate controls and the authors have included a very detailed analysis of the phenotypes.<br /> The manuscript significantly advances our understanding of Thermobia development and the role of JH in Thermobia development.<br /> The authors interpret this data to present some hypotheses regarding the role of JH in the evolution of metamorphosis, some aspects of which can be addressed by future studies.

Weaknesses:<br /> The results are based on using inhibitors and mimetics of JH and there was no attempt to discern immediate effects of JH from downstream effects. The authors show, for instance, that the transcription of myoglianin is responsive to JH levels, it would have been interesting to see if any of the phenotypic effects are due to myoglianin upregulation/suppression (using RNAi for example). These kinds of experiments will be necessary to fully work out if and how the JH regulatory network has been co-opted into metamorphosis.

The results generally support the authors' conclusions. However, the discussion contains a lot of speculation and some far-reaching conclusions are made about the role of JH and how it became co-opted into controlling metamorphosis. There are some interesting hypotheses presented and the author's speculations are consistent with the data presented. However, it is difficult to make evolutionary inferences from a single data point as although Thermobia is a basally branching insect, the lineage giving rise to Thermobia diverged from the lineages giving rise to the holo- and hemimetabolous insects approx.. 400 mya and it is possible that the effects of JH seen in Thermobia reflect lineage-specific effects rather than the 'ancestral state'. The authors ignore the possibility that there has been substantial rewiring of the networks that are JH responsive across these 400 my. I would encourage the authors to temper some of the discussion of these hypotheses and include some of the limitations of their inferences regarding the role of JH in the evolution of metamorphosis in their discussion.

Reviewer #3 (Public Review):

Summary:<br /> The paper aims to provide a neurocomputational account of how social perception translates into prosocial behaviors. Participants first completed a novel social perception task during fMRI scanning, in which they were asked to judge the merit or need of people depicted in different situations. Secondly, a separate altruistic choice task was used to examine how the perception of merit and need influences the weights people place on themselves, others, and fairness when deciding to provide help. Finally, a link between perception and action was drawn in those participants who completed both tasks.

Strengths:<br /> The paper is overall very well written and presented, leaving the reader at ease when describing complex methods and results. The approach used by the author is very compelling, as it combines computational modeling of behavior and neuroimaging data analyses. Despite not being able to comment on the computational model, I find the approach used (to disentangle sensitivity and biases, for merit and need) very well described and derived from previous theoretical work. Results are also clearly described and interpreted.

Weaknesses:<br /> My main concern relates to the selection of the social perception task, which to me is the weakest point. Such weakness has been also addressed by the same authors in the limitation section, and related to the fact that merit and need are evaluated by means of very different cues that rely on different cognitive processes (more abstract thinking for merit than need). I wonder whether and how such difference can bias the overall computational model and interpretation of the results (e.g. ideal you vary merit and need to leave all other aspects invariant).

A second weakness is related to the sample size which is quite small for study 2. I wonder, given that study 2 fRMI data are not analyzed, whether is possible to recover some of the participants' behavioral results, at least the ones excluded because of bad MR image quality.

Finally, on a theoretical note, I would elaborate more on the distinction of merit and need. These concepts tap into very specific aspects of morality, which I suspect have been widely explored. At the moment I am missing a more elaborate account of this.

Reviewer #3 (Public Review):

Kang, Huang, and colleagues investigated the impact of LRRK1 and LRRK2 deletion, specifically in dopaminergic neurons, using a novel cDKO mouse model. They observed a significant reduction in DAergic neurons in the substantia nigra in their conditional LRRK1 and LRRK2 KO mice and a corresponding increase in markers of apoptosis and gliosis. This work set out to address a long-standing question within the field around the role and importance of LRRK1 and LRRK2 in DAergic neurons and suggests that the loss of both proteins triggers some neurodegeneration and glial activation.

The studies included in this work are carefully performed and clearly communicated, but additional studies are needed to strengthen further the authors' claims around the consequences of LRRK2 deletion in DAergic neurons.

1) In Figures 2E and F, the authors assess the protein levels of LRRK1 and LRRK2 in their cDKO mouse model to confirm the deletion of both proteins. They observe a mild loss of LRRK1 and LRRK2 signals in the ventral midbrain compared to wild-type animals. While this is not surprising given other cell types that still express LRRK1 and LRRK2 would be present in their dissected ventral midbrain samples, it does not sufficiently confirm that LRRK1 and LRRK2 are not expressed in DAergic neurons. Additional data is needed to more directly demonstrate that LRRK1 and LRRK2 protein levels are reduced in DAergic neurons, including analysis of LRRK1 and LRRK2 protein levels via immunohistochemistry or FACS-based analysis of TH+ neurons.

2) The authors observed a significant but modest effect of LRRK1 and LRRK2 deletion on the number of TH+ neurons in the substantia nigra (12-15% loss at 20-24 months of age). It is unclear whether this extent of neuron loss is functionally relevant. To strengthen the impact of these data, additional studies are warranted to determine whether this translates into any PD-relevant deficits in the mice, including motor deficits or alterations in alpha-synuclein accumulation/aggregation.

3) The authors demonstrate that, unlike in the germline LRRK DKO mice, they do not observe any alterations in electron-dense vacuoles via EM. Given their data showing increased apoptosis and gliosis, it remains unclear how the loss of LRRK proteins leads to DAergic neuronal cell loss. Mechanistic studies would be insightful to understand better potential explanations for how the loss of LRRK1 and LRRK2 may impair cellular survival, and additional text should be added to the discussion to discuss potential hypotheses for how this might occur.

4) The authors discuss the potential implications of the neuronal cell loss observed in cDKO mice for LRRK1 and LRRK2 for therapeutic approaches targeting LRRK2 and suggest this argues that LRRK2 variants may exert their effects through a loss-of-protein function. However, all of the data generated in this work focus on a mouse in which both LRRK1 and LRRK2 have been deleted, and it is therefore difficult to make any definitive conclusions about the consequences of specifically targeting LRRK2. The authors note potential redundancy between the two LRRK proteins, and they should soften some of their conclusions in the discussion section around implications for the effects of LRRK2 variants. Human subjects that carry LRRK2 loss-of-function alleles do not have an increased risk for developing PD, which argues against the author's conclusions that LRRK2 variants associated with PD are loss-of-function. Additional text should be included in their discussion to better address these nuances and caution should be used in terms of extrapolating their data to effects observed with PD-linked variants in LRRK2.

Reviewer #3 (Public Review):

Summary:<br /> In this study, Jin et al. report the first evidence of CFAP52 mutations in human male infertility by identifying deleterious compound heterozygous mutations of CFAP52 in infertile human patients with acephalic and multiple morphological abnormalities in flagella (MMAF) phenotypes but without other abnormalities in motile cilia. They validated the pathogenicity of the mutations by an in vitro minigene assay and the absence of proteins in the patient's spermatozoa. Using a Cfap52 knockout mouse model they generated, the authors showed that the animals are hydrocephalic and the sperm have coupling defects, head decapitation, and axonemal structure disruption, supporting what was observed in human patients.

Strengths:<br /> The major strengths of the study are the rigorous phenotypic and molecular analysis of normal and patient spermatozoa and the demonstration of infertility treatment by ICSI. The authors demonstrated the interaction between CFAP52 and SPATA6, a head-tail coupling regulator and structural protein, and showed that CFAP52 can interact with components of the microtubule inner protein (MIP), radial spoke, and outer dynein arm proteins.

Weaknesses:<br /> The weakness of the study is some inconsistency in the localization of the CFAP52 protein in human spermatozoa in the figures and the lack of such localization information completely missing in mouse spermatozoa. Putting their findings in the context of the newly available structural information from the recent series of unambiguous and unequivocal identification of CFAP52 as an MIP in the B tubule will not only greatly benefit the interpretation of the study, but also resolve the inconsistent sperm phenotypes reported by an independent study. Since the mouse model is not designed to exactly recapitulate the human mutations but a complete knockout and the knockout mice show hydrocephaly phenotype as well, some of the claims of causality and ICSI as a treatment need to be tempered. Discussing the frequency of acephaly and MMAF in primary male infertility will be beneficial to justify CFAP52 as a practical diagnostic tool.

Reviewer #3 (Public Review):

Summary:<br /> The authors perform a thorough examination of the phenotypes of a newly generated Trip13 null allele in mice, noting defects in chromosome synapsis and impact on localization of other key proteins (namely HORMADs) on meiotic chromosomes. The vast majority of data confirms observations of several prior studies of Trip13 alleles (moderate and severe hypomorphs). The original or primary aims of the study aren't clear, but it can be assumed that the authors wanted to better study the role of this protein in evicting HORMADs upon synapsis by studying phenotypes of mutants and better characterizing TRIP13 localization data (which they find localizes to the central element of synapsed chromosomes using a new epitope-tagged allele). Their data confirm prior reports and are consistent with localization data of the orthologous Pch2 protein in many other organisms.

Strengths:<br /> The quality of data is high. Probably the most important data the authors find is that TRIP13 is localized along the CE of synapsed chromosomes. However, this was not unexpected because PCH2 is also similarly localized. Also, the authors use a clear null (deletion allele), whereas prior studies used hypomorphs.

Weaknesses:<br /> There is limited new data; most are confirmatory or expected (i.e., SC localization), and thus the impact of this report is not high. The claim that TRIP13 "functions as a dosage-sensitive regulator of meiosis" is exaggerated in my opinion. Indeed, the authors make the observation that hets have a phenotype, but numerous genes have haploinsufficient phenotypes. In my opinion, it is a leap to extrapolate this to infer that TRIP13 is a "regulator" of meiosis. What is the definition of a meiosis regulator? Is it at the apex of the meiosis process, or is it a crucial cog of any aspect of meiosis?

Reviewer #3 (Public Review):

In this study, Lazar-Contes and colleagues aimed to determine whether chromatin accessibility changes in the spermatogonial population during different phases of postnatal mammalian testis development. Because actions of the spermatogonial population set the foundation for continual and robust spermatogenesis and the gene networks regulating their biology are undefined, the goal of the study has merit. To advance knowledge, the authors used mice as a model and isolated spermatogonia from three different postnatal developmental age points using a cell sorting methodology that was based on cell surface markers reported in previous studies and then performed bulk RNA-sequencing and ATAC-sequencing. Overall, the technical aspects of the sequencing analyses and computational/bioinformatics seem sound but there are several concerns with the cell population isolated from testes and lack of acknowledgment for previous studies that have also performed ATAC-sequencing on spermatogonia of mouse and human testes. The limitations, described below, call into question the validity of the interpretations and reduce the potential merit of the findings.

I suggest changing the acronym for spermatogonial cells from SC to SPG for two reasons. First, SPG is the commonly used acronym in the field of mammalian spermatogenesis. Second, SC is commonly used for Sertoli Cells.

The authors should provide a rationale for why they used postnatal day 8 and 15 mice.

The FACS sorting approach used was based on cell surface proteins that are not germline-specific so there were undoubtedly somatic cells in the samples used for both RNA and ATAC sequencing. Thus, it is essential to demonstrate the level of both germ cell and undifferentiated spermatogonial enrichment in the isolated and profiled cell populations. To achieve this, the authors used PLZF as a biomarker of undifferentiated spermatogonia. Although PLZF is indeed expressed by undifferentiated spermatogonia, there have been several studies demonstrating that expression extends into differentiating spermatogonia. In addition, PLZF is not germ-cell specific and single-cell RNA-seq analyses of testicular tissue have revealed that there are somatic cell populations that express Plzf, at least at the mRNA level. For these reasons, I suggest that the authors assess the isolated cell populations using a germ-cell specific biomarker such as DDX4 in combination with PLZF to get a more accurate assessment of the undifferentiated spermatogonial composition. This assessment is essential for the interpretation of the RNA-seq and ATAC-seq data that was generated.

A previous study by the Namekawa lab (PMID: 29126117) performed ATAC-seq on a similar cell population (THY1+ FACS sorted) that was isolated from pre-pubertal mouse testes. It was surprising to not see this study referenced in the current manuscript. In addition, it seems prudent to cross-reference the two ATAC-seq datasets for commonalities and differences. In addition, there are several published studies on scATAC-seq of human spermatogonia that might be of interest to cross-reference with the ATAC-seq data presented in the current study to provide an understanding of translational merit for the findings.

Reviewer #3 (Public Review):

Summary:<br /> As SMAD1/5 activities have previously been indistinguishable, these studies provide a new mouse model to finally understand unique downstream activation of SMAD1/5 target genes, a model useful for many scientific fields. Using CUT&RUN analyses with gene overlap comparisons and signaling pathway analyses, specific targets for SMAD1 versus SMAD5 were compared, identified, and interpreted. These data validate previous findings showing strong evidence that SMADs directly govern critical genes required for endometrial receptivity and decidualization, including cell adhesion and vascular development. Further, SMAD targets were overlapped with progesterone receptor binding sites to identify regions of potential synergistic regulation of implantation. The authors report strong correlations between progesterone receptor and SMAD1/5 direct targets to cooperatively promote embryo implantation. Finally, the authors validated SMAD1/5 gene regulation in primary human endometrial stromal cells. These studies provide a data-rich survey of SMAD family transcription, defining its role as a governor of early pregnancy.

Strengths:<br /> This manuscript provides a valuable survey of SMAD1/5 direct transcriptional events at the time of receptivity. As embryo implantation is controlled by extensive epithelial to stromal molecular crosstalk and hormonal regulation in space and time, the authors state a strong, descriptive narrative defining how SMAD1/5 plays a central role at the site of this molecular orchestration. The implementation of cutting-edge techniques and models and simple comparative analyses provide a straightforward, yet elegant manuscript.

Although the progesterone receptor exists as a major regulator of early pregnancy, the authors have demonstrated clear evidence that progesterone receptor with SMAD1/5 work in concert to molecularly regulate targets such as Sox17, Id2, Tgfbr2, Runx1, Foxo1 and more at embryo implantation. Additionally, the authors pinpoint other critical transcription factor motifs that work with SMADs and the progesterone receptor to promote early pregnancy transcriptional paradigms.

Weaknesses:<br /> Although a wonderful new tool to ascertain SMAD1 versus SMAD5 downstream signaling, the importance of these factors in governing early pregnancy is not novel. Furthermore, functional validation studies are needed to confirm interactions at promoter regions. Addtionally, the authors presume that all overlapped genes are shared between progesterone receptor and SMAD1/5, yet some peak representations do not overlap. Although, transcriptional activation can occur at the same time, they may not occur in the same complex. Thus, further confirmation of these transcriptional events is warranted.

Since whole murine uterus was used for these studies, the specific functions of SMAD1/5 in the stroma versus the epithelium (versus the myometrium) remain unknown. Specific roles for SMAD1/5 in the uterine stroma and epithelial compartments still need to be examined. Also, further work is needed to delineate binding and transcriptional activation of SMAD1/5 and the progesterone receptor in stromal versus epithelial uterine compartments.

There are asynchronous gene responses in the SMAD1/5 ablated mouse model compared to the siRNA-treated human endometrial stromal cells. These differences can be confounding, and more clarity is required in understanding the meaning of these differences and as they relate to the entire SMAD transcriptome.

Reviewer #3 (Public Review):

This simulation study presents a valuable finding on the load-dependence (i.e., dependence on a pulling force) of the recognition of a peptide-bound major histocompatibility complex (pMHC) antigen by a T cell receptor (TCR). The evidence supporting the claims of the authors is solid, although inclusion of a larger number of simulations would have strengthened the study. The work will be of interest to computational structural biologists and immunologists.

Reviewer #3 (Public Review):

This study tackles a major problem with replay detection, which is that different methods can produce vastly different results. It provides compelling evidence that the source of this inconsistency is that biological data often violates assumptions of independent samples. This results in false positive rates that can vary greatly with the precise statistical assumptions of the chosen replay measure, the detection parameters, and the dataset itself. To address this issue, the authors propose to empirically estimate the false positive rate and control for it by adjusting the significance threshold. Remarkably, this reconciles the differences in replay detection methods, as the results of all the replay methods tested converge quite well (see Figure 6B). This suggests that by controlling for the false positive rate, one can get an accurate estimate of replay with any of the standard methods.

When comparing different replay detection methods, the authors use a sequence-independent log-odds difference score as a validation tool and an indirect measure of replay quality. This takes advantage of the two-track design of the experimental data, and its use here relies on the assumption that a true replay event would be associated with good (discriminable) reactivation of the environment that is being replayed. The other way replay "quality" is estimated is by the number of replay events detected once the false positive rate is taken into account. In this scheme, "better" replay is in the top right corner of Figure 6B: many detected events associated with congruent reactivation.

There are two possible ways the results from this study can be integrated into future replay research. The first, simpler, way is to take note of the empirically estimated false positive rates reported here and simply avoid the methods that result in high false positive rates (weighted correlation with a place bin shuffle or all-spike Spearman correlation with a spike-id shuffle). The second, perhaps more desirable, way is to integrate the practice of estimating the false positive rate when scoring replay and to take it into account. This is very powerful as it can be applied to any replay method with any choice of parameters and get an accurate estimate of replay.

How does one estimate the false positive rate in their dataset? The authors propose to use a cell-ID shuffle, which preserves all the firing statistics of replay events (bursts of spikes by the same cell, multi-unit fluctuations, etc.) but randomly swaps the cells' place fields, and to repeat the replay detection on this surrogate randomized dataset. Of course, there is no perfect shuffle, and it is possible that a surrogate dataset based on this particular shuffle may result in one underestimating the true false positive rate if different cell types are present (e.g. place field statistics may differ between CA1 and CA3 cells, or deep vs. superficial CA1 cells, or place cells vs. non-place cells if inclusion criteria are not strict). Moreover, it is crucial that this validation shuffle be independent of any shuffling procedure used to determine replay itself (which may not always be the case, particularly for the pre-decoding place field circular shuffle used by some of the methods here) lest the true false-positive rate be underestimated. Once the false positive rate is estimated, there are different ways one may choose to control for it: adjusting the significance threshold as the current study proposes, or directly comparing the number of events detected in the original vs surrogate data. Either way, with these caveats in mind, controlling for the false positive rate to the best of our ability is a powerful approach that the field should integrate.

Which replay detection method performed the best? If one does not control for varying false positive rates, there are two methods that resulted in strikingly high (>15%) false positive rates: these were weighted correlation with a place bin shuffle and Spearman correlation (using all spikes) with a spike-id shuffle. However, after controlling for the false positive rate (Figure 6B) all methods largely agree, including those with initially high false positive rates. There is no clear "winner" method, because there is a lot of overlap in the confidence intervals, and there also are some additional reasons for not overly interpreting small differences in the observed results between methods. The confidence intervals are likely to underestimate the true variance in the data because the resampling procedure does not involve hierarchical statistics and thus fails to account for statistical dependencies on the session and animal level. Moreover, it is possible that methods that involve shuffles similar to the cross-validation shuffle ("wcorr 2 shuffles", "wcorr 3 shuffles" both use a pre-decoding place field circular shuffle, which is very similar to the pre-decoding place field swap used in the cross-validation procedure to estimate the false positive rate) may underestimate the false positive rate and therefore inflate adjusted p-value and the proportion of significant events. We should therefore not interpret small differences in the measured values between methods, and the only clear winner and the best way to score replay is using any method after taking the empirically estimated false positive rate into account.

The authors recommend excluding low-ripple power events in sleep, because no replay was observed in events with low (0-3 z-units) ripple power specifically in sleep, but that no ripple restriction is necessary for awake events. There are problems with this conclusion. First, ripple power is not the only way to detect sharp-wave ripples (the sharp wave is very informative in detecting awake events). Second, when talking about sequence quality in awake non-ripple data, it is imperative for one to exclude theta sequences. The authors' speed threshold of 5 cm/s is not sufficient to guarantee that no theta cycles contaminate the awake replay events. Third, a direct comparison of the results with and without exclusion is lacking (selecting for the lower ripple power events is not the same as not having a threshold), so it is unclear how crucial it is to exclude the minority of the sleep events outside of ripples. The decision of whether or not to select for ripples should depend on the particular study and experimental conditions that can affect this measure (electrode placement, brain state prevalence, noise levels, etc.).

Finally, the authors address a controversial topic of de-novo preplay. With replay detection corrected for the false positive rate, none of the detection methods produce evidence of preplay sequences nor sequenceless reactivation in the tested dataset. This presents compelling evidence in favour of the view that the sequence of place fields formed on a novel track cannot be predicted by the sequential structure found in pre-task sleep.

Reviewer #3 (Public Review):

This study tackles a major problem with replay detection, which is that different methods can produce vastly different results. It provides compelling evidence that the source of this inconsistency is that biological data often violates assumptions of independent samples. This results in false positive rates that can vary greatly with the precise statistical assumptions of the chosen replay measure, the detection parameters, and the dataset itself. To address this issue, the authors propose to empirically estimate the false positive rate and control for it by adjusting the significance threshold. Remarkably, this reconciles the differences in replay detection methods, as the results of all the replay methods tested converge quite well (see Figure 6B). This suggests that by controlling for the false positive rate, one can get an accurate estimate of replay with any of the standard methods.

When comparing different replay detection methods, the authors use a sequence-independent log-odds difference score as a validation tool and an indirect measure of replay quality. This takes advantage of the two-track design of the experimental data, and its use here relies on the assumption that a true replay event would be associated with good (discriminable) reactivation of the environment that is being replayed. The other way replay "quality" is estimated is by the number of replay events detected once the false positive rate is taken into account. In this scheme, "better" replay is in the top right corner of Figure 6B: many detected events associated with congruent reactivation.

There are two possible ways the results from this study can be integrated into future replay research. The first, simpler, way is to take note of the empirically estimated false positive rates reported here and simply avoid the methods that result in high false positive rates (weighted correlation with a place bin shuffle or all-spike Spearman correlation with a spike-id shuffle). The second, perhaps more desirable, way is to integrate the practice of estimating the false positive rate when scoring replay and to take it into account. This is very powerful as it can be applied to any replay method with any choice of parameters and get an accurate estimate of replay.

How does one estimate the false positive rate in their dataset? The authors propose to use a cell-ID shuffle, which preserves all the firing statistics of replay events (bursts of spikes by the same cell, multi-unit fluctuations, etc.) but randomly swaps the cells' place fields, and to repeat the replay detection on this surrogate randomized dataset. Of course, there is no perfect shuffle, and it is possible that a surrogate dataset based on this particular shuffle may result in one underestimating the true false positive rate if different cell types are present (e.g. place field statistics may differ between CA1 and CA3 cells, or deep vs. superficial CA1 cells, or place cells vs. non-place cells if inclusion criteria are not strict). Moreover, it is crucial that this validation shuffle be independent of any shuffling procedure used to determine replay itself (which may not always be the case, particularly for the pre-decoding place field circular shuffle used by some of the methods here) lest the true false-positive rate be underestimated. Once the false positive rate is estimated, there are different ways one may choose to control for it: adjusting the significance threshold as the current study proposes, or directly comparing the number of events detected in the original vs surrogate data. Either way, with these caveats in mind, controlling for the false positive rate to the best of our ability is a powerful approach that the field should integrate.

Which replay detection method performed the best? If one does not control for varying false positive rates, there are two methods that resulted in strikingly high (>15%) false positive rates: these were weighted correlation with a place bin shuffle and Spearman correlation (using all spikes) with a spike-id shuffle. However, after controlling for the false positive rate (Figure 6B) all methods largely agree, including those with initially high false positive rates. There is no clear "winner" method, because there is a lot of overlap in the confidence intervals, and there also are some additional reasons for not overly interpreting small differences in the observed results between methods. The confidence intervals are likely to underestimate the true variance in the data because the resampling procedure does not involve hierarchical statistics and thus fails to account for statistical dependencies on the session and animal level. Moreover, it is possible that methods that involve shuffles similar to the cross-validation shuffle ("wcorr 2 shuffles", "wcorr 3 shuffles" both use a pre-decoding place field circular shuffle, which is very similar to the pre-decoding place field swap used in the cross-validation procedure to estimate the false positive rate) may underestimate the false positive rate and therefore inflate adjusted p-value and the proportion of significant events. We should therefore not interpret small differences in the measured values between methods, and the only clear winner and the best way to score replay is using any method after taking the empirically estimated false positive rate into account.

The authors recommend excluding low-ripple power events in sleep, because no replay was observed in events with low (0-3 z-units) ripple power specifically in sleep, but that no ripple restriction is necessary for awake events. There are problems with this conclusion. First, ripple power is not the only way to detect sharp-wave ripples (the sharp wave is very informative in detecting awake events). Second, when talking about sequence quality in awake non-ripple data, it is imperative for one to exclude theta sequences. The authors' speed threshold of 5 cm/s is not sufficient to guarantee that no theta cycles contaminate the awake replay events. Third, a direct comparison of the results with and without exclusion is lacking (selecting for the lower ripple power events is not the same as not having a threshold), so it is unclear how crucial it is to exclude the minority of the sleep events outside of ripples. The decision of whether or not to select for ripples should depend on the particular study and experimental conditions that can affect this measure (electrode placement, brain state prevalence, noise levels, etc.).

Finally, the authors address a controversial topic of de-novo preplay. With replay detection corrected for the false positive rate, none of the detection methods produce evidence of preplay sequences nor sequenceless reactivation in the tested dataset. This presents compelling evidence in favour of the view that the sequence of place fields formed on a novel track cannot be predicted by the sequential structure found in pre-task sleep.

DOI: 10.7554/eLife.85930

Resource: ZFIN_ZDB-GENO-100618-3

Curator: @evieth

SciCrunch record: RRID:ZFIN_ZDB-GENO-100618-3

What is this?

Reviewer #3 (Public Review):

This work is carried out by the research group led by Shuiqiao Yuan, who has a long interest in and significant contribution to the field of male germ cell development. The authors study a protein for which limited information existed prior to this work, a component of the E3 ubiquitin ligase complex, FBXO24. The authors generated the first FBXO24 KO mouse model reported in the literature using CRISPR, which they complement with HA-tagged FBXO24 transgenic model in the KO background. The authors begin their study with a very careful examination of the expression pattern of the FBXO24 gene at the level of mRNA and the HA-tagged transgene, and they provide conclusive evidence that the protein is expressed exclusively in the mouse testis and specifically in post-meiotic spermatids of stages VI to IX, which include early stages of spermatid elongation and nuclear condensation. The authors report a fully sterile phenotype for male mice, while female mice are normal. Interestingly, the testis size and the populations of spermatogenic cells in the KO mutant mice show a small (but significant) reduction compared to the WT testis. Importantly, the mature sperm from KO animals show a series of defects that were very thoroughly documented in this work by scanning and transmission electron microscopy; this data constitutes a very strong point in this paper. FBXO24 KO sperm have severe defects in the mitochondrial sheath with missing mitochondria near the annulus, and missing outer dense fibers. Collectively these defects cause abnormal bending of the flagellum and severely reduced sperm motility. Moreover, defects in nuclear condensation are observed with faint nuclear staining of elongating and elongated spermatids, and reduction of protein levels of protamine 2 combined with increased levels of histones and transition protein 1. All of the above are in line with the observed male sterility phenotype.

The authors also performed RNAseq in the KO animal, and found profound changes in the abundance of thousands of mRNAs; and changes in mRNA splicing patterns as well. The data reveal deeply affected gene expression patterns in the FBXO24 KO testis, which further supports the essential role that this factor serves in spermiogenesis. Unfortunately, a molecular explanation of what causes these changes is missing; it is still possible that they are an indirect consequence of the KO and not directly caused by the KO.

A well-reasoned narrative on if and how the absence of FBXO24 as an E3 ubiquitin ligase is responsible for the observed mRNA and protein differential expression is missing. If FBXO24-mediated ubiquitination is required for normal protein degradation during spermiogenesis, protein level increase should be the direct consequence of genuine FBXO24 targets in the KO testis. Importantly, besides the Miwi ubiquitination experiment which is performed in a heterologous and therefore may not be ideal for extracting conclusions, the possible involvement of ubiquitination was not shown for any other proteins that the authors found that interact with FBXO24. For example splicing factors SRSF2, SRSF3, SRSF9, or any of the other proteins whose levels were found to be changed (reduced, thus the change in the KO is less likely due to the absence of ubiquitination) such as ODF2, AKAP3, TSSK4, PHF7, TSSK6, and RNF8. Interestingly, the authors do observe increased amounts of histones and transition proteins, but reduced amounts of protamines, which directly shows that histone to protamine transition is indeed affected in the FBXO24 KO testis, and is in agreement with the observed less condensed nuclei of spermatozoa. Could histones and transition proteins be targets of the proposed ubiquitin ligase activity of FBXO24, and in its absence, histone replacement is abrogated? Providing experimental evidence to address this possibility would greatly expand our understanding of why FBXO24 is essential during spermiogenesis.

Regarding the results on Miwi protein and piRNAs, the following remarks can be made:

The finding that the Miwi protein is upregulated is an important point in this work, and it is in agreement with the observed increased size of the chromatoid body, where most of the Miwi protein is accumulated in round spermatids. This finding needs to be further supported and verified with experiments done in WT and KO mice. Miwi should be immunoprecipitated and Miwi ubiquitination should be detected (with WB or mass spec) in WT testis. It should be expected that Miwi ubiquitination is reduced in KO testis. The experiments that the authors performed in HEK293T cells are informative but experiments with tissue/cells normally expressing Miwi and FBXO24 are missing. With regard to piRNA expression, it is an exaggeration to call the observed increase in piRNA expression remarkable, especially since one replicate small RNA library per condition was sequenced. Although the library is constructed from total small RNA (which includes Mili-bound piRNAs as well), it does seem that the upregulated piRNAs are Miwi-bound piRNAs because the size of the upregulated piRNAs is mostly 29-32 bases. However, the direct comparison of the number of upregulated piRNAs with upregulated miRNAs is not in support of the claim that the increase in piRNA expression is higher compared to miRNAs: there are approximately a few hundred miRNAs expressed in mice, but hundreds of thousands of different piRNA sequences, so upregulation of ~10 times more piRNA species than miRNAs is a smaller proportional increase. Moreover, the observed increase in the overall piRNA levels could be just an epiphenomenon of the increased abundance of the Miwi protein; it has been documented that Piwi proteins stabilize their piRNA cargo, so most likely the increase in iRNA levels in 29-32 nt sizes is probably not a result of altered biogenesis, but increased half-life of the piRNAs as a result of Miwi upregulation. Therefore, the claim that FBXO24 is essential for piRNA biogenesis/production (lines 308, 314) is not appropriately supported.

Reviewer #3 (Public Review):

Summary:

The paper points out that non-significance in both the original study and a replication does not ensure that the studies provide evidence for the absence of an effect. Also, it can not be considered a "replication success". The main point of the paper is rather obvious. It may be that both studies are underpowered, in which case their non-significance does not prove anything. The absence of evidence is not evidence of absence! On the other hand, statistical significance is a confusing concept for many, so some extra clarification is always welcome.

One might wonder if the problem that the paper addresses is really a big issue. The authors point to the "Reproducibility Project: Cancer Biology" (RPCB, Errington et al., 2021). They criticize Errington et al. because they "explicitly defined null results in both the original and the replication study as a criterion for replication success." This is true in a literal sense, but it is also a little bit uncharitable. Errington et al. assessed replication success of "null results" with respect to 5 criteria, just one of which was statistical (non-)significance.

It is very hard to decide if a replication was "successful" or not. After all, the original significant result could have been a false positive, and the original null-result a false negative. In light of these difficulties, I found the paper of Errington et al. quite balanced and thoughtful. Replication has been called "the cornerstone of science" but it turns out that it's actually very difficult to define "replication success". I find the paper of Pawel, Heyard, Micheloud, and Held to be a useful addition to the discussion.

Strengths:

This is a clearly written paper that is a useful addition to the important discussion of what constitutes a successful replication.

Weaknesses:

To me, it seems rather obvious that non-significance in both the original study and a replication does not ensure that the studies provide evidence for the absence of an effect. I'm not sure how often this mistake is made.

Reviewer #3 (Public Review):

Summary<br /> This study investigated the role of the exonuclease Xrn1 in regulating autophagy in response to methionine deprivation in the budding yeast (S. cerevisiae). As a model system, wild-type and xrn1-deletion cells are switched from a nutrient-rich, lactate-based media (YPL) to a synthetic, minimal, lactate media with or without re-addition of methionine. Autophagy is measured by a previously reported Idh-GFP cleavage assay, and in some cases by quantification of alkaline phosphatase activity. The authors conclude that Xrn1 suppresses autophagy in response to methionine depletion based on the results of the Idh-GFP assay. However, the alkaline phosphatase assay could potentially suggest the opposite conclusion, with xnr1 deletion blocking the induction of autophagy relative to baseline in those cells, an interpretation which is complicated by higher basal autophagy induction upon xnr1 deletion. To address the mechanism of Xrn1 regulation of autophagy, a model is presented in which Xrn1 activates Target of Rapamycin Complex 1 (TORC1), which suppresses autophagy. This regulation is proposed to occur through physical association of Xrn1 with known upstream regulators of TORC1 activity, the SEACIT/GATOR1 and Gtr/Rag complexes. However, TORC1 activity is not measured under many key experimental conditions, making it difficult to determine the accuracy of this model. If the model ultimately proves correct, this would be an important finding that establishes a new player in the critical TORC1 pathway that controls cell growth and metabolism in response to changes in nutrient availability.

Strengths<br /> Clear and highly reproducible results using the Idh-GFP cleavage assay to measure apoptosis.

Detailed characterization of the metabolic and transcriptomic effects of Xrn1 deletion through metabolomics and RNA-seq.

Use of a catalytically inactive Xrn1 mutant to demonstrate that its effects on autophagy require its catalytic activity.

Weaknesses<br /> Predominant use of a single autophagy assay (Idh-GFP cleavage), with potentially conflicting results in another assay (alkaline phosphatase activity).

TORC1 activity is not measured under many key experimental conditions.

Protein-protein interactions are studied by overexpression of tagged proteins. While this may be essential for detection, the level of overexpression relative to endogenous protein is unclear, as well as whether this recapitulates the endogenous interactions and regulation.

Results from some experiments have several possible interpretations.

Reviewer #3 (Public Review):

The authors use a full-likelihood multispecies coalescent (MSC) approach to identify major introgression events throughout the radiation of Heliconius butterflies, thereby improving estimates of the phylogeny. First, the authors conclude that H. aoede is the likely outgroup relative to other Heliconius species; miocene introgression into the ancestor of H. aoede makes it appear to branch later. Topologies at most loci were not concordant with this scenario, though 'aoede-early' topologies were enriched in regions of the genome where interspecific introgression is expected to be reduced: the Z chromosome and larger autosomes. The revised phylogeny is interesting because it would mean that no extant Heliconius species has reverted to a non-pollen-feeding ancestral state. Second, the authors focus on a particularly challenging clade in which ancient and ongoing gene flow is extensive, concluding that silvaniform species are not monophyletic. Building on these results, a third set of analyses investigates the origin of the P1 inversion, which harbours multiple wing patterning loci, and which is maintained as a balanced polymorphism in H. numata. The authors present data supporting a new scenario in which P1 arises in H. numata or its ancestor and is introduced to the ancestor of H. pardilinus and H. elevatus - introgression in the opposite direction to what has previously been proposed using a smaller set of taxa and different methods.

The analyses were extensive and methodologically sound. Care was taken to control for potential sources of error arising from incorrect genotype calls and the choice of a reference genome. The argument for H. aoede as the earliest-diverging Heliconius lineage was compelling, and analyses of the melpomene-silvaniform clade were thorough.

The authors have demonstrated the strengths of a full-likelihood MSC approach when reconstructing the evolutionary history of "difficult" clade. This approach, however, can quickly become intractable in large species complexes where there is extensive gene flow or significant shifts in population size. In such cases, there may be hundreds of potentially important parameters to estimate, and alternate introgression scenarios may be impossible to disentangle. This is particularly challenging in systems unlike Heliconius, where fewer data are available and there is little a priori knowledge of reproductive isolation, genome evolution, and the unique life history traits of each species.

Reviewer #3 (Public Review):

This study investigated the role of Zn2+ on the maintenance of Ca2+ oscillation upon fertilization. TPEN was used to reduce the level of available Zn2+ in fertilized oocytes and different inhibitors were used to pinpoint the mechanistic involvement of intracellular Zn2+ on the maintenance of Ca2+ oscillation. As also stated in the manuscript, previous studies have demonstrated the role of Zn2+ for the successful completion of meiosis/fertilization. The manuscript expands our understanding of fertilization process by describing how the level of Zn2+ regulates Ca2+ channels and stores. The manuscript is well-organized and the topic is important in early embryo development fields.

The authors added more information to the manuscript based on reviewers' comments. The quality of the manuscript has been improved and the study addresses important questions in mammalian fertilization.

Reviewer #3 (Public Review):

Summary:

The paper investigates which aspects of neural activity in LIP of the macaque give rise to individual decisions (specificity of choice and reaction times) in single trials, by recording simultaneously from hundreds of neurons. Using a variety of dimensionality reduction and decoding techniques, they demonstrate that a population-based drift-diffusion signal, which relies on a small subset of neurons that overlap choice targets, is responsible for the choice and reaction time variability. Analysis of direction-selective neurons in LIP and their correlation with decision-related neurons (T con in neurons ) suggests that evidence integration occurs within area LIP.

Strengths:

This is an important and interesting paper, which resolves conflicting hypotheses regarding the mechanisms that underlie decision-making in single trials. This is made possible by exploiting novel technology (Primatepixels recordings), in conjunction with state-of-the-art analyses and well-established dynamic random dot motion discrimination tasks.

Reviewer #3 (Public Review):

Summary:

The authors address a very important issue of going beyond a single-copy model obtained by the two principal experimental methods of structural biology, macromolecular crystallography and cryo electron microscopy (cryo-EM). Such multiconformer model is based on the fact that experimental data from both these methods represent a space- and time-average of a huge number of the molecules in a sample, or even in several samples, and that the respective distributions can be multimodal. Different from structure prediction methods, this approach is strongly based on high-resolution experimental information and requires validated single-copy high-quality models as input. Overall, the results support the authors' conclusions.

In fact, the method addresses two problems which could be considered separately:

- An automation of construction of multiple conformations when they can be identified visually;<br /> - A determination of multiple conformations when their visual identification is difficult or impossible.

The first one is a known problem, when missing alternative conformations may cost a few percent in R-factors. While these conformations are relatively easy to detect and build manually, the current procedure may save significant time being quite efficient, as the test results show.

The second problem is important from the physical point of view and has been addressed first by Burling & Brunger (1994; https://doi.org/10.1002/ijch.199400022). The new procedure deals with a second-order variation in the R-factors, of about 1% or less, like placing riding hydrogen atoms, modeling density deformation or variation of the bulk solvent. In such situations, it is hard to justify model improvement. Keeping Rfree values or their marginal decreasing can be considered as a sign that the model is not overfitted data but hardly as a strong argument in favor of the model.

In general, overall targets are less appropriate for this kind of problem and local characteristics may be better indicators. Improvement of the model geometry is a good choice. Indeed, yet Cruickshank (1956; https://doi.org/10.1107/S0365110X56002059) showed that averaged density images may lead to a shortening of covalent bonds when interpreting such maps by a single model. However, a total absence of geometric outliers is not necessarily required for the structures solved at a high resolution where diffraction data should have more freedom to place the atoms where the experiments "see" them.

The key local characteristic for multi conformer models is a closeness of the model map to the experimental one. Actually, the procedure uses a kind of such measure, the Bayesian information criteria (BIC). Unfortunately, there is no information about how sharply it identifies the best model, how much it changes between the initial and final models; in overall there is not any feeling about its values. The Q-score (page 17) can be a tool for the first problem where the multiple conformations are clearly separated and not for the second problem where the contributions from neighboring conformations are merged. In addition to BIC or to even more conventional target functions such as LS or local map correlation, the extreme and mean values of the local difference maps may help to validate the models.

This method with its results is a strong argument for a need in experimental data and information they contain, differently from a pure structure prediction. At the same time, absence of strong density-based proofs may limit its impact.

Strengths:

Addressing an important problem and automatization of model construction for alternative conformations using high-resolution experimental data.

Weaknesses:

An insufficient validation of the models when no discrete alternative conformations are visible and essentially missing local real-space validation indicators.

Reviewer #3 (Public Review):

This paper has high significance because it addresses a prevalent parasitic infection of the nervous system, Neurocysticercosis (NCC). The infection is caused by larvae of the parasitic cestode Taenia solium It is a leading cause of epilepsy in adults worldwide

To address the effects of cestode larvae, homogenates and excretory/secretory products of larvae were added to organotypic brain slice cultures of rodents or layer 2/3 of human cortical brain slices from patients with refractory epilepsy.

A self-made pressure ejection system was used to puff larvae homogenate (20 ms puff) onto the soma of patched neurons. The mechanical force could have caused depolarizaton so a vehicle control is critical. On line 150 they appear to have used saline in this regard, and clarification would be good. Were the controls here (and aCSF elsewhere) done with the low Mg2+o aCSF like the larvae homogenates?

They found that neurons depolarized after larvae homogenate exposure and the effect was mediated by glutamate but not nicotinic receptors for acetylcholine (nAChRs), acid-sensing channels or substance P. To address nAChRs, they used 10uM mecamyline, and for ASICs 2mM amiloride which seems like a high concentration. Could the concentrations be confirmed for their selectivity? Glutamate receptor antagonists, used in combination, were 10uM CNQX, 50uM DAP5, and 2mM kynurenic acid. These concentrations are twice what most use. Please discuss. Also, it would be very interesting to know if the glutamate receptor is AMPA, Kainic acid, or NMDA. Were metabotropic antagonists ever tested? That would be logical because CNQX/DAPR/Kynurenic acid did not block all of the depolarization.

They also showed the elevated K+ in the homogenate (~11 mM) could not account for the depolarization. However, the experiment with K+ was not done in a low Mg2+o buffer (Or was it -please clarify). They also confirmed that only small molecules led to the depolarization after filtering out very large molecules. That supports the conclusion that glutamate - which is quite small - could be responsible.

It is logical to test substance P because the Intro points out prior work links the larvae and seizures by inflammation and implicates substance P. However, why focus on nAChRs and ASIC?

The depolarizations caused seizure-like events in slices. The slices were exposed to a proconvulant buffer though- low Mg2+o. This buffer can cause spontaneous seizure-like events so it is important to know what the buffer did alone.

They suggest the effects could underlie seizure generation in NCC. However, there is only one event that is seizure-like in the paper and it is just an inset. Were others similar? How frequency were they? How long?

Using Glutamate-sensing fluorescent reporters they found the larvae contain glutamate and can release it, a strength of the paper.

Fig. 4. Could an inset be added to show the effects are very fast? That would support an effect of glutamate.

Why is aspartate relatively weak and glutamate relatively effective as an agonist?

Could some of the variability in Fig 4G be due to choice of different cell types? That would be consistent with Fig 5B where only a fraction of cells in the culture showed a response to the larvae nearby.

On what basis was the ROI drawn in Fig. 5B.

Also in 5B, I don't see anything in the transmitted image. What should be seen exactly?

Human brain slices were from temporal cortex of patients with refractory epilepsy. Was the temporal cortex devoid of pathology and EEG abnormalities? This area may be quite involved in the epilepsy because refractory epilepsy that goes to surgery is often temporal lobe epilepsy. Please discuss the liitations of studying the temporal cortex of humans with epilepsy since it may be more susceptible to depolarizations of many kinds, not just larvae.

Please discuss the limitations of the cultures - they are from very young animals and cultured for 6-14 days.

Reviewer #3 (Public Review):

General comments:

(1) While Dynasore and Pitstop-2 may impede release site clearance due to an arrest of membrane retrieval, neither Latrunculin-B nor ML-141 specifically acts on AZ scaffold proteins. Interference with actin polymerization may have a number of consequences many of which may be unrelated to release site clearance. Therefore, neither Latrunculin-B nor ML-141 can be considered suitable tools for specifically identifying the role of AZ scaffold proteins (i.e. ELKS family proteins, Piccolo, Bassoon, α-liprin, Unc13, RIM, RBP, etc) in release site clearance which was defined as one of the principal aims of this study.

(2) Initial EPSC amplitudes more than doubled in the presence of Dynasor at hippocampal SC->CA1 synapses (Figure S2). This unexpected result raises doubts about the specificity of Dynasor as a tool to selectively block SV endocytosis.

(3) In this study, the application of Dynasore and Pitstop-2 strongly decreases 100 Hz steady-state release at calyx synapses while - quite unexpectedly - strongly accelerates recovery from depression. A previous study found that genetic ablation of dynamin-1 actually enhanced 300 Hz steady-state release while only little affecting recovery from depression (Mahapatra et al., 2016). A similar scenario holds for the Latrunculin-B effects: In this study, Latrunculin-B strongly increased steady-state depression while in Babu et al. (2020), Latrunculin-B did not affect steady-state depression. In Mahapatra et al. (2016), Latrunculin-B marginally enhanced steady-state depression. The authors need to make a serious attempt to explain all these seemingly contradicting results.

(4) The experimental conditions need to be better specified. It is not clear which recordings were obtained in 1.3 mM and which (if any?) in 2 mM external Ca. It is also unclear whether 'pooled data' are presented (obtained from control recordings and from separate recordings after pre-incubation with the respective drugs), or whether the data actually represent 'before'/'after' comparisons obtained from the same synapses after washing in the respective drugs. The exact protocol of drug application (duration of application/pre-incubation?, measurements after wash-out or in the continuous presence of the drugs?) needs to be clearly described in the methods and needs to be briefly mentioned in Results and/or Figure legends.

(5) The authors compare results obtained in calyx with those obtained in SC->CA1 synapses which they considered examples for 'fast' and 'slow' synapses, respectively. There is little information given to help readers understand why these two synapse types were chosen, what the attributes 'fast' and 'slow' refer to, and how that may matter for the questions studied here. I assume the authors refer to the maximum frequency these two synapse types are able to transmit rather than to EPSC kinetics?

(6) Strong presynaptic stimuli such as those illustrated in Figures 1B and C induce massive exocytosis. The illustrated Cm increase of 2 to 2.5 pF represents a 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. Since the data shown in Figs. 1 and 3 are central to the argumentation, illustration of the corresponding conductance traces is mandatory. Merely mentioning that the first 450 ms after stimulation were skipped during analysis is insufficient.

(7) It is essential for this study to preclude a contamination of the results with postsynaptic effects (AMPAR saturation and desensitization). AMPAR saturation limits the amplitudes of initial responses in EPSC trains and hastens the recovery from depression due to a 'ceiling effect'. AMPAR desensitization occludes paired-pulse facilitation and reduces steady-state responses during EPSC trains while accelerating the initial recovery from depression. The use of, for example, 1 mM kynurenic acid in the bath is a well-established strategy to attenuate postsynaptic effects at calyx synapses. All calyx EPSC recordings should have been performed under such conditions. Otherwise, recovery time courses and STP parameters are likely contaminated by postsynaptic effects. Since the effects of AMPAR saturation on EPSC_1 and desensitization on EPSC_ss may partially cancel each other, an unchanged relative STD in the presence of kynurenic acid is not necessarily a reliable indicator for the absence of postsynaptic effects. The use of kynurenic acid in the bath would have had the beneficial side effect of massively improving voltage-clamp conditions. For the typical values given in this MS (10 nA EPSC, 3 MOhm Rs) the expected voltage escape is ~30 mV corresponding to a change in driving force of 30 mV/80 mV=38%, i.e. initial EPSCs in trains are likely underestimated by 38%. Such large voltage escape usually results in unclamped INa(V) which was suppressed in this study by routinely including 2 mM QX-314 in the pipette solution. That approach does, however, not reduce the voltage escape.

(8) In the Results section (pages 7 and 8), the authors analyze the time course into STD during 100 Hz trains in the absence and presence of drugs. In the presence of drugs, an additional fast component is observed which is absent from control recordings. Based on this observation, the authors conclude that '... the mechanisms operate predominantly at the beginning of synaptic depression'. However, the consequences of blocking or slowing site clearing are expected to be strongly release-dependent. Assuming a probability of <20% that a fusion event occurs at a given release site, >80% of the sites cannot be affected at the arrival of the second AP even by a total arrest of site clearance simply because no fusion has yet occurred. That number decreases during a train according to (1-0.2)^n, where n is the number of the AP, such that after 10 APs, ~90% of the sites have been used and may potentially be unavailable for new rounds of release after slowing site clearance. Perhaps, the faster time course into STD in the presence of the drugs isn't related to site clearance?

(9) In the Discussion (page 10), the authors present a calculation that is supposed to explain the reduced size of the second calyx EPSC in a 100 Hz train in the presence of Dynasore or Pitstop-2. Does this calculation assume that all endocytosed SVs are immediately available for release within 10 ms? Please elaborate.

(10) It is not clear, why the bafilomycin/folimycin data is presented in Fig. S5. The data is also not mentioned in the Discussion. Either explain the purpose of these experiments or remove the data.

(11) The scheme in Figure 7 is not very helpful.

Reviewer #3 (Public Review):

Summary:<br /> This study aims to investigate the stoichiometric effect between core factors and partners forming the heterodimeric transcription factor network in living cells at endogenous expression levels. Using state-of-the-art single-molecule analysis techniques, the authors tracked individual RARα and RXRα molecules labeled by HALO-tag knock-in. They discovered an asymmetric response to the overexpression of counter-partners. Specifically, the fact that an increase in RARα did not lead to an increase in RXRα chromatin binding is incompatible with the previous competitive core model. Furthermore, by using a technique that visualizes only molecules proximal to partners, they directly linked transcription factor heterodimerization to chromatin binding.

Strengths:<br /> The carefully designed experiments, from knock-in cell constructions to single-molecule imaging analysis, strengthen the evidence of the stoichiometric perturbation response of endogenous proteins. The novel finding that RXR, previously thought to be a target of competition among partners, is in excess provides new insight into key factors in dimerization network regulation. By combining the cutting-edge single-molecule imaging analysis with the technique for detecting interactions developed by the authors' group, they have directly illustrated the relationship between the physical interactions of dimeric transcription factors and chromatin binding. This has enabled interaction analysis in live cells that was challenging in single-molecule imaging, proving it is a powerful tool for studying endogenous proteins.

Weaknesses:<br /> As the authors have mentioned, they have not investigated the effects of other T2NRs or RXR isoforms. These invisible factors leave room for interpretation regarding the origin of chromatin binding of endogenous proteins (Recommendations 4). In the PAPA experiments, overexpressed factors are visualized, but changes in chromatin binding of endogenous proteins due to interactions with the overexpressed proteins have not been investigated. This might be tested by reversing the fluorescent ligands for the Sender and Receiver. Additionally, the PAPA experiments are likely to be strengthened by control experiments (Recommendations 5).

Reviewer #3 (Public Review):

The authors revisit an old question of how MCAK goes to microtubule ends, partially answered by many groups over the years. The authors seem to have omitted the literature on MCAK in the past 10-15 years. The novelty is limited due to what has previously been done on the question. Previous work showed MCAK targets to microtubule plus-ends in cells through association with EB proteins and Kif18b (work from Wordeman, Medema, Walczak, Welburn, Akhmanova) but none of their work is cited.

It is not obvious in the paper that these in vitro studies only reveal microtubule end targeting, rather than plus end targeting. MCAK diffuses on the lattice to both ends and its conformation and association with the lattice and ends has also been addressed by other groups-not cited here. I want to particularly highlight the work from Friel's lab where they identified a CDK phosphomimetic mutant close to helix4 which reduces the end preference of MCAK. This residue is very close to the one mutated in this study and is highly relevant because it is a site that is phosphorylated in vivo. This study and the mutant produced here suggest a charge-based recognition of the end of microtubules.

Here the authors analyze this MCAK recognition of the lattice and microtubule ends, with different nucleotide states of MCAK and in the presence of different nucleotide states for the microtubule lattice. The main conclusion is that MCAK affinity for microtubules varies in the presence of different nucleotides (ATP and analogs) which was partially known already. How different nucleotide states of the microtubule lattice influence MCAK binding is novel. This information will be interesting to researchers working on the mechanism of motors and microtubules. However, there are some issues with some experiments. In the paper, the authors say they measure MCAK residency of growing end microtubules, but in the kymographs, the microtubules don't appear dynamic- in addition, in Figure 1A, MCAK is at microtubule ends and does not cause depolymerization. I would have expected to see depolymerization of the microtubule after MCAK targeting. The MCAK mutants are not well characterized. Do they still have ATPase activity? Are they folded? Can the authors also highlight T537 and discuss this?

Finally, a few experiments are done with MCAK and XMAP215, after the authors say they have demonstrated the binding sites overlap. The data supporting this statement were not obvious and the conclusions that the effect of the two molecules are additive would argue against competing binding sites. Overall, while there are some interesting quantitative measurements of MCAK on microtubules - in particular in relation to the nucleotide state of the microtubule lattice - the insights into end-recognition are modest and do not address or discuss how it might happen in cells. Often the number of events is not recorded. Histograms with large SEM bars are presented, so it is hard to get a good idea of data distribution and robustness. Figures lack annotations. This compromises therefore their quantifications and conclusions. The discussion was hard to follow and needs streamlining, as well as putting their work in the context of what is known from other groups who produced work on this in the past few years.

Reviewer #3 (Public Review):

Summary:<br /> This manuscript details the characterization of ClpL from L. monocytogenes as a potent and autonomous AAA+ disaggregase. The authors demonstrate that ClpL has potent and DnaK-independent disaggregase activity towards a variety of aggregated model substrates and that this disaggregase activity appears to be greater than that observed with the canonical DnaK/ClpB co-chaperone. Furthermore, Lm ClpL appears to have greater thermostability as compared to Lm DnaK, suggesting that ClpL-expressing cells may be able to withstand more severe heat stress conditions. Interestingly, Lm ClpP can provide thermotolerance to E. coli that have been genetically depleted of either ClpB or in cells expressing a mutant DnaK103. The authors further characterized the mechanisms by which ClpL interacts with protein aggregates, identifying that the N-terminal domain of ClpL is essential for disaggregase function. Lastly, by EM and mutagenesis analysis, the authors report that ClpL can exist in a variety of larger macromolecular complexes, including dimer or trimers of hexamers/heptamers, and they provide evidence that the N-terminal domains of ClpL prevent dimer ring formation, thus promoting an active and substrate-binding ClpL complex. Throughout this manuscript the authors compare Lm ClpL to ClpG, another potent and autonomous disaggregase found in gram-negative bacteria that have been reported on previously, demonstrating that these two enzymes share homologous activity and qualities. Taken together this report clearly establishes ClpL as a novel and autonomous disaggregase.

Strengths:<br /> The work presented in this report amounts to a significant body of novel and significant work that will be of interest to the protein chaperone community. Furthermore, by providing examples of how ClpL can provide in vivo thermotolerance to both E. coli and L. gasseri the authors have expanded the significance of this work and provided novel insight into potential mechanisms responsible for thermotolerance in food-borne pathogens.

Weaknesses:<br /> The figures are clearly depicted and easy to understand, though some of the axis labeling is a bit misleading or confusing and may warrant revision. While I do feel that the results and discussion as presented support the authors' hypothesis and overall goal of demonstrating ClpL as a novel disaggregase, interpretation of the data is hindered as no statistical tests are provided throughout the manuscript. Because of this only qualitative analysis can be made, and as such many of the concluding statements involving pairwise comparisons need to be revisited or quantitative data with stats needs to be provided. The addition of statistical analysis is critical and should not be difficult, nor do I anticipate that it will change the conclusions of this report.

Reviewer #3 (Public Review):

Summary: The authors are trying to find out whether the levels of omega-6 and omega-3 fatty acids in the blood are linked to the likelihood of dying from anything, of dying from cancer and of dying from cardiovascular disease. They use a large dataset called UK Biobank where fatty acid levels were measured in blood at the start of the study and what happened to the participants over the following years (average of 12.7 years) was followed. They find that both omega-6 AND omega-3 fatty acids were linked with less likelihood of dying from anything, from cancer and from cardiovascular disease. The effects of omega-3s were stronger. They then made a ratio of omega-6 to omega-3 fatty acids and found that as that ratio increased risk of dying also increased,. This supports the idea that omega-3s have stronger effects than omega-6s.

Strengths: This is a large study (over 85,000 participants) with a good follow up period (average 12.7 years). Using blood levels of fatty acids is superior to using estimated dietary intakes. The authors take account of many variables that could interfere with the findings (confounding variables) - they do this using statistical methods.

Weaknesses: There are several omega-6 and omega-3 fatty acids - it is not clear which ones were actually measured in this study.

Reviewer #3 (Public Review):

Freire and co-authors examine the role of the exocyst complex during the formation and secretion of mucins from secretory granules in the larval salivary gland of Drosophila melanogaster. Using transgenic lines with a tagged Sgs3 mucin the authors KD expression of exocyst subunit members and observe a defect in secretory granules with a heterogeneity of phenotypes. By carefully controlling RNAi expression using a Gal4-based system the authors can KD exocyst subunit expression to varying degrees. The authors find that the stronger the inhibition of expression of exocyst the earlier in the secretory pathway the defect. The manuscript is well written, the model system is physiological, and the techniques are innovative.

My major concern is that the evidence underlying the fundamental claim of the manuscript that "the exocyst complex participates" in multiple secretory processes lacks direct evidence. It is clear from multiple lines of evidence, which are discussed by the authors, that exocyst is essential for an array of exocytic events. The fundamental concern is that loss of homeostasis on the plasma membrane proteome and lipidome might have severe pleiotropic effects on the cell. Indeed exocyst is essential, even in tissue culture conditions, and loss is lethal. Therefore, is an alternative explanation not that they are observing varying degrees of pleiotropic defect on the secretory pathway? Perhaps the authors have more evidence that exocyst is important for homeotypic fusion of the SGs, as supported by the localisation of Sec15 on the fusion sites.

The second question that I think is important to address is, what exactly do the varying RNAi levels correspond to in terms of experiments, and have these been validated? Due to the fundamental claim being that the severity of the phenotype being correlated with the level of KD, I think validation of this model is absolutely essential.

Reviewer #3 (Public Review):

Summary:

This study uses a state-of-the-art artificial skin assay to determine the quantity of P. falciparum sporozoites expelled during feeding using mosquito infection (by standardised membrane feeding assay SMFA) using both cultured gametocytes and natural infection. Sporozoite densities in salivary glands and expelled into the skin are quantified using a well-validated molecular assay. These studies show clear positive correlations between mosquito infection levels (as determined by oocyst numbers), sporozoite numbers in salivary glands, and sporozoites expelled during feeding. This indicates potentially significant heterogeneity in infectiousness between mosquitoes with different infection loads and thus challenges the often-made assumption that all infected mosquitoes are equally infectious.

Strengths:

Very rigorously designed studies using very well validated, state-of-the-art methods for studying malaria infections in the mosquito and quantifying load of expelled sporozoites. This resulted in very high-quality data that was well-analyzed and presented. Both sources of gametocytes (cultures vs. natural infection) show consistent results further strengthening the quality of the results obtained.

Weaknesses:

As is generally the case when using SMFAs, the mosquito infections levels are often relatively high compared to wild-caught mosquitoes (e.g. Bombard et al 2020 IJP: median 3-4 ), and the strength of the observed correlations between oocyst sheet and salivary gland sporozoite load even more so between salivary gland sporozoite load and expelled sporozoite number may be dominated by results from mosquitoes with infection levels rarely observed in wild-caught mosquitoes. This could result in an overestimation of the importance of these well-observed positive relationships under natural transmission conditions.

The results obtained from these excellently designed and executed studies very well supported their conclusion - with a slight caveat regarding their application to natural transmission scenarios

This work very convincingly highlights the potential for significant heterogeneity in the infectiousness between individual P. falciparum-infected mosquitoes. Such heterogeneity needs to be further investigated and if again confirmed taken into account both when modelling malaria transmission and when evaluating the importance of low-density infections in sustaining malaria transmission.

Reviewer #3 (Public Review):

Summary:

This important paper describes improvements to the measurement of enkephalins in vivo using microdialysis and LC-MS. The key improvement is the oxidation of met- to prevent having a mix of reduced and oxidized methionine in the sample which makes quantification more difficult. It then shows measurements of enkephalins in the nucleus accumbens in two different stress situations - handling and exposure to predator odor. It also reports the ratio of released met- and leu-enkephalin matching what is expected from the digestion of proenkephalin. Measurements are also made by photometry of Ca2+ changes for the fox odor stressor. Some key takeaways are the reliable measurement of met-enkephalin, the significance of directly measuring peptides as opposed to proxy measurements, and the opening of a new avenue into the research of enkephalins due to stress based on these direct measurements.

Strengths:

-Improved methods for measurement of enkephalins in vivo.

-Compelling examples of using this method.

-Opening a new area of looking at stress responses through the lens of enkephalin concentrations.

Weaknesses:

1) It is not clear if oxidized met-enk is endogenous or not and this method eliminates being able to discern that.

2) It is not clear if the spatial resolution is really better as claimed since other probes of similar dimensions have been used.

3) Claims of having the first concentration measurement are not quite accurate.

4) Without a report of technical replicates, the reliability of the method is not as well-evaluated as might be expected.

Reviewer #3 (Public Review):

Summary: This is a careful examination of the distribution of mitochondria in the basal dendrites of ferret visual cortex in a previously published volume electron microscopy dataset. The authors report that mitochondria are sparsely, as opposed to continuously distributed in the dendritic shafts, and that they tend to cluster near dendritic spines with heterogeneous orientation selectivity.

Strengths: Volume EM is the gold standard for quantification of organelle morphology. An unusual strength of this particular dataset is that the orientation selectivity of the dendritic spines was measured by calcium imaging prior to EM reconstruction. This allowed the authors to assess how spines with varying selectivity are organized relative to mitochondria, leading to an intriguing observation that they localize to heterogeneous spine clusters. The analysis is carefully performed. An additional strength is the use of a carnivore with a sophisticated visual system.

Weaknesses: Using threshold distances between mitochondria and synapses as opposed to absolute distances may overlook important relationships in the data.

Reviewer #3 (Public Review):

In this manuscript, Millard et al. investigate the effects of nicotine on pain sensitivity and peak alpha frequency (PAF) in resting state EEG. To this end, they ran a pre-registered, randomized, double-blind, placebo-controlled experiment involving 62 healthy adults who received either 4 mg nicotine gum (n=29) or placebo (n=33). Prolonged heat and pressure were used as pain models. Resting state EEG and pain intensity (assessed with a visual analog scale) were measured before and after the intervention. Additionally, several covariates (sex at birth, depression and anxiety symptoms, stress, sleep quality, among others) were recorded. Data was analyzed using ANCOVA-equivalent two-wave latent change score models, as well as repeated measures analysis of variance. Results do not show *experimentally relevant* changes of PAF or pain intensity scores for either of the prolonged pain models due to nicotine intake.

The main strengths of the manuscript are its solid conceptual framework and the thorough experimental design. The researchers make a good case in the introduction and discussion for the need to further investigate the association of PAF and pain sensitivity. Furthermore, they proceed to carefully describe every aspect of the experiment in great detail, which is excellent for reproducibility purposes. Finally, they analyze the data from almost every possible angle and provide an extensive report of their results.<br /> The main weakness of the manuscript is the interpretation of these results. Even though some of the differences are statistically significant (e.g., global PAF, pain intensity ratings during heat pain), these differences are far from being experimentally or clinically relevant. The effect sizes observed are not sufficiently large to consider that pain sensitivity was modulated by the nicotine intake, which puts into question all the answers to the research questions posed in the study.

Reviewer #3 (Public Review):

Summary: in this manuscript, Hansen and co-authors investigated the role of R-coils in the multimerization and ice nucleation activity of PbINP, an ice nucleation protein identified in Pseudomonas borealis. The results of this work suggest that the length, localization, and amino acid composition of R-coils are crucial for the formation of PbINP multimers.

Strengths: The authors use a rational mutagenesis approach to identify the role of the length, localisation, and amino acid composition of R-coils in ice nucleation activity. Based on these results, the authors hypothesize a multimerization model. Overall, this is a multidisciplinary work that provides new insights into the molecular mechanisms underlying ice nucleation activity.

Weaknesses: Several parts of the work appear cryptic and unsuitable for non-expert readers. The results of this work should be better described and presented.

Reviewer #3 (Public Review):

Summary:

The authors generate and characterize two phosphospecific antisera for FFA2 receptor and claim a "bar code" difference between white fat and Peyers patches.

Strengths:

The question is interesting and the antibody characterization is convincing.

Weaknesses:

The mass spectrometry analysis is not convincing because the method is not quantitative (no SILAC, TMT, internal standards etc). Figure 1 shows single tryptic peptides with one and two phosphorylation fragmentations as claimed, but there is no data testing the abundance of these so the differences claimed between cell treatment conditions are not established.

The blot analysis cannot distinguish 296/7 but it does convincingly show an agonist increase. Can the authors clarify why the amount of constitutive phosphorylation is much higher in the example blot in Figure 2 than in Figure 3? It would be helpful to quantify this across more than one example, like in Figures 4 and 5 for tissue.

Compound 101 is shown in Figure 2 to block barrestin recruitment. I agree this suggests phosphorylation mediated by GRK2/3 but this is not tested. The new antibodies should be good for this so I don't understand why the indirect approach.

The conditions used to inhibit dephosphorylation are not specified, the method only says "phosphatase inhibitors". How do the authors know that low P at 306/7 in white fat is not a result of dephosphorylation during sample preparation? If these sites are GRK2/3 dependent (see above) then does adipose tissue lack this GRK?

Reviewer #3 (Public Review):

In this manuscript, Zhou et al. demonstrate that the pseudokinase ULK4 has an important role in Hedgehog signaling by scaffolding the active kinase Stk36 and the transcription factor Gli2, enabling Gli2 to be phosphorylated and activated.<br /> Through nice biochemistry experiments, they show convincingly that the N-terminal pseudokinase domain of ULK4 binds Stk36 and the C-terminal Heat repeats bind Gli2.

Lastly, they show that upon Sonic Hedgehog signaling, ULK4 localizes to the cilia and is needed to localize Stk36 and Gli2 for proper activation.

This manuscript is very solid and methodically shows the role of ULK4 and STK36 throughout the whole paper, with well controlled experiments. The phosphomimetic and incapable mutations are very convincing as well.<br /> I think this manuscript is strong and stands as is, and there is no need for additional experiments.

Overall, the strengths are the rigor of the methods, and the convincing case they bring for the formation of the ULK4-Gli2-Stk36 complex. There are no weaknesses noted. I think a little additional context for what is being observed in the immunofluorescence might benefit readers who are not familiar with these cell types and structures.

The revised manuscript has improved some of the unclear areas.

Reviewer #3 (Public Review):

The manuscript by Boyd and co-authors "A Vibrio cholerae viral satellite maximizes its spread and inhibits phage by remodelling hijacked phage coat proteins into small capsids" reports important results related to self-defending mechanisms that bacteria are used against phages that infect them. It has been shown previously that bacteria produce phage-inducible chromosomal island-like elements (PLE) that encode proteins that are integrated into bacterial genome. These proteins are used by bacteria to amend the phage capsids and to create phage-like particles (satellites) that move between cells and transfer the genetic material of PLE to another bacteria. That study highlights the interactions between a PLE-encoded protein, TcaP, and capsid proteins of the phage ICP1.

The manuscript is well written, provides a lot of new information and the results are supported by biochemical analysis.

Reviewer #3 (Public Review):

This paper describes an algorithm that provides a general mechanism for goal-directed behaviour in a biologically plausible neural form.

The method depends on substantial simplifying assumptions. The simulated animal effectively moves through an environment consisting of discrete locations and can reliably detect when it is in each location. Whenever it moves from one location to an adjacent location, it perfectly learns the connectivity between these two locations (changes the value in an adjacency matrix to 1). This creates a graph of connections that reflects the explored environment. In this graph, the current location gets input activation and this spreads to all connected nodes multiplied by a constant decay (adjusted to the branching number of the graph) so that as the number of connection steps increases the activation decreases. Some locations will be marked as goals through experiencing a resource of a specific identity there and subsequently will be activated by an amount proportional to their distance in the graph from the current location, i.e., their activation will increase if the agent moves a step closer and decrease if it moves a step further away. Hence by making such exploratory movements, the animal can decide which way to move to obtain a specified goal.

Although the algorithm is presented within a conceptual framework of chemotaxis, I.e., making movements to sample a local gradient and move up it, the approach relates closely to previous models of exploration, learning, and navigation that similarly establish (through experience) a graph structure to represent how locations are connected and use some form of activity-propagation from the current node or goal node to identify a (shortest) route between them. Many of these similarly claim to be plausible neural circuits. The current authors argue that the current algorithm has several desirable features with respect to such previous work: for example, the 'readout' of the path does not require explicit 'look-up' and activation of the goal node (although it does require a choice of which goal node is currently connected to behavior); and does not require any separate control or rules for learning vs. navigation phases. By comparison to the successor representation method used in RL, which also appears related, they note that the gain (decay) factor is not equivalent to a temporal discount and that their method learns only state-state transitions, allowing the value of actions to be externalised, I.e., calculated by trying alternative actions to see which increases the activation at the goal node the most. On the other hand, it should be noted that some issues addressed in previous models, such as uncertainty over the current state or probabilistic state(-action) transitions are not addressed in this work.

The algorithm presents some elegant features with respect to previous work such as conceptually separating the 'goal' nodes from the state (location) graph (I.e. 'goals' are not just special target states within the graph) so that a small number of goals can become associated to (potentially) multiple regions of the state graph where they are satisfied, or near to being satisfied. This architecture is suggested, in the discussion, to resemble the insect mushroom body (MB), where it is known that a small number of output neurons (MBONs, putative goal neurons) are activated by plastic connections from Kenyon cells (KCs, putative state neurons). However, it goes substantially beyond any available evidence to claim that KC connectivity could support the acquisition of a graph (in the form of an adjacency matrix) representing the structure of the environment: KCs show sparse distributed activity (not one active node per state); it seems unlikely that any two arbitrary KCs can (rapidly) become connected; and as yet has not been demonstrated that KC connectivity is plastic at all.

The results presented are fairly straightforward given the simplification of the tasks, as described above. They show 1) in practical terms, the spreading signal travels further for a larger decay but becomes erratic as the decay parameter (map neuron gain) approaches its theoretical upper bound and decreases below noise levels beyond a certain distance. Both follow the theory but it is perhaps helpful to see that there is a viable range of values of the gain for which the mechanism works, that is, it is not highly dependent on precise tuning. 2) That different graph structures can be acquired and used to approach goal locations (not surprising). 3) That simultaneous learning and exploitation of the graph only minimally affects the performance over starting with perfect knowledge of the graph. 4) That the parameters interact in expected ways. 5) That the separation of goals from states can be used flexibly e.g. the homing behaviour (a goal state is learned before any of the map is learned) and the patrolling behaviour (a goal cell that monitors all states for how recently they were visited). It is also interesting to link the mechanism of exploration of neighbouring states to observed scanning behaviours in navigating animals. It would have been interesting to explore whether the parameters could be dynamically tuned, based on the overall graph activity.

Reviewer #3 (Public Review):

In this manuscript, Lewis et al. investigate the role of tetraspanins in the formation of discs- the key structure of vertebrate photoreceptors essential for light reception. Two tetraspanin proteins play a role in this process: PRPH2 and ROM1. The critical contribution of PRPH2 has been well established and loss of its function is not tolerated and result in gross anatomical pathology and degeneration in both mice and humans. However, the role of ROM1 is much less understood and has been considered somewhat redundant. This paper provides a definitive answer about the long-standing uncertainty regarding the contribution of ROM1 firmly establishing its role in outer segment morphogenesis. First, using ingenious quantitative proteomic technique the authors show PRPH2 compensatory increase in ROM1 knockout explaining the redundancy of its function. Second, they uncover that despite this compensation, ROM1 is still needed and its loss delays disc enclosure and result in the failure to form incisures. Third, the authors used a transgenic mouse model and show that deficits seen in ROM1 KO could be completely compensated by the overexpression of PRPH2. Finally, they analyzed yet another mouse model based on double manipulation with both ROM1 loss and expression of PRPH2 mutant unable to form dimerizing disulfide bonds further arguing that PRPH2-ROM1 interactions are not required for disc enclosure. To top it off the authors complement their in vivo studies by series of biochemical assays done upon reconstitution of tetraspanins in transfected cultured cell as well as fractionations of native retinas. This report is timely, addresses significant questions in cell biology of photoreceptors and pushes the field forward in a classical area of photoreceptor biology and mechanics of membrane structure as well. The manuscript is executed at the top level of technical standard, exceptionally well written and does not leave much more to desire. It also pushes standards of the field- one such domain is quantitative approach to analysis of the EM images which is notoriously open to alternative interpretations - yet this study does an exceptional job unbiasing this approach.

Reviewer #3 (Public Review):

Summary:<br /> Ishii et al used molecular genetics and behavioral analyses in mice to study the functional role of a subset of MPOA neurons in the regulation of female sexual drive. They first employed a self-paced mating assay during which a female could control the amount of interaction time with a male to assess female sexual drive after completion of mating. The authors observed that after mating completion females spent significantly less time interacting with the mated males, indicating that their sexual drive was reduced. Next, the authors performed a brain-wide analysis of neurons activated during the completion of mating and identified the MPOA as a strong candidate region. However, their activity labeling was not exclusive to neurons activated during mating completion but included all neurons activated before, during, and after the mating encounter. This makes it difficult to interpret these data. Importantly, the authors do provide in vivo calcium imaging data showing that a subset of MPOA neurons responds significantly and specifically to mating completion and not other behaviors during the social encounter. The authors performed these studies in both excitatory and inhibitory populations of the MPOA. Their analysis identified a subpopulation of inhibitory neurons that exhibit sustained increased activity for 90 sec following mating completion. Finally, the authors used chemogenetics to activate MPOA neurons during home cage mating, condition place preference, pup retrieval, and the self-paced mating assay. They found that activation of these neurons significantly reduced mating behaviors and time spent interacting with a male during the self-paced mating assay. The authors suggest that their chemogenetic activation is restricted to neurons activated during mating completion, but their activity-dependent labeling strategy resulted in chemogenetic activation of all MPOA neurons activated either before, during, or after mating.

The authors' experimental execution is rigorous and well-performed. Their data identify inhibitory neurons in the female MPOA as a neural locus that is activated following the completion of mating and potentially a key neural population in the regulation of female sexual motivation. However, the conclusions and interpretation of the data extend beyond what is reasonable given the limitations of the activity-dependent labeling strategy employed.

Strengths:<br /> 1) The use of the self-paced mating assay in combination with neural imaging and manipulation to assess female sexual drive is innovative. The authors correctly assert that relatively little is known about how mating completion affects sexual motivation in females as compared to males. Therefore, the data collected from these studies is important and valuable.

2) The authors provide convincing histological data and analyses to verify and validate their brain-wide activity labeling, neural imaging, and chemogenetic studies.

3) The single-cell in vivo calcium imaging data are well performed and analyzed. They provide key insights into the activity profiles of both excitatory and inhibitory neurons in the female MPOA during mating encounters. The authors' identification of an inhibitory subpopulation of female MPOA neurons that are selectively activated following the completion of mating is fundamental for future experiments which could potentially find a molecular marker for this population and specifically manipulate these neurons to understand their role in female sexual motivation in greater detail.

Weaknesses:<br /> 1) Their activity-dependent labeling strategy is not exclusive to mating completion but instead includes all neurons active before, during, and after the social encounter. In the manuscript, the authors did not discuss the time course of Fos activation or the timeframe of the FosTRAP labeling strategy. Fos continues to be expressed and is detectable for hours following neural activation. Therefore, the FosTRAP strategy also labels neurons that were activated 3 hours before the injection of 4-OHT. The original FosTRAP2 paper which is cited in this manuscript (DeNardo et al, 2019) performed a detailed analysis of the labeling window in Supplementary Figure 2 of that paper. Here is quoted text from that paper: "Resultant patterns of tdTomato expression revealed that the majority of TRAPing occurred within a 6-hour window centered around the 4-OHT injection." Thus, the FosTRAP "mating completion" groups throughout this manuscript also include neurons activated 3 hours before mating completion, which includes neurons activated during appetitive and consummatory mating behaviors.

This makes all of the FosTRAP data very difficult to interpret. Compounding this is the issue that the two groups the authors compare in their experiments are females administered 4-OHT following appetitive investigation behaviors (with the male removed before mating behaviors occurred) and females administered 4-OHT following mating completion. The "appetitive" group labeled neurons activated only during appetitive investigation, but the "completion" group labeled neurons activated during appetitive investigations, consummatory mating bouts, and mating completion. Therefore, in the brain-wide analysis of Figure 2, it is impossible to identify brain regions that were activated exclusively by mating completion and not by consummatory mating behaviors. This could have been achieved if the "completion" group was compared to a group of females that had commenced consummatory mating behaviors but were separated from the male before mating was completed. Then, any neurons labeled by the "completion" FosTRAP but not the "consummatory" FosTRAP would be neurons specifically activated by mating completion. In the current brain-wide analysis experiments, neurons activated by consummatory behaviors and mating completion can not be disassociated.

This same issue is present in the interpretation of the chemogenetic activation data in Figure 6. In the experiments of Figure 6, the authors are activating neurons naturally activated during consummatory mating behaviors as well as those activated during mating completion.

2) This study does not definitively show that the female mice used in this study display decreased sexual motivation after the completion of mating. The females exhibit reduced interaction with males that had also just completed mating, but it is unclear if the females would continue to show reduced interaction time if given the choice to interact with a male that was not in the post-ejaculatory refractory period. Perhaps, these females have a natural preference to interact more with sexually motivated males compared to recently mated (not sexually motivated) males. To definitively show that these females exhibit decreased sexual motivation the authors should perform two control experiments: 1) provide the females with access to a fully sexually motivated male after the females have completed mating with a different male to see if interaction time changes, and 2) compare interaction time toward mated and non-mated males using the self-paced mating assay. These controls would show that the reduction in the interaction time is because the females have reduced sexual motivation and not because these females just naturally interact with sexually motivated males more than males in the post-ejaculatory refractory period.

3) It is unclear how the transient 90-second response of these MPOA neurons following the completion of mating causes the prolonged reduction in female sexual motivation that is at the minutes to hours timeframe. No molecular or cellular mechanism is discussed.

4) The authors discuss potential cell types and neural population markers within the MPOA and go into some detail in Figure S3. However, their experiments are performed with only the larger excitatory and inhibitory MPOA neural populations.

Reviewer #3 (Public Review):

This is an interesting work reporting ferroptosis that is involved in the tooth morphogenesis. The authors showed that Gpx4, the core anti-lipid peroxidation enzyme in ferroptosis, is upregulated in tooth development using ex vivo culture system.

Reviewer #3 (Public Review):

Summary:<br /> Grob and colleagues investigated the causal role of the angular gyrus in insight-driven memory reconfiguration. Participants watched unrelated movie scenes while EEG was recorded prior to receiving either active or sham continuous theta burst stimulation (cTBS) over the left angular gyrus. Following stimulation, participants either observed or imagined links or non-links between scenes watched before stimulation. Next, participants rated their comprehension of the links. Following this part, participants completed questionnaires for 30 minutes, followed by a free recall test of details from the videos. Subjects then watched the videos again while EEG was recorded and engaged in a recognition test to determine whether they retained information about the linking events. Participants showed strong evidence of insight-driven linking between videos. The results indicate that overall memory of video details was stronger for the Sham group compared to the cTBS group, but only for the linked videos. An RSA analysis using pre- and post-video observation indicated that similarity increased for imagined and linked videos for the sham group, but not for the cTBS group, in sensors in parieto-temporal regions. Similarity for imagined, non-linked videos increased for the cTBS group, but not for the sham group, in frontal sensors. Coherence between fronto-parietal sensors decreased during the viewing of videos linked by imagination for the cTBS group, but not the sham group. Coherence between the same sensors increased while watching videos that were linked by observation in the cTBS group, but decreased for the sham group. The authors conclude that the angular gyrus is causally related to memory-insight reconfiguration.

Strengths:<br /> The paper is nicely written, and the rigor of the experimental design is strong. The paper is pre-registered, and the authors used a double-blind sham-controlled design to eliminate the possibility of bias and non-specific effects of rTMS on their results. The behavioral results are striking and provide strong evidence that their intervention significantly decreased memory for details of linked events. The authors also took care to leave time between stimulation and recall to reduce the influence of carry-over rTMS effects on memory. There are also strong behaviorally-relevant neural changes.

Weaknesses:<br /> My major criticism relates to the main claim of the paper regarding causality between the angular gyrus and the authors' behavior of interest. Specifically, I am not convinced by the evidence that the effects of stimulation noted in the paper are attributable specifically to the angular gyrus, and not other regions/networks.

Reviewer #3 (Public Review):

Summary:<br /> The current manuscript shows that 14-3-3 are binding partners of spastin, preventing its degradation. It is additionally shown, using complementary methods, that both 14-3-3 and spastin are necessary for axon regeneration in vitro and in vivo. While interesting in vitro and vivo data is provided, some of the claims of the authors are not convincingly supported.

Major strengths:<br /> Very interesting effect of FC-A in functional recovery after spinal cord injury.

Major Weaknesses:<br /> Some of the in vitro data, including colocalizations, and analysis of microtubule severing fall short to support the claims of the authors.<br /> The in vivo selectivity of FC-A towards spastin is not adequately supported by the data presented.<br /> There are aspects of the spinal cord injury site histology that are unclear.

Reviewer #3 (Public Review):

The authors attempt to dissociate differences in resting vs active vs perturbed movement biases in people with motor deficits resulting from stroke. The analysis of movement utilizes techniques that are similar to previous motor control in both humans and non-human primates, to assess impairments related to sensorimotor injuries. In this regard, the authors provide additional support to the extensive literature describing movement abnormalities in patients with hemiparesis both at rest and during active movement. The authors describe their intention to separate out the contribution of holding still at a position vs active movement as a demonstration that these two aspects of motor control are controlled by two separate control regimes.

Strengths:<br /> 1. The authors utilize a device that is the same or similar to devices previously used to investigate motor control of movement in normal and impaired conditions in humans and non-human primates. This allows comparisons to existing motor control studies.<br /> 2. Experiment 1 demonstrates resting flexion biases both in supported and unsupported forelimb conditions. These biases show a correlated relationship with FM-UE scores, suggesting that the degree of motor impairment and the degree of resting bias are related.<br /> 3. The stroke patient participant population had a wide range of both levels of impairment and time since stroke, including both sub-acute and chronic cases allowing the results to be compared across impairment levels.

The authors describe several results from their study: 1. Postural biases were systematically toward the body (flexion) and increased with distance from the body (when the arm was more extended) and were stronger when the arm was unsupported. 2. These postural biases were correlated with FM-UE score. 3. They found no evidence of postural biases impacting movement, even when that movement was perturbed. 4. When holding a position at the end of a movement, if the position was perturbed opposite of the direction of bias, movement back to the target was improved compared to the perturbation in the direction of bias. Taken together, the authors suggest that there are at least two separate motor controls for tasks at rest versus with motion. Further, the authors propose that these results indicate that there is an imbalance between cortical control of movement (through the corticospinal tracts) and postural control (through the reticulospinal tract). There are several weaknesses related to the interpretation of the results:

In Experiment 1, the participants are instructed to keep their limbs in a passive position after being moved. The authors show that, in the impaired limb, these resting biases are significantly higher when the limb is unsupported and increase when the arm is moved to a more extended position.

When supported by the air sled, the arm is in a purely passive position, not requiring the same anti-gravity response so will have less RST but also less CST involvement. While the unsupported task invokes more involvement of the reticulospinal tract (RST), it likely also has significantly higher CST involvement due to the increased difficulty and novelty of the task.

If there were an imbalance in CST regulating RST as proposed by the authors, the bias should be higher in the supported condition as there should be relatively less CST activation/involvement/modulation leading to less moderating input onto the RST and introducing postural biases. In the unsupported condition, there is likely more CST involvement, potentially leading to an increased modulatory effect on RST. If the proportion of CST involvement significantly outweighs the RST activation in the unsupported task, then it isn't obvious that there is a clear differentiation of motor control. As the degree of resting force bias and FM-UE score are correlated, an argument could be made that they are both measuring the impairment of the CST unrelated to any RST output. If it is purely the balance of CST integrity compared to RST, then the degree of bias should have been the same in both conditions. In this idea of controller vs modulator, it is unclear when this switch occurs or how to weigh individual contributions of CST vs. extrapyramidal tracts. Further, it isn't clear why less modulation on the RST would lead only to abnormal flexion.

This resting bias could be explained by an imbalance in the activation of flexors vs extensors which follows the results that this bias is larger as the arm is extended further, and/or in a disconnect in sensory integration that is overcome during active movement. Neither would necessitate separate motor control for holding vs active movement.

In Experiment 2, the participants are actively moving to and holding at targets for all trials while being supported by the air sled. Even with the support, the paretic participants all showed start- and end-point force biases around the movement despite not showing systematic deviations in force direction during active movement start or stop. There could be several factors that limit systematic deviations in force direction. The most obvious is that the measured biases are significantly higher when the limb is unsupported and by testing with a supported limb the authors are artificially limiting any effect of the bias. It is also possible that significant adaptation or plasticity with the CST or rubrospinal tracts could give rise to motor output that already accounts for any intrinsic resting bias. In any case, the results from the reaching phase of Experiment 2 do not definitively show that directional biases are not present during active reaching, just that the authors were unable to detect them with their design. The authors do acknowledge the limitations in this design (a 2D constrained task) in explaining motor impairment in 3D unconstrained tasks.

It would have been useful, in Experiment 2, to use FM-UE scores (and time from injury) as a factor to determine the relationship between movement and rest biases. Using a GLMM would have allowed a similar comparison to Experiment 1 of how impairment level is related to static perturbation responses. While not a surrogate for imaging tractography data showing a degree of CST involvement in stroke, FM-UE may serve as an appropriate proxy so that this perturbation at hold responses may be put into context relative to impairment.

It is not clear that even in the static perturbation trials that the hold (and subsequent move from perturbation) is being driven by reticulospinal projections. Given a task where ~20% of the trials are going to be perturbed, there is likely a significant amount of anticipatory or preparatory signaling from the CST. How does this balance with any proposed contribution that the RST may have with increased grip?

In general, the weakness of the interpretation of the results with respect to the CST/RST framework is that it is necessary to ascribe relative contributions of different tracts to different phases of movement and hold using limited or indirect measures. Barring any quantification of this data during these tasks, different investigators are likely to assess these contributions in different ways and proportions limiting the framework's utility.

Reviewer #3 (Public Review):

Observers make judgements about expected stimuli faster and more accurately. How expectations facilitate such perceptual decisions remains an ongoing area of investigation, however, as expectations may exert their effects in multiple ways. Expectations may directly influence the encoding of sensory signals. Alternatively (or additionally), expectations may influence later stages of decision-making, such as motor preparation, when they bear on the appropriate behavioral response.

In the present study, Walsh and colleagues directly measured the effect of expectations on sensory and motor signals by making clever use of the encephalogram (EEG) recorded from human observers performing a contrast discrimination task. On each trial, a predictive cue indicated which of two superimposed stimuli would likely be higher contrast and, therefore, whether a left or right button press was likely to yield a correct response. Deft design choices allowed the authors to extract both contrast-dependent sensory signals and motor preparation signals from the EEG. The authors provide compelling evidence that, when predictive cues provide information about both a forthcoming stimulus and the appropriate behavioral response, expectation effects are immediately manifest in motor preparation signals and only emerge in sensory signals after extensive training.

Future work should attempt to reconcile these results with related investigations in the field. As the authors note, several groups have reported expectation-induced modulation of sensory signals (using both fMRI and EEG/MEG) on shorter timescales (e.g. just one or two sessions of a few hundred trials, versus the intensive multi-session study reported here). One interesting possibility is that perceptual expectations are not automatic but demand the deployment of feature-based attention, while motor preparation is comparatively less effortful and so dominates when both sources of information are available, as in the present study. This hypothesis is consistent with the authors' thoughtful analysis showing decreased neural signatures of attention over posterior electrodes following predictive cues. Therefore, observing the timescale of sensory effects using the same design and methods (facilitating direct comparison with the present work), but altering task demands slightly such that cues are no longer predictive of the appropriate behavioral response, could be illuminating.

Reviewer #3 (Public Review):

In their study, Bolivar et al. set out to explore whether four distinct neuronal subtypes within the peripheral nervous system exhibit varying potentials for axon regeneration following nerve injury. To investigate this question, they harnessed the power of four distinct reporter mouse models featuring fluorescent labeling of these neuronal subtypes. Their findings reveal that axons of nociceptor neurons exhibit faster regeneration than those of motor neurons, with mechanoreceptors, and proprioceptors displaying the slowest regeneration rate.

To delve into the molecular mechanisms underlying this divergence in regeneration potential, the authors employed the Ribotag technique in mice. This approach enabled them to dissect the differential translatomes of these four neuronal populations after nerve injury, comparing them to uninjured neurons. Their comprehensive expression profiling data uncovers a remarkably heterogeneous response among these neuron subtypes to axon injury.

To focus on one identified target with a mechanistic experiment as a proof of concept, their analysis highlights a striking upregulation of MED12 in proprioceptors, leading to the hypothesis that this molecule may play an inhibitory role, contributing to the comparatively slower regeneration of proprioceptor axons when compared to other neuronal subtypes. This hypothesis gains support from their in vitro model, where siRNA-mediated downregulation of MED12 results in a significant increase in neurite outgrowth in proprioceptive neurons after plating in cell culture dishes.

Overall, this is an interesting study, and in conjunction with similar work from others will be highly valuable for neurobiologists studying how to modulate the regeneration of axons from distinct neuronal subtypes. The quality of data presentation appears to be very good in general, and the manuscript is appropriately written.

Reviewer #3 (Public Review):

The study of Weber et al. provides a thorough investigation of the roles of four individual dopamine neurons for aversive associative learning in the Drosophila larva. They focus on the neurons of the DL-1 cluster which already have been shown to signal aversive teaching signals. However, the authors go far beyond the previous publications and test whether each of these dopamine neurons responds to salt or sugar, is necessary for learning about salt, bitter, or sugar, and is sufficient to induce a memory when optogenetically activated. In addition, previously published connectomic data is used to analyze the synaptic input to each of these dopamine neurons. The authors conclude that the aversive teaching signal induced by salt is distributed across the four DL-1 dopamine neurons, with two of them, DAN-f1 and DAN-g1, being particularly important. Overall, the experiments are well designed and performed, support the authors' conclusions, and deepen our understanding of the dopaminergic punishment system.

Strengths:<br /> 1. This study provides, at least to my knowledge, the first in vivo imaging of larval dopamine neurons in response to tastants. Although the selection of tastants is limited, the results close an important gap in our understanding of the function of these neurons.

2. The authors performed a large number of experiments to probe for the necessity of each individual dopamine neuron, as well as combinations of neurons, for associative learning. This includes two different training regimens (1 or 3 trials), three different tastants (salt, quinine, and fructose) and two different effectors, one ablating the neuron, the other one acutely silencing it. This thorough work is highly commendable, and the results prove that it was worth it. The authors find that only one neuron, DAN-g1, is partially necessary for salt learning when acutely silenced, whereas a combination of two neurons, DAN-f1 and DAN-g1, are necessary for salt learning when either being ablated or silenced.

3. In addition, the authors probe whether any of the DL-1 neurons is sufficient for inducing an aversive memory. They found this to be the case for three of the neurons, largely confirming previous results obtained by a different learning paradigm, parameters, and effector.

4. This study also takes into account connectomic data to analyze the sensory input that each of the dopamine neurons receives. This analysis provides a welcome addition to previous studies and helps to gain a more complete understanding. The authors find large differences in inputs that each neuron receives, and little overlap in input that the dopamine neurons of the "aversive" DL-1 cluster and the "appetitive" pPAM cluster seem to receive.

5. Finally, the authors try to link all the gathered information in order to describe an updated working model of how aversive teaching signals are carried by dopamine neurons to the larva's memory center. This includes important comparisons both between two different aversive stimuli (salt and nociception) and between the larval and adult stages.

Weaknesses:<br /> 1. The authors repeatedly claim that they found/proved salt-specific memories. I think this is problematic to some extent.

1a. With respect to the necessity of the DL-1 neurons for aversive memories, the authors' notion of salt-specificity relies on a significant reduction in salt memory after ablating DAN-f1 and g1, and the lack of such a reduction in quinine memory. However, Fig. 5K shows a quite suspicious trend of an impaired quinine memory which might have been significant with a higher sample size. I therefore think it is not fully clear yet whether DAN-f1 and DAN-g1 are really specifically necessary for salt learning, and the conclusions should be phrased carefully.

1b. With respect to the results of the optogenetic activation of DL-1 neurons, the authors conclude that specific salt memories were established because the aversive memories were observed in the presence of salt. However, this does not prove that the established memory is specific to salt - it could be an unspecific aversive memory that potentially could be observed in the presence of any other aversive stimuli. In the case of DAN-f1, the authors show that the neuron does not even get activated by salt, but is inhibited by sugar. Why should activation of such a neuron establish a specific salt memory? At the current state, the authors clearly showed that optogenetic activation of the neurons does induce aversive memories - the "content" of those memories, however, remains unknown.

2. In many figures (e.g. figures 4, 5, 6, supplementary figures S2, S3, S5), the same behavioural data of the effector control is plotted in several sub-figures. Were these experiments done in parallel? If not, the data should not be presented together with results not gathered in parallel. If yes, this should be clearly stated in the figure legends.

Reviewer #3 (Public Review):

Summary:<br /> ISR contributes to the pathogenesis of multiple neurodegenerative diseases, such as ALS, FTD, VWMD, etc. Targeting ISR is a promising avenue for potential therapeutics. However, previously identified ways to target ISR present some challenges. PERK inhibitors suppress ISR by inhibiting eIF2alpha phosphorylation and cause pancreatic toxicity in mice. In order to bypass eIF2alpha, previous studies have identified ISR suppressors that target eIF2B, such as ISRIB and 2BAct. These molecules suppress neurodegeneration but do not cause detrimental effects in mouse models. However, ISRIB is water-insoluble, and 2BAct causes cardiovascular complications in dogs, preventing their use in clinics. Here, the authors showed that DNL343, a new ISR inhibitor targeting eIF2B, suppresses neurodegeneration in mouse models. Combined with their previous results of a clinical phase I trial showing the safety of DNL343, these findings suggest the promise of DNL343 as a potential drug for neurodegenerative diseases in which ISR contributes to pathogenesis.

Strengths:<br /> The finding is important and has disease implications, and the conclusion is not surprising.

Weaknesses:<br /> The experimental design and data are hard to comprehend for an audience with a basic research background. This reviewer suggests that the authors use the same way that previous studies on ISRIB and 2BAct (e.g., Wong et al; eLife, 2019) designed experiments and interpret data.