Reviewer #2 (Public Review):

Summary:

This is an important study characterizing striatal dysfunction and behavioral deficits in Cntnap2-/- mice. There is growing evidence suggesting that striatal dysfunction underlies core symptoms of ASD but the specific cellular and circuit level abnormalities disrupted by different risk genes remain unclear. This study addresses how the deletion of Cntnap2 affects the intrinsic properties and synaptic connectivity of striatal spiny projection neurons (SPN) of the direct (dSPN) and indirect (iSPN) pathways. Using Thy1-ChR2 mice and optogenetics the authors found increased firing of both types of SPNs in response to cortical afferent stimulation. However, there was no significant difference in the amplitude of optically-evoked excitatory postsynaptic currents (EPSCs) or spine density between Cntnap2-/- and WT SPNs, suggesting that the increased corticostriatal coupling might be due to changes in intrinsic excitability. Indeed, the authors found Cntnap2-/- SPNs, particularly dSPNs, exhibited higher intrinsic excitability, reduced rheobase current, and increased membrane resistance compared to WT SPNs. The enhanced spiking probability in Cntnap2-/- SPNs is not due to reduced inhibition. Despite previous reports of decreased parvalbumin-expressing (PV) interneurons in various brain regions of Cntnap2-/- mice, the number and function (IPSC amplitude and intrinsic excitability) of these interneurons in the striatum were comparable to WT controls.

This study also includes a comprehensive behavioral analysis of striatal-related behaviors. Cntnap2-/- mice demonstrated increased repetitive behaviors (RRBs), including more grooming bouts, increased marble burying, and increased nose poking in the holeboard assay. MoSeq analysis of behavior further showed signs of altered grooming behaviors and sequencing of behavioral syllables. Cntnap2-/- mice also displayed cognitive inflexibility in a four-choice odor-based reversal learning assay. While they performed similarly to WT controls during acquisition and recall phases, they required significantly more trials to learn a new odor-reward association during reversal, consistent with potential deficits in corticostriatal function.

Strengths:

This study provides significant contributions to the field. The finding of altered SPN excitability, the detailed characterization of striatal inhibition, and the comprehensive behavioral analysis are novel and valuable to understanding the pathophysiology of Cntnap2-/- mice.

Weaknesses:

(1) The approach based on Thy-ChR2 mice has the advantage of overcoming issues caused by injection efficiency and targeting variability. However, the spread of oEPSC amplitudes across mice shown in panels of Figure 1 G/I is very high with almost one order of magnitude difference between some mice. Given this is one of the most important points of the study it will be important to further analyze and discuss what this variability might be due to. Typically, in acute slice recordings, the within-animal variability is larger than the variability across animals. From the sample sizes reported it seems the authors sampled a large number of animals, but with a relatively low number of neurons per animal (per condition). Could this be one of the reasons for this variability?

(2) This is particularly important because the analysis of corticostriatal evoked APs in panels C and E is performed on pooled data without considering the variability in evoked current amplitudes across animals shown in G and I. Were the neurons in panels C/E recorded from the same mice as shown in G/I? If so, it would be informative to regress AP firing data (say at 20% LED) to the average oEPSC amplitude recorded on those mice at the same light intensity. However, if the low number of neurons recorded per mouse is due to technical limitations, then increasing the sample size of these experiments would strengthen the study.

(3) On a similar note, there is no discussion of why iSPNs also show increased corticostriatal evoked firing in Figure 1E, despite the difference in intrinsic excitability shown in Figure 3. This suggests other potential mechanisms that might underlie altered corticostriatal responses. Given the role of Caspr2 in clustering K channels in axons, altered presynaptic function or excitability could also contribute to this phenotype, but potential changes in PPR have not been explored in this study.

(4) Male and female SPNs have different intrinsic properties but the number and/or balance of M/F mice used for each experiment is not reported.

(5) There is no mention of how membrane resistance was calculated, and no I/V plots are shown.

(6) It would be interesting to see which behavior transitions most contribute to the decrease in entropy. Are these caused by repeated or perseverative grooming bouts? Or is this inflexibility also observed across other behaviors? The transition map in Figure S5 shows the overall number of syllables and transitions but not their sequence during behavior. Can this be analyzed by calculating the ratio of individual 𝑢𝑖 × 𝑝𝑖,𝑗 × log2 𝑝𝑖,𝑗 factors across genotypes?

Reviewer #2 (Public Review):

Summary:

Boldt et al. investigated whether previously established relationships between transdiagnostic psychiatric symptom dimensions and confidence distortions would result in downstream influences on the confidence-related behaviour of reminder setting. 600 individuals from the general population completed a battery of psychiatric symptom questionnaires and an online reminder-setting task. In line with previous studies, individuals high in compulsivity (CIT) showed over-confidence in their task performance, whereas individuals high in anxious depression (AD) tended to be under-confident. Crucially, the over-confidence associated with CIT partially mediated a decreased tendency to use external reminders during task performance, whereas the under-confidence associated with AD did not result in any alteration in the external reminder setting. The authors suggest that metacognitive monitoring is impaired in CIT which has a knock-on effect on reminder setting behaviour, but that a direct link also exists between CIT and reduced reminder setting independently of confidence.

Strengths:

The study combines the latest advances in transdiagnostic approaches to psychopathology with a cleverly designed external reminder-setting task. The approach allows for investigation of what some of the downstream consequences associated with impaired metacognition in sub-clinical psychopathology may be.

The experimental design and hypotheses were pre-registered prior to data collection.

The manuscript is well written and rigorous analysis approaches are used throughout.

Weaknesses:

Participants only performed a single task so it remains unclear if the observed effects would generalise to reminder-setting in other cognitive domains.

The sample consisted of participants recruited from the general population. Future studies should investigate whether the effects observed extend to individuals with the highest levels of symptoms (including clinical samples).

Reviewer #2 (Public Review):

Summary:

This paper attempts to examine how rare, extreme events impact decision-making in rats. The paper used an extensive behavioural study with rats to evaluate how the probability and magnitude of outcomes impact preference. The paper, however, provides limited evidence for the conclusions because the design did not allow for the isolation of the rare, extreme events in choice. There are many confounding factors, including the outcome variance and presence of less-rare, and less-extreme outcomes in the same conditions.

Strengths:

(1) The major strength of the paper is the significant volume of behavioural data with a reasonable sample size of 20 rats.

(2) The paper attempts to examine losses with rats (a notoriously tricky problem with non-human animals) by substituting time-outs as a proxy for losses. This allows for mixed gambles that have both gain and loss possible outcomes.

(3) The paper integrates both a behavioural and a modelling approach to get at the factors that drive decision-making.

(4) The paper takes seriously the question of what it means for an event to be rare, pushing to less frequent outcomes than usually used with non-human animals.

Weaknesses:

(1) The primary issue with this work is that the primary experimental manipulation fails to isolate the rare, extreme events in choice. As I understand the task, in all the conditions with a rare extreme event (e.g., 80 pellets with probability epsilon), there is also a less-rare, less-extreme event (e.g., 12 pellets with probability 5). In addition, the variance differs between the two conditions. So, any impact attributable to the rare, extreme event could be due to the less rare event or due difference in the variance. The design does not support the conclusions. Finally, by deliberately confounding rarity and extremity, the design does not allow for assessing the impact of either aspect.

(2) The RL-modelling work also fails to show a specific impact of the rare extreme event. As best as I can understand Eq 2, the model provides a free parameter that adds a bonus to the value of either the two options with high-variance gains (A and V in the paper) or to the two options with high-variance losses (F and V in the paper). This parameter only depends on whether this option could have possibly yielded the rare, extreme outcome (i.e., based on the generative probability) and was not connected to its actual appearance. That makes it a free parameter that just bumps up (or down) the probability of selecting a pair of options. In the case of the "black swan" or high-variance loss conditions, this seems very much like a loss aversion parameter, but an additive one instead of a multiplicative one.

(3) The paper presented the methods and results with lots of neologisms and fairly obscure jargon (e.g., fragility, total REE sensitivity). That made it very hard to decipher exactly what was done and what was found. For example, on p. 4, the use of concave and convex was very hard to decipher; the text even has to repeat itself 3 times (i.e., "to repeat" and "in other words") and is still not clear. It would be much clearer (and probably accurate) to say that the options varied along the variance dimension, separately for gains and losses. Option A was low-variance gains and losses. Option B was low-variance losses and high-variance gains. Option C was high-variance losses and low-variance gains, and Option D was high-variance losses and gains. That tells much more clearly what the animals experienced without the reader having to master a set of new terminologies around fragility and robustness, which brings a set of theoretical assumptions unnecessarily into the description of the experimental design. In terms of results, "Black Swan" avoidance is more simply known as risk aversion for losses.

(4) Were the probabilities shuffled or truly random (seem to be fixed sequences, so neither)? What were the experienced probabilities? Given the fixed sequences, these experienced ("ex-post") probabilities, could differ tremendously from the scheduled ("ex ante") probabilities. It's quite possible that an animal never experienced the rare, extreme event for a specific option. It's even possible (if they only picked it on the 10th/60th choices by chance), that they only ever experienced that rare extreme event. This cannot be known given the information provided. The Supplemental info on p.55 only gives gross overall numbers but does not indicate what the rats experienced for each choice/option-which is what matters here. A simple table that indicates for each of the 4 options, how often they were selected, and how often the animals experienced each of the 6-8 possible outcome would make it much clearer how closely the experience matched the planned outcomes. In addition, by restricting the rare outcome to either the 10th or 60th activations in a session, these are not random. Did the animals learn this association?

(5) The choice data are only presented in an overprocessed fashion with a sum and a difference (in both figures and tables). The basic datum (probability/frequency of selecting each of the 4 options) is not provided directly, even if it can theoretically be inferred from the sum and the difference. To understand what the rats actually do, we first need to see how often they select each option, without these transformations.

(6) There is insufficient detail provided on the inferential statistical tests (e.g., no degrees of freedom or effect sizes), and only limited information on exactly what tests were run and how (bootstrapping, but little detail). Without code or data (only summary information is provided in the supplement), this is difficult to evaluate. In addition, the studies seem not to be pre-registered in any way, leaving many researchers with degrees of freedom. Were any alternative analysis pipelines attempted? Similarly, there were many sub-groupings of the animals, and then comparisons between them - were these post-hoc?

(7) On p. 17, there is an attempt to look at the impact of a rare, extreme event by plotting a measure of preference for the 10 trials before/after the rare, extreme event. In the human literature, the main impact of experiencing a rare, extreme event is what is known as the wavy recency effect (See Plonsky et al. 2015 in Psych Review for example). What this means is that there tends to be some immediate negative recency (e.g., avoiding a rare gain) followed by positive recency (e.g., chasing the rare gain). Using a 10-trial window would thus obscure any impact of this rare, extreme event. An analysis that looks at a time course trial-by-trial could reveal any impact.

(8) As I understood the method (p. 31), the assignment of options to physical locations was not random or counterbalanced, but deliberately biased to have one of the options in the preferred location. This would seem to create a bias towards a particular option and a bias away from the other options, which confounds the preference data in subsequent analyses.

(9) Are delays really losses? This is a big assumption. Magnitude and delay are different aspects of experience, which are not necessarily commensurable and can be manipulated independently. And, for the model, how were these delays transformed into outcomes for the model? Eq 1 skips over that. Is there an assumption of linearity? In addition, I was not wholly clear if the delays meant fewer trials in a session or if the delays merely extended the session and meant longer delays until the next choice period.

(10) The paper does not sufficiently accurately represent the existing literature on human risky decision-making (with and without rare events). Here are a few examples of misrepresented and/or missing literature:<br /> -Most studies on decision-making do not only rely on p > 10% (as per p. 2). Maybe that is true with animals, but not a fair statement generally. Some do, and some don't. There is substantial literature looking at rarer events in both descriptions (most famously with Kahneman & Tversky's work), but also in experience (which is alluded to in reference 19). That reference is not only about the situation when choices are not repeated (e.g. the sampling paradigm), but also partial feedback and full-feedback situations.

The literature on learning from rewarding experiences in humans is obliquely referenced but not really incorporated. In short, there are two main findings - firstly people underweight rare events in experience; second, people overweight extreme outcomes in experience (both contrary to description). Some related papers are cited, but their content is not used or incorporated into the logic of the manuscript.

One recent study systematically examined rarity and extremity in human risky decision-making, which seems very relevant here: Mason et al. (2024). Rare and extreme outcomes in risky choice. Psychonomic Bulletin & Review, 31, 1301-1308.

There is a fair bit of research on the human perception of the risk of rare events (including from experience) and important events like climate. One notable paper is Newell et al (2015) in Nature Climate Change.

Reviewer #2 (Public Review):

The manuscript investigates the relationship between sleep, DNA damage, and aging in the Mexican cavefish (Astyanax mexicanus), a species that exhibits significant differences in sleep patterns between surface-dwelling and cave-dwelling populations. The authors aim to understand whether these evolved sleep differences influence the DNA damage response (DDR) and oxidative stress levels in the brain and gut of the fish.

Summary of the Study:

The primary objective of the study is to determine if the reduced sleep observed in cave-dwelling populations is associated with increased DNA damage and altered DDR. The authors compared levels of DNA damage markers and oxidative stress in the brains and guts of surface and cavefish. They also analyzed the transcriptional response to UV-induced DNA damage and evaluated the DDR in embryonic fibroblast cell lines derived from both populations.

Strengths of the Study:

Comparative Approach:<br /> The study leverages the unique evolutionary divergence between surface and cave populations of A. mexicanus to explore fundamental biological questions about sleep and DNA repair.

Multifaceted Methodology:<br /> The authors employ a variety of methods, including immunohistochemistry, RNA sequencing, and in vitro cell line experiments, providing a comprehensive examination of DDR and oxidative stress.

Interesting Findings:

The study presents intriguing results showing elevated DNA damage markers in cavefish brains and increased oxidative stress in cavefish guts, alongside a reduced transcriptional response to UV-induced DNA damage.

Weaknesses of the Study:

Link to Sleep Physiology:<br /> The evidence connecting the observed differences in DNA damage and DDR directly to sleep physiology is not convincingly established. While the study shows distinct DDR patterns, it does not robustly demonstrate that these are a direct result of sleep differences.

Causal Directionality:<br /> The study fails to establish a clear causal relationship between sleep and DNA damage. It is possible that both sleep patterns and DDR responses are downstream effects of a common cause or independent adaptations to the cave environment.

Environmental Considerations:<br /> The lab conditions may not fully replicate the natural environments of the cavefish, potentially influencing the results. The impact of these conditions on the study's findings needs further consideration.

Photoreactivity in Albino Fish:<br /> The use of UV-induced DNA damage as a primary stressor may not be entirely appropriate for albino, blind cavefish. Alternative sources of genotoxic stress should be explored to validate the findings.

Assessment of the Study's Achievements:

The authors partially achieve their aims by demonstrating differences in DNA damage and DDR between surface and cavefish. However, the results do not conclusively support the claim that these differences are driven by or directly related to the evolved sleep patterns in cavefish. The study's primary claims are only partially supported by the data.

Impact and Utility:

The findings contribute valuable insights into the relationship between sleep and DNA repair mechanisms, highlighting potential areas of resilience to DNA damage in cavefish. While the direct link to sleep physiology remains unsubstantiated, the study's data and methods will be useful to researchers investigating evolutionary biology, stress resilience, and the molecular basis of sleep.

Reviewer #2 (Public Review):

Summary:

Gu and Liang et. al investigated how auditory information is mapped and transformed as it enters and exits an auditory cortex. They use anterograde transsynaptic tracers to label and perform calcium imaging of thalamorecipient neurons in A1 and retrograde tracers to label and perform calcium imaging of corticothalamic output neurons. They demonstrate a degradation of tonotopic organization from the input to output neurons.

Strengths:

The experiments appear well executed, well described, and analyzed.

Weaknesses:

(1) Given that the CT and TR neurons were imaged at different depths, the question as to whether or not these differences could otherwise be explained by layer-specific differences is still not 100% resolved. Control measurements would be needed either by recording (1) CT neurons in upper layers, (2) TR in deeper layers, (3) non-CT in deeper layers and/or (4) non-TR in upper layers.

(2) What percent of the neurons at the depths are CT neurons? Similar questions for TR neurons?

(3) V-shaped, I-shaped, or O-shaped is not an intuitively understood nomenclature, consider changing. Further, the x/y axis for Figure 4a is not labeled, so it's not clear what the heat maps are supposed to represent.

(4) Many references about projection neurons and cortical circuits are based on studies from visual or somatosensory cortex. Auditory cortex organization is not necessarily the same as other sensory areas. Auditory cortex references should be used specifically, and not sources reporting on S1, and V1.

Reviewer #2 (Public Review):

Summary:

In this manuscript, "Cryo-EM structure of the bicarbonate receptor GPR30," the authors aimed to enrich our understanding of the role of GPR30 in pH homeostasis by combining structural analysis with a receptor function assay. This work is a natural development and extension of their previous work (PMID: 38413581). In the current body of work, they solved the first cryo-EM structure of the human GPR30-G-protein (mini-Gsqi) complex in the presence of bicarbonate ions at 3.21 Å resolution. From the atomic model built based on this map, they observed the overall canonical architecture of class A GPCR and also identified 4 extracellular pockets created by extracellular loops (ECLs) (Pockets A-D). Based on the polarity, location, and charge of each pocket, the authors hypothesized that pocket D is a good candidate for the bicarbonate binding site. To verify their structural observation, on top of the 10 mutations they generated in the previous work, the authors introduced another 11 mutations to map out the essential residues for the bicarbonate response on hGPR30. In addition, the human GPR30-G-protein complex model also allowed the authors to untangle the G-protein coupling mechanism of this special class A GPCR that plays an important role in pH homeostasis.

Strengths:

As a continuation of their recent Nature Communication publication (PMID: 38413581), this study was carefully designed, and the authors used mutagenesis and functional studies to confirm their structural observations. This work provided high-resolution structural observations for the receptor in complex with G-protein, allowing us to explore its mechanism of action, and will further facilitate drug development targeting GPR30. There were 4 extracellular pockets created by ECLs (Pockets A-D). The authors were able to filter out 3 of them and identified that pocket D was a good candidate for the bicarbonate binding site based on the polarity, location, and charge of each pocket. From there, the authors identified the key residues on GPR30 for its interaction with the substrate, bicarbonate. Together with their previous work, they carefully mapped out nine amino acids that are critical for receptor reactivity.

Weaknesses:

It is unclear how novel the aspects presented in the new paper are compared to the most recent Nature Communications publication (PMID: 38413581). Some areas of the manuscript appear to be mixed with the previous publication. The work is still impactful to the field. The new and novel aspects of this manuscript could be better highlighted.

I also have some concerns about the TGFα shedding assay the authors used to verify their structural observation. I understand that this assay was also used in the authors' previous work published in Nature Communications. However, there are still several things in the current data that raised concerns:

(1) The authors confirmed the "similar expression levels of HA-tagged hGPR30" mutants by WB in Supplemental Figure 1A and B. However, compared to the hGPR30-HA (~6.5 when normalized to the housekeeping gene, Na-K-ATPase), several mutants of the key amino acids had much lower surface expression: S134A, D210A, C207A had ~50% reduction, D125A had ~30% reduction, and Q215A and P71A had ~20% reduction. This weakens the receptor reactivity measured by the TGFα shedding assay.

(2) In the previous work, the authors demonstrated that hGPR30 signals through the Gq signaling pathway and can trigger calcium mobilization. Given that calcium mobilization is a more direct measurement for the downstream signaling of hGPR30 than the TGFα shedding assay, pairing the mutagenesis study with the calcium assay will be a better functional validation to confirm the disruption of bicarbonate signaling.

(3) It was quite confusing for Figure 4B that all statistical analyses were done by comparing to the mock group. It would be clearer to compare the activity of the mutants to the wild-type cell line.

Additional concerns about the structural data include:

(1) E218 was in close contact with bicarbonate in Figure 4D. However, there is no functional validation for this observation. Including the mutagenesis study of this site in the cell-based functional assay will strengthen this structural observation.

(2) For the flow chart of the cryo-EM data processing in Supplemental data 2, the authors started with 10,148,422 particles after template picking, then had 441,348 Particles left after 2D classification/heterogenous refinement, and finally ended with 148,600 particles for the local refinement for the final map. There seems to be a lot of heterogeneity in this purified sample. GPCRs usually have flexible and dynamic loop regions, which explains the poor resolution of the ECLs in this case. Thus, a solid cell-based functional validation is a must to assign the bicarbonate binding pocket to support their hypothesis.

Reviewer #2 (Public Review):

Summary:

In this manuscript, Fujita et al. characterized the neutrality indexes of several protein mutants in S. cerevisiae and uncovered that mox-YG and Gpm1-CCmut can be expressed as abundant as 40% of total proteins without causing severe growth defects. The authors then looked at the transcriptome and proteome of cells expressing excess mox-YG to investigate how protein burden affects yeast cells. Based on RNA-seq and mass-spectrometry results, the authors uncover that cells with excess mox-YG exhibit nitrogen starvation, respiration increase, inactivated TORC1 response, and decreased ribosomal abundance. The authors further showed that the decreased ribosomal amount is likely due to nucleoli defects, which can be partially rescued by nuclear exosome mutations.

Strengths:

Overall, this is a well-written manuscript that provides many valuable resources for the field, including the neutrality analysis on various fluorescent proteins and glycolytic enzymes, as well as the RNA-seq and proteomics results of cells overexpressing mox-YG. Their model on how mox-YG overexpression impairs the nucleolus and thus leads to ribosomal abundance decline will also raise many interesting questions for the field.

Weaknesses:

The authors concluded from their RNA-seq and proteomics results that cells with excess mox-YG expression showed increased respiration and TORC1 inactivation. I think it will be more convincing if the authors can show some characterization of mitochondrial respiration/membrane potential and the TOR responses to further verify their -omic results.

In addition, the authors only investigated how overexpression of mox-YG affects cells. It would be interesting to see whether overexpressing other non-toxic proteins causes similar effects, or if there are protein-specific effects. It would be good if the authors could at least discuss this point considering the workload of doing another RNA-seq or mass-spectrum analysis might be too heavy.

Reviewer #2 (Public Review):

Satouh et al report the presence of spherical structures composed of endosomes, lysosomes, and autophagosomes within immature mouse oocytes. These endolysosomal compartments have been named as Endosomal-LYSosomal organellar Assembly (ELYSA). ELYSAs increase in size as the oocytes undergo maturation. ELYSAs are distributed throughout the oocyte cytoplasm of GV stage immature oocytes but these structures become mostly cortical in the mature oocytes. Interestingly, they tend to avoid the region which contains metaphase II spindle and chromosomes. They show that the endolysosomal compartments in oocytes are less acidic and therefore non-degradative but their pH decreases and becomes degradative as the ELYSAs begin to disassemble in the embryos post-fertilization. This manuscript shows that lysosomal switching does not happen during oocyte development, and the formation of ELYSAs prevents lysosomes from being activated. Structures similar to these ELYSAs have been previously described in mouse oocytes (Zaffagnini et al, 2024) and these vesicular assemblies are important for sequestering protein aggregates in the oocytes but facilitate proteolysis after fertilization. The current manuscript, however, provides further details of endolysosomal disassembly post-fertilization. Specifically, the V1-subunit of V-ATPase targeting the ELYSAs increases the acidity of lysosomal compartments in the embryos. This is a well-conducted study and their model is supported by experimental evidence and data analyses.

Reviewer #2 (Public Review):

Summary:

Adhikari and colleagues developed a new technique, rapamycin-induced proximity assay (RiPA), to identify E3-ubiquitin (ub) ligases of a protein target, aiming at identifying additional E3 ligases that could be targeted for PROTAC generation or ligases that may degrade a protein target. The study is timely, as expanding the landscape of E3-ub ligases for developing targeted degraders is a primary direction in the field.

Strengths:

The study's strength lies in its practical application of the FRB:FKBP12 system. This system is used to identify E3-ub ligases that would degrade a target of interest, as evidenced by the reduction in luminescence upon the addition of rapamycin. This approach effectively mimics the potential action of a PROTAC.

Weaknesses:

(1) While the technique shows promise, its application in a discovery setting, particularly for high-throughput or unbiased E3-ub ligase identification, may pose challenges. The authors should provide more detailed insights into these potential difficulties to foster a more comprehensive understanding of RiPA's limitations.

(2) While RiPA will help identify E3 ligases, PROTAC design would still be empirical. The authors should discuss this limitation. Could the technology be applied to molecular glue generation?

(3) Controls to verify the intended mechanism of action are missing, such as using a proteasome inhibitor or VHL inhibitors/siRNA to verify on-target effects. Verification of the target E3 ligase complex after rapamycin addition via orthogonal approaches, such as IP, should be considered.

Minor concern:

The graphs in Figure 1E are missing.

Reviewer #2 (Public Review):

Summary:

The investigation provides computational as well as biochemical insights into the (un)binding mechanisms of a pair of psychoactive substances into cannabinoid receptors. A combination of molecular dynamics simulation and a set of state-of-the art statistical post-processing techniques were employed to exploit GPCR-ligand dynamics.

Strengths:

The strength of the manuscript lies in the usage and comparison of TRAM as well as Markov state modelling (MSM) for investigating ligand binding kinetics and thermodynamics. Usually, MSMs have been more commonly used for this purpose. But as the authors have pointed out, implicit in the usage of MSMs lies the assumption of detailed balance, which would not hold true for many cases especially those with skewed binding affinities. In this regard, the author's usage of TRAM which harnesses both biased and unbiased simulations for extracting the same, provides a more appropriate way out.

Weaknesses:

(1) While the authors have used TRAM (by citing MSM to be inadequate in these cases), the thermodynamic comparisons of both techniques provide similar values. In this case, one would wonder what advantage TRAM would hold in this particular case.

(2) The initiation of unbiased simulations from previously run biased metadynamics simulations would almost surely introduce hysteresis in the analysis. The authors need to address these issues.

(3) The choice of ligands in the current work seems very forced and none of the results compare directly with any experimental data. An ideal case would have been to use the seminal D.E. Shaw research paper on GPCR/ligand binding as a benchmark and then show how TRAM, using much lesser biased simulation times, would fare against the experimental kinetics or even unbiased simulated kinetics of the previous report

(4) The method section of the manuscript seems to suggest all the simulations were started from a docked structure. This casts doubt on the reliability of the kinetics derived from these simulations that were spawned from docked structure, instead of any crystallographic pose. Ideally, the authors should have been more careful in choosing the ligands in this work based on the availability of the crystallographic structures.

(5) The last part of using a machine learning-based approach to analyse allosteric interaction seems to be very much forced, as there are numerous distance-based more traditional precedent analyses that do a fair job of identifying an allosteric job.

(6) While getting busy with the methodological details of TRAM vs MSM, the manuscript fails to share with sufficient clairty what the distinctive features of two ligand binding mechanisms are.

Reviewer #2 (Public Review):

Summary of goals:

The aims of the study were to identify new lineage trajectories for the cardiac lineages of the heart, and to use computational and cell and animal studies to identify and validate new gene regulatory mechanisms involved in these trajectories.

Strengths:

The study addresses the long-standing yet still not fully answered questions of what drives the earliest specification mechanisms of the heart lineages. The introduction demonstrates a good understanding of the relevant lineage trajectories that have been previously established, and the significance of the work is well described. The study takes advantage of several recently published data sets and attempts t use these in combination to uncover any new mechanisms underlying early mesoderm/cardiac specification mechanisms. A strength of the study is the use of an in vitro model system (mESCs) to assess the functional relevance of the key players identified in the computational analysis, including innovative technology such as CRISPR-guided enhancer modulations. Lastly, the study generates mesoderm-specific Hand1 LOF embryos and assesses the differentiation trajectories in these animals, which represents a strong complementary approach to the in vitro and computational analysis earlier in the paper. The manuscript is clearly written and the methods section is detailed and comprehensive.

Comments and Weaknesses:

Overall: The computational analysis presented here integrates a large number of published data sets with one new data point (E7.0 single cell ATAC and RNA sequencing). This represents an elegant approach to identifying new information using available data. However, the data presentation at times becomes rather confusing, and relatively strong statements and conclusions are made based on trajectory analysis or other inferred mechanisms while jumping from one data set to another. The cell and in vivo work on Hand1 and Foxf1 is an important part of the study. Some additional experiments in both of these model systems could strongly support the novel aspects that were identified by the computational studies leading into the work.

(1) Definition of MJH and PSH trajectory:<br /> The study uses previously published data sets to identify two main new differentiation trajectories: the MJH and the PSH trajectory (Figure 1). A large majority of subsequent conclusions are based on in-depth analysis of these two trajectories. For this reason, the method used to identify these trajectories (WTO, which seems a highly biased analysis with many manually chosen set points) should be supported by other commonly used methods such as for example RNA velocity analysis. This would inspire some additional confidence that the MJH and PSH trajectories were chosen as unbiased and rigorous as possible and that any follow-up analysis is biologically relevant.

(2) Identification of MJH and PSH trajectory progenitors:<br /> The study defines various mesoderm populations from the published data set (Figure 1A-E), including nascent mesoderm, mixed mesoderm, and extraembryonic mesoderm. It further assigns these mesoderm populations to the newly identified MJH/PSH trajectories. Based on the trajectory definition in Figure 1A it appears that both trajectories include all 3 mesoderm populations, albeit at different proportions and it seems thus challenging to assign these as unique progenitor populations for a distinct trajectory, as is done in the epigenetic study by comparing clusters 8 (MJH) and s (PSH)(Figure 2). Along similar lines, the epigenetic analysis of clusters 2 and 8 did not reveal any distinct differences in H3K4m1, H3K27ac, or H3K4me3 at any of the time points analyzed (Figure 2F). While conceptually very interesting, the data presented do not seem to identify any distinct temporal patterns or differences in clones 2 and 8 (Figure 2H), and thus don't support the conclusion as stated: "the combined transcriptome and chromatin accessibility analysis further supported the early lineage segregation of MJH and the epigenetic priming at gastrulation stage for early cardiac genes".

(3) Function of Hand1 and Foxf1 during early cardiac differentiation:<br /> The study incorporated some functional studies by generating Hand1 and Foxf1 KO mESCs and differentiated them into mesoderm cells for RNA sequencing. These lines would present relevant tools to assess the role of Hand1 and Foxf1 in mesoderm formation, and a number of experiments would further support the conclusions, which are made for the most part on transcriptional analysis. For example, the study would benefit from quantification of mesoderm cells and subsequent cardiomyocytes during differentiation (via IF, or more quantitatively, via flow cytometry analysis). These data would help interpret any of the findings in the bulk RNAseq data, and help to assess the function of Hand1 and Foxf1 in generating the cardiac lineages. Conclusions such as "the analysis indicated that HAND1 and FOXF1 could dually regulate MJH specification through directly activating the MJH specific genes and inhibiting PSH specific genes" seem rather strong given the data currently provided.

(4) Analysis of Hand1 cKO embryos:<br /> Adding a mouse model to support the computational analysis is a strong way to conclude the study. Given the availability of these early embryos, some of the findings could be strengthened by performing a similar analysis to Figure 7B&C and by including some of the specific EEM markers found to be differentially regulated to complement the structural analysis of the embryos.

(5) Current findings in the context of previous findings:<br /> The introduction carefully introduces the concept of lineage specification and different progenitor pools. Given the enormous amount of knowledge already available on Hand1 and Foxf1, and their role in specific lineages of the early heart, some of this information should be added, ideally to the discussion where it can be put into context of what the present findings add to the existing understanding of these transcription factors and their role in early cardiac specification.

Reviewer #2 (Public Review):

Summary:

James et al, in this study, build on their previous work investigating tau as a drug target. The authors identify tryptanthrin (TA) and its analogs as powerful inhibitors of tau4RD aggregation, even at low concentrations (nanomolar range). Interestingly, these analogs specifically target the initial stages of aggregation, where tau self-association first begins. This targeted approach effectively explains why such small amounts of tryptanthrin analogs are sufficient for inhibition. The study further shows that slight modifications to the structure of these molecules can significantly impact their effectiveness.

Strengths:

The experiments are well-designed and executed. The reviewer, in particular, appreciates the authors for the simple yet intelligent study design to understand the mechanism of aggregation inhibition by TA analogs.

Weaknesses:

Certain areas in the manuscript need clarifications, revisions, or additional supporting studies to strengthen the outcomes. For example, the authors mostly apply a single approach to assess tau aggregation or aggregation inhibition. Using additional techniques as suggested below will be helpful.

Reviewer #2 (Public Review):

The authors addressed the question of how mitochondrial proteins that are dually localized or only to a minor fraction localized to mitochondria can be visualized on the whole genome scale. For this, they used an established and previously published method called BiG split-GFP, in which GFP strands 1-10 are encoded in the mitochondrial DNA and fused the GFP11 strand C-terminally to the yeast ORFs using the C-SWAT library. The generated library was imaged under different growth and stress conditions and yielded positive mitochondrial localization for approximately 400 proteins. The strength of this method is the detection of proteins that are dually localized with only a minor fraction within mitochondria, which so far has hampered their visualization due to strong fluorescent signals from other cellular localizations. The weakness of this method is that due to the localization of the GFP1-10 in the mitochondrial matrix, only matrix proteins and IM proteins with their C-termini facing the matrix can be detected. Also, proteins that are assembled into multimeric complexes (which will be the case for probably a high number of matrix and inner membrane-localized proteins) resulting in the C-terminal GFP11 being buried are likely not detected as positive hits in this approach. Taking these limitations into consideration, the authors provide a new library that can help in the identification of eclipsed protein distribution within mitochondria, thus further increasing our knowledge of the complete mitochondrial proteome. The approach of global tagging of the yeast genome is the logical consequence after the successful establishment of the BiG split-GFP for mitochondria. The authors also propose that their approach can be applied to investigate the topology of inner membrane proteins, however, for this, the inherent issue remains that it cannot be excluded that even the small GFP11 tag can impact on protein biogenesis and topology. Thus, the approach will not overcome the need to assess protein topology analysis via biochemical approaches on endogenous untagged proteins.

DOI: 10.1038/s41598-021-84180-2

Resource: RRID:BDSC_64349

Curator: @mpairish

SciCrunch record: RRID:BDSC_64349

What is this?

DOI: 10.1038/s41467-021-25445-2

Resource: RRID:BDSC_27275

Curator: @anisehay

SciCrunch record: RRID:BDSC_27275

What is this?

DOI: 10.1038/s41467-020-18185-2

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @mzhang007

SciCrunch record: RRID:SCR_006457

What is this?

Reviewer #2 (Public Review):

The manuscript by Harry and Zakas determined the extent to which gene expression differences contribute to developmental divergence by using a model that has two distinct developmental morphs within a single species. Although the authors did collect a valuable dataset and trends in differential expression between the two morphs of S. benedicti were presented, we found limitations about the methods, system, and resources that the authors should address.

We have two major points:

(1) Background information about the biological system needs to be clarified in the introduction of this manuscript. The authors stated that F1 offspring can have intermediate larval traits compared to the parents (Line 81). However, the authors collected F1 offspring at the same time as the mother in the cross. If offspring have intermediate larval traits, their developmental timeline might be different than both parents and necessitate the collection of offspring at different times to obtain the same stages as the parents. Could the authors (1) explain why they collected offspring at the same time as parents given that other literature and Line 81 state these F1 offspring develop at intermediate rates, and (2) add the F1 offspring to Figure 1 to show morphological and timeline differences in development?

Additionally, the authors state (Lines 83-85) that they detail the full-time course of embryogenesis for both the parents and the F1 crosses. However, we do not see where the authors have reported the full-time course for embryogenesis of the F1 offspring. Providing this information would shape the remaining results of the manuscript.

(2) We have several concerns about the S. benedicti genome and steps regarding the read mapping for RNA-seq:

The S. benedicti genome used (Zakas et al. 2022) was generated using the PP morph. The largest scaffolds of this assembly correspond to linkage groups, showing the quality of this genome. The authors should point out in the Methods and/or Results sections that the quality of this genome means that PP-specific gene expression can be quantified well. However, the challenges and limitations of mapping LL-specific expression data to the PP genome should be discussed.

It is possible that the authors did not find exclusive gene expression in the LL morph because they require at least one gene to be turned on in one morph as part of the data-cleaning criteria. Because the authors are comparing all genes to the PP morph, they could be missing true exclusive genes responsible for the biological differences between the two morphs. Did they make the decision to only count genes expressed in one stage of the other morph because the gene models and mapping quality led to too much noise?

The authors state that the mapping rates between the two morphs are comparable (Supplementary Figure 1). However, there is a lot of variation in mapping the LL individuals (~20% to 43%) compared to the PP individuals. What is the level of differentiation within the two morphs in the species (pi and theta)? The statistical tests for this comparison should be added and the associated p-value should be reported. The statistical test used to compare mapping rates between the two morphs may be inappropriate. The authors used Salmon for their RNA alignment and differential expression analysis, but it is possible that a different method would be more appropriate. For example, Salmon has some limitations as compared to Kallisto as others have noted. The chosen statistical test should be explained, as well as how RNA-seq data are processed and interpreted.

What about the read mapping rate and details for the F1 LP and PL individuals? How did the offspring map to the P genome? These details should be included in Supplementary Figure 1. Could the authors also provide information about the number of genes expressed at each stage in the F1 LP and PL samples in S Figure 2? How many genes went into the PCA? Many of these details are necessary to evaluate the F1 RNA-seq analyses.

Generally, the authors need to report the statistics used in data processing more thoroughly. The authors need to report the statistics used to (1) process and evaluate the RNA-seq data and (2) determine the significance between the two morphs (Supplementary Figures 1 and 2).

Reviewer #2 (Public Review):

Summary:

The endocannabinoid system (ECS) regulates many critical functions, including reproductive function. Recent evidence indicates that dysregulated ECS contributes to endometriosis pathophysiology and microenvironment. Therefore, the authors further examined the dysregulated ECS and its mechanisms in endometriosis lesion establishment and progression using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. The authors presented differential gene expressions and altered pathways, especially those related to the adaptive immune response in CNR1 and CNR2 ko lesions. Interstingly, the T-cell population was dramatically reduced in the peritoneal cavity lacking CNR2, and the loss of proliferative activity of CD4+ T helper cells. Imaging mass cytometry analysis provided spatial profiling of cell populations and potential relationships among immune cells and other cell types. This study provided fundamental knowledge of the endocannabinoid system in endometriosis pathophysiology.

Strengths:

Dysregulated ECS and its mechanisms in endometriosis pathogenesis were assessed using two different endometrial sources of mouse models of endometriosis with CNR1 and CNR2 knockout mice. Not only endometriotic lesions but also peritoneal exudate (and splenic) cells were analyzed to understand the specific local disease environment under the dysregulated ECS.

Providing the results of transcriptional profiles and pathways, immune cell profiles, and spatial profiles of cell populations support altered immune cell population and their disrupted functions in endometriosis pathogenesis via dysregulation of ECS.

L386: Role of CNR2 in T cells: Finding nearly absent CD3+ T cells in the peritoneal cavity of CNR2 ko mice is intriguing.

Interpretation of the results is well-described in discussion.

Weaknesses:

The study was terminated and characterized 7 days after EM induction surgery without the details for selecting the time point to perform the experiments.

The authors also mentioned that altered eutopic endometrium contributes to the establishment and progression of endometriosis. This reviewer agrees L324-325. If so, DEGs are likely identified between eutopic endometrium (with/without endometriosis lesion induction) and ectopic lesions. It would be nice to see the data (even though using publicly available data sets).

Figure 7 CDEF. Please add the results of the statistical analyses and analyzed sample numbers. L444-450 cannot be reviewed without them.

This reviewer agrees L498-500. In contrast, retrograded menstrual debris is not decidualized. The section could be modified to avoid misunderstanding.

The authors addressed all my concerns. I do not have any comments.

Reviewer #2 (Public Review):

The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.

Comments on revised version:

The authors demonstrate the correlation between overexertion of atg6 and higher stability and activity of npr1. They claim a novel activity of atg6 in the nucleus.<br /> Overall, the experimental scope of the study is solid, however, the over-interpretation of the results substantially reduces the significance and value of this study for the target plant immunity readership.

Reviewer #2 (Public Review):

Summary:

The authors set out to determine which lipid transfer proteins impact the lipids of Golgi apparatus, and they identified a reasonable number of "hits" where the lack of one lipid transfer protein affected a particular Golgi lipid or class of lipids. They then carried out something close to a "proof of concept" for one lipid (sphingomyelin) and two closely related lipid transfer proteins (ORP9/ORP11). They looked into that example in great detail and found a previous unknown relationship between the level of phosphatidylserine in the Golgi (presumably trans-Golgi, trans-Golgi Network) and function of the sphingomyelin synthase enzyme. This was all convincingly done - results support their conclusions - showing that the authors achieved their aims.

Impact:

There are likely to be 2 types of impact:

(I) cell biology: sphoingomyelin synthase, ORP9/11 will be studied in future in more informed ways to understand (a) the role of different Golgi lipids - this work opens that out and produces a to more questions than answers (b) the role of different ORPs: what distinguishes ORP11 from its paralogy ORP10?

(ii) molecular biochemistry: combining knockdown miniscreen with organelle lipidomics must be time-consuming, but here it is shown to be quite a powerful way to discover new aspects of lipid-based regulation of protein function. This will be useful to others as an example, and if this kind of workflow could be automated, then the possible power of the method could be widely applied.

Strengths:

Nicely controlled data;

Wide-ranging lipidomics dataset with repeats and SDs - all data easily viewed.

Simple take home message that PS traffic to the TGN by ORP9/11 is required for some aspect of SMS1 function.

Weaknesses:

Model and Discussion:

Despite the authors saying that this has been addressed in their rebuttal, I still struggle to find any ideas about the aspect of SMS1 function that is being affected.

As I mentioned before, even if no further experiments were carried out the authors could discuss possibilities. one might speculate what the PS is being used for. For example, is it a co-factor for integral membrane proteins, such as flippases? Is it a co-factor for peripheral membrane proteins, such as yet more LTPs? The model could include the work of Peretti et al (2008), which linked Nir2 activity exchanging PI:PA (Yadav et al, 2015) to the eventual function of CERT. Could the PS have a role in removing/reducing DAG produced by CERT?

Reviewer #2 (Public Review):

Salt stress is a significant and growing concern for agriculture in some parts of the world. While the effects of sodium excess have been studied in Arabidopsis and (many) crop species, most studies have focused on Na uptake, toxicity, and overall effects on yield, rather than on developmental responses to excess Na, per se. The work by Ishka and colleagues aims to fill this gap.

Working from an existing dataset that exposed a diverse panel of A. thaliana accessions to control, moderate, and severe salt stress, the authors identify candidate loci associated with altering the root:shoot ratio under salt stress. Following a series of molecular assays, they characterize a DUF247 protein which they dub SR3G, which appears to be a negative regulator of root growth under salt stress.

Overall, this is a well-executed study that demonstrates the functional role played by a single gene in plant response to salt stress in Arabidopsis.

The abstract and beginning of the Discussion section highlight the "new tool" developed here for measuring biomass accumulation. I feel that this distracts from the central aims of the study, which is really about the role of a specific gene in root development under salt stress. I would suggest moving the tool description to less prominent parts of the manuscript.

Reviewer #2 (Public Review):

Summary:

This study integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points post-MI. They identified four transcriptionally distinct subtypes of lymphatic endothelial cells and localized them in space. They observed that LECs subgroups are localized in different zones of infarcted heart with functions. Specifically, they demonstrated that LEC ca III may be involved in directly regulating myocardial injuries in the infarcted zone concerning metabolic stress, while LEC ca II may be related to the rapid immune inflammatory responses of the border zone in the early stage of MI. LEC ca I and LEC collection mainly participate in regulating myocardial tissue edema resolution in the middle and late stages post-MI. Finally, cell trajectory and Cell-Chat analyses further identified that LECs may regulate myocardial edema through Aqp1, and likely affect macrophage infiltration through the galectin9-CD44 pathway. The authors concluded that their study revealed the dynamic transcriptional heterogeneity distribution of LECs in different regions of the infarcted heart and that LECs formed different functional subgroups that may exert different bioeffects in myocardial tissue post-MI.

Strengths:

The study addresses a significant clinical challenge, and the results are of great translational value. All experiments were carefully performed, and their data support the conclusion.

Weaknesses:

(1) Language expression must be improved. Many incomplete sentences exist throughout the manuscript. A few examples: Lines 70-71: In order to further elucidate the effects and regulatory mechanisms of the lymphatic vessels in the repair process of myocardial injury following MI. Lines 71-73: This study, integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points after MI from publicly available data (E-MTAB-7895, GSE214611) in the ArrayExpress and gene expression omnibus (GEO) databases. Line 88-89: Since the membrane protein LYVE1 can present lymphatic vessel morphology more clearly than PROX1.

(2) The type of animal models (i.e., permeant MI or MI plus reperfusion) included in ArrayExpress and gene expression omnibus (GEO) databases must be clearly defined as these two models may have completely different effects on lymphatic vessel development during post-MI remodeling.

(3) Lines 119-120: Caution must be taken regarding Cav1 as a lymphocyte marker because Cav1 is expressed in all endothelial cells, not limited to LEC.

(4) Figure 1 legend needs to be improved. RZ, BZ, and IZ need to be labeled in all IF images. Day 0 images suggest that RZ is the tissue section from the right ventricle. Was RZ for all other time points sampled from the right ventricular tissue section?

(5) The discussion section needs to be improved and better focused on the findings from the current study.

Reviewer #2 (Public Review):

Summary:

Transcriptomics technologies play important roles in biological studies. Technologies based on second-generation sequencing, such as mRNA-seq, face some serious obstacles, including isoform analysis, due to short read length. Third-generation sequencing technologies perfectly solve these problems by having long reads, but they are much more expensive. The authors presented a useful real-time strategy to minimize the cost of sequencing with Oxford Nanopore Technologies (ONT). The authors performed three sets of experiments to illustrate the utility of the real-time strategy. However, due to the problems in experimental design and analysis, their aims are not completely achieved. If the authors can significantly improve the experiments and analysis, the strategy they proposed will guide biologists to conduct transcriptomics studies with ONT in a fast and cost-effective way and help studies in both basic research and clinical applications.

Strengths:

The authors have recently developed a computational tool called NanopoReaTA to perform real-time analysis when cDNA/RNA samples are sequenced with ONT (Wierczeiko et al., 2023). The advantage of real-time analysis is that the sequencing can be stopped once enough data is collected to save cost. Here, they described three sets of experiments: a comparison between two human cell lines, a comparison among RNA preparation procedures, and a comparison between genetically modified yeasts. Their results show that the real-time strategy works for different species and different RNA preparation methods.

Weaknesses:

However, especially considering that the computational tool NanopoReaTA is their previous work, the authors should present more helpful guidelines to perform real-time ONT analysis and more advanced analysis methods. There are four major weaknesses:

(1) For all three sets of experiments, the authors focused on sample clustering and gene-level differential expression analysis (DEA), and only did little analysis on isoform level and even nothing in any figures in the main text. Sample clustering and gene-level DEA can be easily and well done using mRNA-seq at a much cheaper cost. Even for initial data quality checking, mRNA-seq can be first done in Illumina MiSeq/NextSeq which is quick, before deep sequencing in HiSeq/NovaSeq. The real power of third-generation RNA sequencing is the isoform analysis due to the long read length. At least for now, PacBio Iso-seq is very expensive and one cannot analyze the data in real-time. Thus, the authors should focus on the real-time isoform analysis of ONT to show the advantages.

(2) The sample sizes are too small in all three sets of experiments: only two for sets 1 and 2, and three for set 3. For DEA, three is the minimal number for proper statistics. But a sample size of three always leads to very poor power. Nowadays, a proper transcriptomics study usually has a larger sample size. Besides the power issue, biological samples always contain many outliers due to many reasons. It is crucial to show whether the real-time analysis also works for larger sample sizes, such as 10, i.e., 20 samples in total. Will the performance still hold when the sample number is increasing? What is the maximum sample number for an ONT run? If the samples need to be split into multiple runs, how the real-time analysis will be adjusted? These questions are quite useful for researchers who plan to use ONT.

(3) According to the manuscript, real-time analysis checks the sequencing data in a few time points, this is usually called sequential analysis or interim analysis in statistics which is usually performed in clinical trials to save cost. Care must be taken while performing these analyses, as repeated checks on the data can inflate the type I error rate. Thus, the authors should develop a sequential analysis procedure for real-time RNA sequencing.

(4) The experimental set 1 (comparison between two completely different human cell lines) and experimental set 2 (comparison among RNA preparation procedures) are not quite biologically meaningful. If it is possible, it is better for the authors to perform an experiment more similar to a real situation for biological discovery. Then the manuscript can attract more researchers to follow its guidelines.

DOI: 10.1186/s13041-024-01117-2

Resource: Bloomington Stock Center

Curator: @maulamb

SciCrunch record: Bloomington Stock Center

What is this?

DOI: 10.1038/s41598-021-00663-2

Resource: RRID:BDSC_9331

Curator: @anisehay

SciCrunch record: RRID:BDSC_9331

What is this?

Reviewer #2 (Public Review):

Summary:

In this manuscript the authors have generated a single-cell atlas of the miracidium, the first free-living stage of an important human parasite, Schistosoma mansoni. Miracidia develop from eggs produced in the mammalian (human) host and are released into freshwater, where they can infect the parasite's intermediate snail host to continue the life cycle. This study adds to the growing single-cell resources that have already been generated for other life-cycle stages and, thus, provides a useful resource for the field.

Strengths:

Beyond generating lists of genes that are differentially expressed in different cell types, the authors validated many of the cluster-defining genes using in situ hybridization chain reaction. In addition to providing the field with markers for many of the cell types in the parasite at this stage, the authors use these markers to count the total number of various cell types in the organism. Because the authors realized that their cell isolation protocols were biasing the cell types they were sequencing, they applied a second method to help them recover additional cell types.

Schistosomes have ZW sex chromosomes and the authors make the interesting observation that the stem cells at this stage are already expressing sex (i.e. W)-specific genes.

Comments on revised version:

The manuscript has been improved after revisions. The methods, data and analyses broadly support the claims with only minor weaknesses.

Reviewer #2 (Public Review):

Summary:

In this study, the authors investigated the release properties of glutamate/GABA co-transmission at the supramammillary nucleus (SuM)-granule cell (GC) synapses using in vitro electrophysiology and anatomical approaches at the light and electron microscopy level. They found that SuM to dentate granule cell synapses, which co-release glutamate and GABA, exhibit distinct differences in paired-pulse ratio, Ca2+ sensitivity, presynaptic receptor modulation, and Ca2+ channel-vesicle coupling configuration for each neurotransmitter. The study shows that glutamate/GABA co-release produces independent glutamatergic and GABAergic synaptic responses, with postsynaptic targets segregated. They show that most SuM boutons form distinct glutamatergic and GABAergic synapses in close proximity, characterized by GluN1 and GABAAα1 receptor labeling, respectively. Furthermore, they demonstrate that glutamate/GABA co-transmission exhibits distinct short-term plasticity, with glutamate showing frequency-dependent depression and GABA showing frequency-independent stable depression.

Their findings suggest that these distinct modes of glutamate/GABA co-release by SuM terminals serve as frequency-dependent filters of SuM inputs.

Strengths:

The conclusions of this paper are mostly well supported by the data.

Weaknesses:

Some aspects of Supplementary Figure 1A and the table need clarification. Specifically, the claim that the authors have stimulated an axon fiber rather than axon terminals is not convincingly supported by the diagram of the experimental setup. Additionally, the antibody listed in the primary antibodies section recognizes the gamma2 subunit of the GABAA receptor, not the alpha1 subunit mentioned in the results and Figure 4.

Reviewer #3 (Public Review):

Summary:

This paper presents a new formulation of a computational model of adaptive learning amid environmental volatility. Using a behavioral paradigm and data set made available by the authors of an earlier publication (Gagne et al., 2020), the new model is found to fit the data well. The model's structure consists of three weighted controllers that influence decisions on the basis of (1) expected utility, (2) potential outcome magnitude, and (3) habit. The model offers an interpretation of psychopathology-related individual differences in decision-making behavior in terms of differences in the relative weighting of the three controllers.

Strengths:

The newly proposed "mixture of strategies" (MOS) model is evaluated relative to the model presented in the original paper by Gagne et al., 2020 (here called the "flexible learning rate" or FLR model) and two other models. Appropriate and sophisticated methods are used for developing, parameterizing, fitting, and assessing the MOS model, and the MOS model performs well on multiple goodness-of-fit indices. Parameters of the model show decent recoverability and offer a novel interpretation for psychopathology-related individual differences. Most remarkably, the model seems to be able to account for apparent differences in behavioral learning rates between high-volatility and low-volatility conditions even with no true condition-dependent change in the parameters of its learning/decision processes. This finding calls into question a class of existing models that attribute behavioral adaptation to adaptive learning rates.

Weaknesses:

The authors have responded to the weaknesses noted previously.

Reviewer #2 (Public Review):

Summary:

In their study titled "Recent evolutionary origin and localized diversity hotspots of mammalian coronaviruses," authors Benoît Perez-Lamarque, Renan Maestri, Anna Zhukova, and Hélène Morlon investigate the complex evolutionary history of coronaviruses, particularly those affecting mammals, including humans. The study focuses on unraveling the evolutionary trajectory of these viruses, which have shown a high propensity for causing pandemics, as evidenced by the SARS-CoV2 outbreak.<br /> The research addresses a significant gap in our understanding of the evolutionary dynamics of coronaviruses, particularly their history, patterns of host-to-host transmission, and geographical spread. These aspects are important for predicting and managing future pandemic scenarios.

Historically, studies have employed cophylogenetic tests to explore virus-host relationships within the Coronaviridae family, often suggesting a long history of virus-host codiversification spanning millions of years. However, the team led by Perez-Lamarque proposes a novel phylogenetic framework that contrasts this traditional view. Their approach, which involves adapting gene tree-species tree reconciliation, is designed to robustly test the validity of two competing scenarios: an ancient origination and codiversification versus a more recent emergence and diversification through host switching.

Upon applying this innovative framework to the study of coronaviruses and their mammalian hosts, the authors' findings challenge the prevailing notion of a deep evolutionary history. Instead, their results strongly support a scenario where coronaviruses have a more recent origin, likely in bat populations, followed by diversification predominantly through host-switching events. This diversification, interestingly, seems to occur preferentially within mammalian orders.

A critical aspect of their findings is the identification of hotspots of coronavirus diversity, particularly in East Asia and Europe. These regions align with the proposed scenario of a relatively recent origin and subsequent localized host-switching events. The study also highlights the rarity of spillovers from bats to other species, yet underscores the relatively higher likelihood of such spillovers occurring towards humans, suggesting a significant role for humans as an intermediate host in the evolutionary journey of these viruses.

The research also points out the high rates of host-switching within mammalian orders, including between humans, domesticated animals, and non-flying wild mammals.<br /> In conclusion, the study by Perez-Lamarque and colleagues presents an important quantitative advance in our understanding of the evolutionary history of mammalian coronaviruses. It suggests that the long-held belief in extensive virus-host codiversification may have been substantially overestimated, paving the way for a reevaluation of how we understand, predict, and potentially control the spread of these viruses.

Strengths:

The study is conceptually robust, and its conclusions are convincing.

Weaknesses:

The authors could only use the "undated" model in ALE, with the dated method (which only allows time-consistent transfers) failing on their dataset. The authors did attempt to address this issue in the revision, albeit with limited success.

Reviewer #2 (Public Review):

Summary:

This article explores the regenerative effects of recombinant PTH analogues on osteogenesis.

Strengths:

Although PTH has known to induce the activity of osteoclasts, accelerating bone resorption, paradoxically its intermittent use has become a common treat for osteoporosis. Previous studies successfully demonstrated this phenomenon in vivo, but most of them used rodent animal models, inevitably having a limitation. In this article, the authors tried to address this, using a beagle model, and assessed the osseointegrative effect of recombinant PTH analogues. As a result, the authors clearly observed the regenerative effects of PTH analogues, and compared the efficacy, using histologic, biochemical, and radiologic measurement for surgical-endocrinal combined large animal models. The data seem to be solid, and has potential clinical implications.

Weaknesses:

All the issues that I raised have been resolved in the revision process.

Overall, this paper is well-written and has clarity and consistency for a broader readership.

Reviewer #2 (Public Review):

Summary:

The article aims to describe a novel methodology for the study of brain organization, in comparison to fMRI functional connectivity, under rest vs. controlled pharmacological stimulation.

Strengths:

Solid study design with pharmacological stimulation applied to assess the biological significance of functional and (novel) molecular connectivity estimates.

Provides relevant information on the multivariate organization of serotoninergic system in the brain.

Provides relevant information on the sensitivity of traditional (univariate PET analysis, fMRI functional connectivity) and novel (molecular connectivity) methods in measuring pharmacological effects on brain function.

Weaknesses:

While the study protocol is referenced in the paper, it would be useful to at least report whether the study uses bolus, constant infusion, or a combination of the two and the duration of the frames chosen for reconstruction. Minimal details on anesthesia should also be reported, clarifying whether an interaction between the pharmacological agent for anesthesia and MDMA can be expected (whole-brain or in specific regions).

Some terminology is used in a bit unclear way. E.g. "seed-based" usually refers to seed-to-voxel and not ROI-to-ROI analysis, or e.g. it is a bit confusing to have IC1 called SERT network when in fact all ICs derived from DASB data are SERT networks. Perhaps a different wording could be used (IC1 = SERT xxxxx network; IC2= SERT salience network) .

The limited sample size for the rats undergoing pharmacological stimulation which might make the study (potentially) not particularly powerful. This could not be a problem if the MDMA effect observed is particularly consistent across rats. Information on inter-individual variability of FC, MC, and BPND could be provided in this regard.

Reviewer #2 (Public Review):

In this study, the authors leverage a large public dataset of intracranial EEG (the University of Pennsylvania RAM repository) to examine electrophysiologic network dynamics involving the participation of salience, frontoparietal, and default mode networks in the completion of several episodic memory tasks. They do this through a focus on the anterior insula (AI; salience network), which they hypothesize may help switch engagement between the DMN and FPN in concert with task demands. By analyzing high-gamma spectral power and phase transfer entropy (PTE; a putative measure of information "flow"), they show that the AI shows higher directed PTE towards nodes of both the DMN and FPN, during encoding and recall, across multiple tasks. They further demonstrate that high-gamma power in the PCC/precuneus is decreased relative to the AI during memory encoding. They interpret these results as evidence of "triple-network" control processes in memory tasks, governed by a key role of the AI.

I commend the authors on leveraging this large public dataset to help contextualize network models of brain function with electrophysiological mechanisms - a key problem in much of the fMRI literature. I also appreciate that the authors emphasized replicability across multiple memory tasks, in an effort to demonstrate conserved or fundamental mechanisms that support a diversity of cognitive processes. However, I believe that their strong claims regarding causal influences within circumscribed brain networks cannot be supported by the evidence as presented. In my efforts to clearly communicate these inadequacies, I will suggest several potential analyses for the authors to consider that might better link the data to their central hypotheses.

(1) As a general principle, the effects that the authors show - both in regards to their high-gamma power analysis and PTE analysis - do not offer sufficient specificity for a reader to understand whether these are general effects that may be repeated throughout the brain, or whether they reflect unique activity to the networks/regions that are laid out in the Introduction's hypothesis. This lack of specificity manifests in several ways, and is best communicated through examples of control analyses.

First, the PTE analysis is focused solely on the AI's interactions with nodes of the DMN and FPN; while it makes sense to focus on this putative "switch" region, the fact that the authors report significant PTE from the AI to nodes of both networks, in encoding and retrieval, across all tasks and (crucially) also at baseline, raises questions about the meaningfulness of this statistic. One way to address this concern would be to select a control region that would be expected to have little/no directed causal influence on these networks and repeat the analysis. Alternatively (or additionally), the authors could examine the time course of PTE as it evolves throughout an encoding/retrieval interval, and relate that to the timing of behavioral events or changes in high-gamma power. This would directly address an important idea raised in their own Discussion, "the AI is well-positioned to dynamically engage and disengage with other brain areas."

Second, the authors state that high-gamma suppression in the PCC/precuneus relative to the AI is an anatomically specific signature that is not present in the FPN. This claim does not seem to be supported by their own evidence as presented in the Supplemental Data (Figures S2 and S3), which to my eye show clear evidence of relative suppression in the MFG and dPPC (e.g. S2a and S3a, most notably) which are notated as "significant" with green bars. I appreciate that the magnitude of this effect may be greater in the PCC/precuneus, but if this is the claim it should be supported by appropriate statistics and interpretation.

(2) I commend the authors on emphasizing replicability, but I found their Bayes Factor (BF) analysis to be difficult to interpret and qualitatively inconsistent with the results that they show. For example, the authors state that BF analysis demonstrates "high replicability" of the gamma suppression effect in Figure 3a with that of 3c and 3d. While it does appear that significant effects exist across all three tasks, the temporal structure of high gamma signals appears markedly different between the two in ways that may be biologically meaningful. Moreover, it appears that the BF analysis did not support replicability between VFR and CATVFR, which is very surprising; these are essentially the same tasks (merely differing in the presence of word categories) and would be expected to have the highest degree of concordance, not the lowest. I would suggest the authors try to analytically or conceptually reconcile this surprising finding.

To aid in interpretability, it would be extremely helpful for the authors to assess across-task similarity in high-gamma power on a within-subject basis, which they are well-powered to do. For example, could they report the correlation coefficient between HGP timecourses in paired-associates versus free-recall tasks, to better establish whether these effects are consistent on a within-subject basis? This idea could similarly be extended to the PTE analysis. Across-subject correlations would also be a welcome analysis that may provide readers with better-contextualized effect sizes than the output of a Bayes Factor analysis.

Reviewer #2 (Public Review):

This paper investigates an interesting question: how do fish react to and avoid thermal disturbances from the optimum that occur on fast timescales? Previous work has identified potential strategies for warm avoidance in fish on short timescales while strategies for cold avoidance are far more elusive. The work combines a clever experimental paradigm with careful analysis to show that trout parr avoid cold water by limiting excursions across a warm-cold thermal interface. While I found the paper interesting and convincing overall, there are a few omissions and choices in the presentation that limit interpretability and clarity.

A main question concerns the thermal interface itself. The authors track this interface using a blue dye that is mixed in with either colder or warmer water before a gate is opened that leads to gravitational flow overlaying the two water temperatures. The dye likely allows to identify convective currents which could lead to rapid mixing of water temperatures. However, it is less clear whether it accurately reflects thermal diffusion. This is problematic as the authors identify upward turning behavior around the interface which appears to be the behavioral strategy for avoiding cold water but not warm water. Without knowing the extent of the gradient across the interface, it is hard to know what the fish are sensing. The authors appear to treat the interface as essentially static, leading them to the conclusion that turning away before the interface is reached is likely related to associative learning. However, thermal diffusion could very likely create a gradient across centimeters which is used as a cue by the fish to initiate the turn. In an ideal world, the authors would use a thermal camera to track the relationship between temperature and the dye interface. Absent that, the simulation that is mentioned in passing in the methods section should be discussed in detail in the main text, and results should be displayed in Figure 1. Error metrics on the parameters used in the simulation could then be used to identify turns in subsequent figures that likely are or aren't affected by a gradient formed across the interface.

The authors assume that the thermal interface triggers the upward-turning behavior. However, an alternative explanation, which should be discussed, is that cold water increases the tendency for upward turns. This could be an adaptive strategy since for temperatures > 4C turning swimming upwards is likely a good strategy to reach warmer water.

The paper currently also suffers from a lack of clarity which is largely created by figure organization. Four main and 38 supplemental figures are very unusual. I give some specific recommendations below but the authors should decide which data is truly supplemental, versus supporting important points made in the paper itself. There also appear to be supplemental figures that are never referenced in the text which makes traversing the supplements unnecessarily tedious.<br /> The N that was used as the basis for statistical tests and plots should be identified in the figures to improve interpretability. To improve rigor, the experimental procedures should be expanded. Specifically, the paper uses two thermal models which are not detailed at all in the methods section.

Reviewer #2 (Public Review):

Summary:

Burdge, Juhmka, et al describe the development and validation of a new automated system for applying plantar stimuli in rodent somatosensory behavior tasks. This platform allows the users to run behavior experiments remotely, removing experimenter effects on animals and reducing variability in the manual application of stimuli. The system integrates well with other automated analysis programs that the lab has developed, providing a complete package for standardizing behavior data collection and analysis. The authors present extensive validations of the system against manual stimulus application. Some proof of concept studies also show how the system can be used to better understand the effect of experimenters on behavior and the effects of how stimuli are presented on the micro features of the animal withdrawal response.

Strengths:

If widely adopted, ARM has the potential to reduce variability in plantar behavior studies across and within labs and provide a means to standardize results. The system is well-validated and results clearly and convincingly presented. Most claims are well supported by experimental evidence.

Weaknesses:

ARM seems like a fantastic system that could be widely adopted, but no details are given on how a lab could build ARM, thus its usefulness is limited.

The ARM system appears to stop short of hitting the desired forces that von Frey filaments are calibrated toward (Figure 2). This may affect the interpretation of results.

The authors mention that ARM generates minimal noise; however, if those sounds are paired with stimulus presentation they could still prompt a withdrawal response. Including some 'catch' trials in an experiment could test for this.

The experimental design in Figure 2 is unclear- did each experimenter have their own cohort of 10 mice, or was a single cohort of mice shared? If shared, there's some concern about repeat testing.

Reviewer #2 (Public Review):

This is a nice paper illustrating the use of equilibrium/non-equilibrium MD simulations to explore allosteric communication in the Spike protein. The results are described in detail and suggest a complex network of signal transmission patterns. The topic is not completely novel as it has been studied before by the same authors and the impact of glycosylation is moderated and localized at the furin site, so not many new conclusions emerge here. It is suggested that mutations are commonly found in the communication pathway which is interesting, but the authors fail to provide evidence that this is related to a positive selection and not simply to a random effect related to mutations at points that are not crucial for stability or function. One interesting point is the connection of the FA site with an additional site binding heme group. It will be interesting to see reversibility, i.e. removal of the ligand at this site is producing perturbation at the FA site?, does it produce other effects suggesting a cascade of allosteric effects? Finally, the paper lacks details to help reproducibility, in particular, I do not see details on D-NEMD calculations. One interesting point is the connection of the FA site with an additional site binding heme group.

Reviewer #2 (Public Review):

Summary:

In this paper, the authors describe a novel function involving the cell cycle protein kinase CDK2, which binds to TBK1 (an essential component of the innate immune response) leading to its degradation in a ubiquitin/proteasome-dependent manner. Moreover, the E3 ubiquitin ligase, Dtx4, is implicated in the process by which CDK2 increases the K48-linked ubiquitination of TBK1. This paper presents intriguing findings on the function of CDK2 in lower vertebrates, particularly its regulation of IFN expression and antiviral immunity.

Strengths:

(1) The research employs a variety of experimental approaches to address a single question. The data are largely convincing and appear to be well executed.

(2) The evidence is strong and includes a combination of in vivo and in vitro experiments, including knockout models, protein interaction studies, and ubiquitination analyses.

(3) This study significantly impacts the field of immunology and virology, particularly concerning the antiviral mechanisms in lower vertebrates. The findings provide new insights into the regulation of IFN expression and the broader role of CDK2 in immune responses. The methods and data presented in this paper are highly valuable for the scientific community, offering new avenues for research into antiviral strategies and the development of therapeutic interventions targeting CDK2 and its associated pathways.

Weaknesses:

(1) While the study focuses on fish, the broader implications for other lower vertebrates and higher vertebrates are not extensively discussed.

(2) The study heavily relies on specific fish models, which may limit the generalizability of the findings across different species.

Reviewer #2 (Public Review):

The study by Bajohr investigates the important question of whether astrocytes can generate oligodendrocytes by direct lineage conversion (DLR). The authors ectopically express three transcription factors - Sox10, Olig2 and Nkx6.2 - in cultured postnatal mouse astrocytes and use a combination of Aldh1|1-astrocyte fate mapping and live cell imaging to demonstrate that Sox10 converts astrocytes to MBP+ oligodendrocytes, whereas Olig2 expression converts astrocytes to PDFRalpha+ oligodendrocyte progenitor cells. Nkx6.2 does not induce lineage conversion. The authors use single-cell RNAseq over 14 days post-transduction to uncover molecular signatures of newly generated iOLs.

The potential to convert astrocytes to oligodendrocytes has been previously analyzed and demonstrated. Despite the extensive molecular characterization of the direct astrocyte-oligodendrocyte lineage conversion, the paper by Bajohr et al. does not represent significant progress. The entire study is performed in cultured cells, and it is not demonstrated whether this lineage conversion can be induced in astrocytes in vivo, particularly at which developmental stage (postnatal, adult?) and in which brain region. The authors also state that generating oligodendrocytes from astrocytes could be relevant for oligodendrocyte regeneration and myelin repair, but they don't demonstrate that lineage conversion can be induced under pathological conditions, particularly after white matter demyelination. Specific issues are outlined below.

(1) The authors perform an extensive characterization of Sox10-mediated DLR by scRNAseq and demonstrate a clear trajectory of lineage conversion from astrocytes to terminally differentiated MBP+ iOLCs. A similar type of analysis should be performed after Olig2 transduction, to determine whether transcriptomics of OPC induction overlaps with any phase of MBP+ oligodendrocyte induction.

(2) A complete immunohistochemical characterization of the cultures should be performed at different time points after Sox10 and Olig2 transduction to confirm OL lineage cell phenotypes.

Reviewer #2 (Public Review):

In this manuscript, Moyse et. al. investigated the origins and potential functions of distinct populations of mononuclear phagocytes (MNPS) in the heart of developing and adult zebrafish. First, the authors demonstrate that the embryonic zebrafish heart contains macrophages early in development and that mpeg1.1 and csf1ra expressing macrophages vary across time and location and present that cardiac tissue macrophages (cTMs) in the juvenile heart are derived by primitive hematopoiesis. By combining the two transgenes, the authors demonstrate that there are 3 distinct (later determined to be 4) subpopulations of MNPs in adult hearts and that the distribution of these subtypes is distinct within the heart consistent with differing distributions of primitive and definitive macrophages in mammalian hearts. Further analysis of these populations demonstrates distinct morphologies of the subpopulations and analysis of markers conserved in mammals demonstrates distinct expression profiles as well. The authors go on to demonstrate that these subpopulations also demonstrate distinct behaviors via ex-vivo imaging. Lastly, the authors investigated the roles of these subpopulations in a model of cardiac injury in adult zebrafish and demonstrated that primitive-derived cTMs proliferate after injury consistent with mammalian models and that the proliferation of these macrophages likely results in reduced scarring in csf1ra mutants which have reduced recruitment of pro-inflammatory definitive macrophages. The data presented in this manuscript provides solid evidence that zebrafish MNPs behave consistently with those in mammals and further solidifies the use of zebrafish models as a useful tool in studying the role of these cells in cardiac repair and regeneration.

The data presented in this manuscript strongly supports the conclusions made by the authors and utilizes novel techniques. The authors appear to have achieved the goals they set out to investigate. The use of ex-vivo imaging to visualize the movement of these macrophage populations within the heart is especially compelling. The combined use of commonly used transgenic reporters for zebrafish macrophages is a very nice use of existing tools to address new questions and highlight the distinct populations of macrophages. While the overall manuscript is very strong and is likely to have a great impact on the field, there are a few weaknesses that should be addressed prior to acceptance:

(1) The reasoning for N used in many of these experiments is not addressed, nor is the question of the number of times experiments were performed. For purposes of rigor and reproducibility, these questions should be addressed in the methods.

(2) In investigating homologs of zebrafish and mammalian genes, the inclusion of additional classical markers and novel markers of subpopulations highlighted in numerous recent studies using single-cell RNA sequencing would greatly add to the impact.

(3) The description of the RT-PCR experiment is not included in the methods. Detailed methods should be provided including probe sequences. Additionally, a quantitative method of presenting this data would strengthen the conclusions presented here as well as the inclusion of additional markers as discussed previously.

DOI: 10.1038/s41467-023-42306-2

Resource: RRID:BDSC_25830

Curator: @maulamb

SciCrunch record: RRID:BDSC_25830

What is this?

DOI: 10.1038/s42255-021-00443-2

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @mpairish

SciCrunch record: RRID:SCR_006457

What is this?

Reviewer #2 (Public Review):

Summary:

In this study, Liu et al. explore the interplay between G-quadruplexes (G4s) and R-loops. The authors developed novel techniques, HepG4-seq and HBD-seq, to capture and map these nucleic acid structures genome-wide in human HEK293 cells and mouse embryonic stem cells (mESCs). They identified dynamic, cell-type-specific distributions of co-localized G4s and R-loops, which predominantly localize at active promoters and enhancers of transcriptionally active genes. Furthermore, they assessed the role of helicase Dhx9 in regulating these structures and their impact on gene expression and cellular functions.

The manuscript provides a detailed catalogue of the genome-wide distribution of G4s and R-loops. However, the conceptual advance and the physiological relevance of the findings are not obvious. Overall, the impact of the work on the field is limited to the utility of the presented methods and datasets.

Strengths:

(1) The development and optimization of HepG4-seq and HBD-seq offer novel methods to map native G4s and R-loops.

(2) The study provides extensive data on the distribution of G4s and R-loops, highlighting their co-localization in human and mouse cells.

(3) The study consolidates the role of Dhx9 in modulating these structures and explores its impact on mESC self-renewal and differentiation.

Weaknesses:

(1) The specificity of the biotinylation process and potential off-target effects are not addressed. The authors should provide more data to validate the specificity of the G4-hemin.

(2) Other methods exploring a catalytic dead RNAseH or the HBD to pull down R-loops have been described before. The superior quality of the presented methods in comparison to existing ones is not established. A clear comparison with other methods (BG4 CUT&Tag-seq, DRIP-seq, R-CHIP, etc) should be provided.

(3) Although the study demonstrates Dhx9's role in regulating co-localized G4s and R-loops, additional functional experiments (e.g., rescue experiments) are needed to confirm these findings.

(4) The manuscript would benefit from a more detailed discussion of the broader implications of co-localized G4s and R-loops.

(5) The manuscript lacks appropriate statistical analyses to support the major conclusions.

(6) The discussion could be expanded to address potential limitations and alternative explanations for the results.

Reviewer #2 (Public Review):

In their manuscript, Medina and colleagues investigate transcriptional differences between mild and severe SARS-CoV-2 infections. Their analyses are very comprehensive incorporating a multitude of bioinformatics tools ranging from PCA plots, GSEA and DEG analysis, protein-protein interaction network, and weighted correlation network analyses. They conclude that in mild COVID-19 infection NK cell functionality is compromised and this is connected to cytokine interactions and Th1/Th2 cell differentiation pathways cross-talk, bridging the innate and the adaptive arms of the immune system. The authors successfully recruited participants with both mild and severe COVID-19 between November 2020 to May 2021. The analyzed cohort is gender and acceptably age-matched and the results reported are promising. Signatures associated with NK cell cytotoxicity in mild and neutrophil functions in the severe group during acute infection are the chief findings reported in this manuscript.

Comments on revised version:

The authors responded appropriately to the previous review critiques.

Reviewer #4 (Public Review):

Summary:

Knecht et al. elucidate a Type VI Secretion System (T6SS) effector-immunity pair in Proteus mirabilis. They demonstrate that the effector protein RdnE exhibits DNase activity in vitro and induces toxicity when ectopically expressed in cells, the latter being neutralized by the cognate immunity protein RdnI. The authors identify major regions within RdnI necessary for the interaction and neutralization of RdnE. Notably, they report cross-talk where both cognate and non-cognate RdnI proteins can neutralize RdnE, mitigating its fitness advantage in bacterial co-swarm assays. A comprehensive metagenomic analysis revealed an abundance of rdnI over rdnE genes in most gut samples, suggesting a potential role of rdnI in providing a fitness advantage against bacteria encoding for RdnE effector.

Strengths:

The authors successfully combined biochemical and microbiological experiments with bioinformatics analysis to advance the understanding of the T6SS-mediated population dynamics in bacteria. The co-swarm functional assay is of particular interest as it demonstrates how bacterial strains carrying only rdnI immunity genes could potentially compete in the same niche with other species armed with toxic rdnE effector genes. The manuscript is well-written, and the figures are self-explanatory.

Weaknesses:

(1) How would the authors explain the discrepancy observed in Figure 4 G and Figure 4 S3 B where two RdnI proteins from Prevotella and Pseudomonas genera do not bind to RdnE_Proteus in BACTH, whereas they co-elute with a RdnE_Proteus-FLAG with efficiency comparable to the cross-neutralizing RdnI_Rothia? Similarly, the interaction results obtained in BACTH with RdnI truncate (Figure 4E) or chimeric RdnI (Figure 4I, lane 4) could be a result of an overexpressed T18-fusion variant.<br /> Alternative in vitro protein binding assay would be beneficial.

(2) Based on the bioinformatic analysis the Rothia and Prevotella species harboring rdnE/I genes co-occurred in 5% of metagenomes tested, suggesting that these bacteria could come into contact. The manuscript would benefit greatly if authors demonstrated that RdnI proteins from Rothia or Prevotella could cross-neutralize its own and its 'neighbor' RdnE effectors, for example in an E. coli viability assay. The cross-neutralizing co-swarming results (Figure 4F) could also be further validated in viability assay as shown in Figure 2 S1.

(3) Little is known about whether RdnE is delivered via T6SS as a full-length protein or as the shorter C-terminal fragment. There is a possibility that immunity proteins could recognize RdnE regions beyond the C-terminal 138 amino acids that authors used in their in vitro assays.

Reviewer #2 (Public Review):

This is an interesting manuscript where a CA-only CG model (Mpipi) was used to examine the critical temperature (Tc) of phase separation of a set of 140 variants of prion-like low complexity domains (PLDs). The key result is that Tc of these PLDs seems to have a linear dependence on substitutions of various sticker and space residues. This is potentially useful for estimating the Tc shift when making novel mutations of a PLD. However, I have strong reservations about the significance of this observation as well as some aspects of the technical detail and writing of the manuscript.

(1) Writing of the manuscript: The manuscript can be significantly shortened with more concise discussions. The current text reads as very wordy in places. It even appears that the authors may be trying a bit too hard to make a big deal out of the observed linear dependence.

The manuscript needs to be toned done to minimize self-promotion throughout the text. Some of the glaring examples include the wording "unprecedented", "our research marks a significant milestone in the field of computational studies of protein phase behavior ..", "Our work explores a new framework to describe, quantitatively, the phase behavior ...", and others.

There is really little need to emphasize the need to manage a large number of simulations for all 140 variants. Yes, some thoughts need to go into designing and managing the jobs and organizing the data, but it is pretty standard in computational studies. For example, large-scale protein ligand-free energy calculations can require one to a few orders of magnitude larger number of runs, and it is pretty routine.

When discussing the agreement with experimental results on Tm, it should be noted that the values of R > 0.93 and RMSD < 14 K are based on only 16 data points. I am not sure that one should refer to this as "extended validation". It is more like a limited validation given the small data size.

Results of linear fitting shown in Eq 4-12 should be summarized in a single table instead of scattering across multiple pages.

The title may also be toned down a bit given the limited significance of the observed linear dependence.

(2) Significance and reliability of Tc: Given the simplicity of Mpipi (a CA-only model that can only describe polymer chain dimension) and the low complexity nature of PLDs, the sequence composition itself is expected to be the key determinant of Tc. This is also reflected in various mean-field theories. It is well known that other factors will contribute, such as patterning (examined in this work as well), residual structures, and conformational preferences in dilute and dense phases. The observed roughly linear dependence is a nice confirmation but really unsurprising by itself. It appears how many of the constructs deviate from the expected linear dependence (e.g., Figure 4A) may be more interesting to explore.

The assumption that all systems investigated here belong to the same universality class as a 3D Ising model and the use of Eqn 20 and 21 to derive Tc is poorly justified. Several papers have discussed this issue, e.g., see Pappu Chem Rev 2023 and others. Muthukumar and coworkers further showed that the scaling of the relevant order parameters, including the conserved order parameter, does not follow the 3D Ising model. More appropriate theoretical models including various mean field theories can be used to derive binodal from their data, such as using Rohit Pappu's FIREBALL toolset. Imposing the physics of the 3D Ising model as done in the current work creates challenges for equivalence relationships that are likely unjustified.

While it has been a common practice to extract Tc when fitting the coexistence densities, it is not a parameter that is directly relevant physiologically. Instead, Csat would be much more relevant to think about if phase separation could occur in cells.

Reviewer #2 (Public Review):

Summary:

This field experiment aimed to assess what motivates macaque monkeys to clean food items prior to consumption and the relative costs and benefits of different cleaning approaches (manually brushing sand from food versus dousing food items in water). The experiment teases apart if/how the benefits of these approaches are mediated by the amount of debris on food and the monkeys' rank in terms of the costs of consuming sand versus the time and energy required to remove it. The authors not only examined the behavioral responses of wild macaques to three conditions of food sand contamination but also tested the relative costs of consuming different levels and sizes of sand particulates. Through this, the authors propose considerations of the macaques' motivations to clean food and the balance they take in energetic gains from consuming food versus the costs of cleaning food and consuming sand. Their data reveal that food washing is more effective in removing sand, but more costly than manually brushing off sand. This study also revealed that only mid-ranked monkeys washed their food, while high and low-ranked monkeys were more likely to remove sand via brushing it off food with their hands.

Strengths:

This study provides a very in-depth consideration of the motivations of macaques to clean their food, and the relative costs and benefits of different food cleaning techniques. Not only did the study test the behavior of wild macaques via a simple yet elegant field study, but they also performed a detailed analysis of the sand particulates to understand the level of potential tooth wear that consuming it could result in. By relying on a wild group of macaques that have been part of a long-term study site, the team also had detailed behavioral data on the population to allow for rank assessments of the animals. This comprehensive study provides important foundational information for a better understanding of how and why macaques clean food, that inform existing and future considerations of this as a potential cultural behavior.

Weaknesses:

As currently written, the paper does not provide sufficient background on this population of animals and their prior demonstrations of food-cleaning behavior or other object-handling behaviors (e.g., stone handling). Moreover, the authors' conclusions focus on the behavior of high-ranked animals, but subordinate animals also showed similar behavioral patterns and they should be considered in more detail too.

DOI: 10.1038/s41419-020-02875-2

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

Curator: @bandrow

SciCrunch record: RRID:BDSC_458

What is this?

DOI: 10.1038/s41598-020-68601-2

Resource: (BDSC Cat# 33832,RRID:BDSC_33832)

Curator: @bandrow

SciCrunch record: RRID:BDSC_33832

What is this?

DOI: 10.1038/s41467-021-27815-2

Resource: (BDSC Cat# 36304,RRID:BDSC_36304)

Curator: @mpairish

SciCrunch record: RRID:BDSC_36304

What is this?

DOI: 10.1038/s41467-020-15593-2

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @mzhang007

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41598-020-64247-2

Resource: (BDSC Cat# 5905,RRID:BDSC_5905)

Curator: @bandrow

SciCrunch record: RRID:BDSC_5905

What is this?

DOI: 10.1038/s41592-022-01417-2

Resource: BDSC_76169

Curator: @mpairish

SciCrunch record: RRID:BDSC_76169

What is this?

Reviewer #2 (Public Review):

Summary:

The paper sets out to understand the mechanisms underlying the colonization and degradation of marine particles using a natural Vibrio isolate as a model. The data are measurements of motility and gene expressing using microfluidic devices and RNA sequencing. The results reveal that degradation products of alginate do stimulate motility but not chemotaxis. In contrast, alginate itself (the polymer) does stimulate chemotaxis. Further, the dispersal from degrading alginate is density dependent, increasing at higher density. The evidence for these claims are strong. From these the authors propose a narrative (Fig. 6) for growth and dispersal cycles in this system. The idea is that cells colonize and degrade alginate, this degradation stimulates motility and dispersal followed by chemotaxis to a new alginate source. This complete narrative has modest support in the data. A quantitative description of these dynamics awaits future studies.

Strengths:

The microfluidic measurements are the central strength of the paper. The density dependence claim is qualitatively supported by the data. The motility and chemotaxis claims are also well supported by the data. The presentation of the experiment and results are well done. The study serves to motivate a unifying picture of growth and dispersal in marine systems. This is a key process in the global carbon cycle.

Weaknesses:

Perhaps not a weakness, but a glimmer that this is not yet the full story. The RNA expression data show alginate lyase expression in response to digested alginate which is unexpected given the narrative articulated above. Why express lyases while leaving the polymer patch via motility? This question is addressed in the Discussion. A holistic and quantitative picture of the proposed process in Figure 6 awaits additional studies.

Reviewer #2 (Public Review):

Kislinger et al. present a method permitting a targeted, multi-scale ultrastructural imaging approach to bridge the resolution gap between large-scale scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The key methodological development consists of an approach to recover sections of resin-embedded material produced by Automated Tape Collecting Ultramicrotomy (ATUM), thereby permitting regions of interest identified by serial section SEM (ATUM-SEM) screening to be subsequently re-examined at higher resolution by TEM tomography (ATUM-Tomo). The study shows that both formvar and permanent marker coatings are in principle compatible with solvent-based release of pre-screened sections from ATUM tape (carbon nanotubule or Kapton tape). However, a comparative analysis of potential limitations and artifacts introduced by these respective coatings revealed permanent marker to provide a superior coating; permanent marker coatings are more easily and reliably applied to tape with only minor contaminants affecting the recovered section-tape interface with negligible influence on tomogram interpretation. Convincing proof-of-principle is provided by integrating this novel ATUMTomo technique into a technically impressive correlated light and electron microscopy (CLEM) approach specifically tailored to investigate ultrastructural manifestations of trauma-induced changes in blood-brain barrier permeability.

Strengths

Schematics and figures are very well-constructed, illustrating the workflow in a logical and easily interpretable manner. Light and electron microscope image data are of excellent quality, and the efficacy of the ATUM-Tomo approach is evidenced by a qualitative assessment of ATUM-SEM performance using coated tape variants and a convincing correlation between scanning and transmission electron microscopy imaging modalities. Potential ultrastructural artifacts induced via solvent exposure and any subsequent mechanical stress incurred during section detachment were thoroughly and systematically investigated using appropriate methods and reported with commendable transparency. In summary, the presented data convincingly support the claims of the study. A major strength of this work includes its general applicability to a broad range of biological questions and ultrastructural targets demanding resolutions exceeding that obtained via serial section and destructive block-face imaging approaches alone. The level of methodological detail provided is sufficient for replication of the ATUM-Tomo technique in other laboratories. Consequently, this relatively simple and cost-effective technique is widely adoptable by electron microscopy laboratories, and its integration into existing ATUM-SEM workflows supports a versatile and non-destructive imaging regime enabling high-resolution details of targeted structures to be interpreted within anatomical and subcellular contexts.

Weaknesses

I find no significant weaknesses in the current version of the manuscript.

Reviewer #2 (Public Review):

Sex peptide (SP) transferred during mating from male to female induces various physiological responses in the receiving female. Among those, the increase in oviposition and decrease in sexual receptivity are very remarkable. Naturally, a long standing and significant question is the identity of the underlying sex peptide target neurons that express the SP receptor and are underlying these responses. Identification of these neurons will eventually lead to the identification of the underlying neuronal circuitry.

The Soller lab has addressed this important question already several years ago (Haussmann et al. 2013), using relevant GAL4-lines and membrane-tethered SP. The results already showed that the action of SP on receptivity and oviposition is mediated by different neuronal subsets and hence can be separated. The GAL4-lines used at that time were, however, broad, and the individual identity of the relevant neurons remained unclear.

In the present paper, Nallasivan and colleagues carried this analysis one step further, using new intersectional approaches and transsynaptic tracing.

Strength:

The intersectional approach is appropriate and state-of-the art. The analysis is a very comprehensive tour-de-force and experiments are carefully performed to a high standard. The authors also produced a useful new transgenic line (UAS-FRTstopFRT mSP). The finding that neurons in the brain (head) mediate the SP effect on receptivity, while neurons in the abdomen and thorax (ventral nerve cord or peripheral neurons) mediate the SP effect on oviposition, is a significant step forward in the endavour to identify the underlying neuronal networks and hence a mechanistic understanding of SP action. Though this result is not entirely unexpected, it is novel as it was not shown before.

Weakness:

Though the analysis identifies a small set of neurons underlying SP responses, it does not go the last step to individually identify at least a few of them. The last paragraph in the discussion rightfully speculates about the neurochemical identity of some of the intersection neurons (e.g. dopaminergic P1 neurons, NPF neurons). At least these suggested identities could have been confirmed by straight-forward immunostainings agains NPF or TH, for which antisera are available. Moreover, specific GAL4 lines for NPF or P1 or at least TH neurons are available which could be used to express mSP to test whether SP activation of those neurons is sufficient to trigger the SP effect.

Reviewer #2 (Public Review):

Summary:

In this study, the authors show that leaf exposure to leafhopper males is required for female attraction in the SAP54-expressing plant. They clarify how SAP54, by degrading SVP, suppresses biotic stress response pathways in leaves exposed to the males, thus facilitating female attraction and plant colonization.

Strengths:

This study suggests the possibility that the attraction of insect vectors to leaves is the major function of SAP54, and the induction of the leaf-like flowers may be a side-effect of the degradation of MTFs and SVP. It is a very surprising discovery that only male insect vectors can effectively suppress the plant's biotic stress response pathway. Although there has been interest in the phyllody symptoms induced by SAP54, the purpose, and advantage of secreting SAP54 were unknown. The results of this study shed light on the significance of secreted proteins in the phytoplasma life cycle and should be highly evaluated.

Weaknesses:

One weakness of this study is that the mechanisms by which male and female leafhoppers differentially affect plant defense responses remain unclear, although I understand that this is a future study.

The authors show that female feeding suppresses female colonization on SAP54-expressing plants. This is also an intriguing phenomenon but this study doesn't explain its molecular mechanism (Figure 7).

Reviewer #2 (Public Review):

Summary:

This is a fascinating study about the behavioral kinematics of prey capture in larvae of several fish species (zebrafish, four cichlid species, and medaka). The authors describe in great detail swimming kinematics, hunting movement, eye movement as well as prey capture kinematics across these species. One striking finding is that cichlids and zebrafish use binocular vision to hunt for prey whereas medaka uses a monocular hunting style with a sideways motion to capture prey. The behavioral variation described in this study forms a strong foundation for future studies on the mechanisms underlying variation in hunting styles.

Strengths:

In general, the paper is well-written and documents very interesting data. The authors used sophisticated analyses that help appreciate the complexity of the behaviors examined. The discussion attempts to place the paper in a broader, comparative context. Overall, this paper reveals novel insight into an important behavior across different teleost species and lays a foundation for future studies on the neural and genetic basis of these distinct swimming and hunting behaviors.

Weaknesses:

The paper is rather descriptive in nature, although more context is provided in the discussion. Most figures are great, but I think the authors could add a couple of visual aids in certain places to explain how certain components were measured.

Reviewer #2 (Public Review):

Summary:

Using a suite of techniques (e.g., RNA seq, proteomics, and functional experiments ex vivo) this paper extensively focuses on the role of PIM1/2 kinases during CD8 T-cell activation and cytokine-driven (i.e., IL-2 or IL-15) differentiation. The authors' key finding is that PIM1/2 enhances protein synthesis in response to IL-2 stimulation, but not IL-15, in CD8+ T cells. Loss of PIM1/2 made T cells less 'effector-like', with lower granzyme and cytokine production, and a surface profile that maintained homing towards secondary lymphoid tissue. The cytokines the authors focus on are IL-15 and Il-2, which drive naïve CD8 T cells towards memory or effector states, respectively. Although PIM1/2 are upregulated in response to T-cell activation and cytokine stimulation (e.g., IL-15, and to a greater extent, IL-2), using T cells isolated from a global mouse genetic knockout background of PIM1/2, the authors find that PIM1/2 did not significantly influence T-cell activation, proliferation, or expression of anything in the proteome under anti-CD3/CD28 driven activation with/without cytokine (i.e., IL-15) stimulation ex vivo. This is perhaps somewhat surprising given PIM1/2 is upregulated, albeit to a small degree, in response to IL-15, and yet PIM1/2 did not seem to influence CD8+ T cell differentiation towards a memory state. Even more surprising is that IL-15 was previously shown to influence the metabolic programming of intestinal intraepithelial lymphocytes, suggesting cell-type specific effects from PIM kinases. What the authors went on to show, however, is that PIM1/2 KO altered CD8 T cell proteomes in response to IL-2. Using proteomics, they saw increased expression of homing receptors (i.e., L-selectin, CCR7), but reduced expression of metabolism-related proteins (e.g., GLUT1/3 & cholesterol biosynthesis) and effector-function related proteins (e.g., IFNy and granzymes). Rather neatly, by performing both RNA-seq and proteomics on the same IL-2 stimulated WT vs. PIM1/2 KO cells, the authors found that changes at the proteome level were not corroborated by differences in RNA uncovering that PIM1/2 predominantly influence protein synthesis/translation. Effectively, PIM1/2 knockout reduced the differentiation of CD8+ T cells towards an effector state. In vivo adoptive transfer experiments showed that PIM1/2KO cells homed better to secondary lymphoid tissue, presumably owing to their heightened L-selectin expression (although this was not directly examined).

Strengths:

Overall, I think the paper is scientifically good, and I have no major qualms with the paper. The paper as it stands is solid, and while the experimental aim of this paper was quite specific/niche, it is overall a nice addition to our understanding of how serine/threonine kinases impact T cell state, tissue homing, and functionality. Of note, they hint towards a more general finding that kinases may have distinct behaviour in different T-cell subtypes/states. I particularly liked their use of matched RNA-seq and proteomics to first suggest that PIM1/2 kinases may predominantly influence translation (then going on to verify this via their protein translation experiment - although I must add this was only done using PIM kinase inhibitors, not the PIM1/2KO cells). I also liked that they used small molecule inhibitors to acutely reduce PIM1/2 activity, which corroborated some of their mouse knockout findings - this experiment helps resolve any findings resulting from potential adaptation issues from the PIM1/2 global knockout in mice but also gives it a more translational link given the potential use of PIM kinase inhibitors in the clinic. The proteomics and RNA seq dataset may be of general use to the community, particularly for analysis of IL-15 or IL-2 stimulated CD8+ T cells.

Weaknesses:

It would be good to perform some experiments in human T cells too, given the ease of e.g., the small molecule inhibitor experiment. Would also be good for the authors to include a few experiments where PIM1/2 have been transduced back into the PIM1/2 KO T cells, to see if this reverts any differences observed in response to IL-2 - although the reviewer notes that the timeline for altering primary T cells via lentivirus/CRISPR may be on the cusp of being practical such that functional experiments can be performed on day 6 after first stimulating T cells. Other experiments could also look at how PIM1/2 KO influences the differentiation of T cell populations/states during ex vivo stimulation of PBMCs or in vivo infection models using (high-dimensional) flow cytometry (rather than using bulk proteomics/RNA seq which only provide an overview of all cell combined). Alongside this, performing a PCA of bulk RNA seq/proteomes or Untreated vs. IL-2 vs. IL-15 of WT and PIM1/2 knockout T cells would help cement their argument in the discussion about PIM1/2 knockout cells being distinct from a memory phenotype.

DOI: 10.1038/s41467-022-32400-2

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @mpairish

SciCrunch record: RRID:SCR_006457

What is this?

Reviewer #2 (Public Review):

Summary and strengths:<br /> This massive paper describes the identity and connectivity of neurons reconstructed from a volumetric EM image volume of the ventral nerve cord (VNC) of a male fruit fly. The segmentation of the EM data was described in one companion paper; the classification of the neurons entering the VNC from the brain (descending neurons or DNs) and the motor neurons leaving the VNC was described in a second companion paper. Here, the authors describe a system for annotating the remaining neurons in the VNC, which include intrinsic neurons, ascending neurons, and sensory neurons, representing the vast majority of neurons in the dataset. Another fundamental contribution of this paper is the identification of the developmental origins (hemilineage) of each intrinsic neuron in the VNC. These comprehensive hemilineage annotations can be used to understand the relationship between development and circuit structure, provide insight into neurotransmitter identity, and facilitate comparisons across insect species.Many sensory neurons are also annotated by comparison to past literature. Overall, defining and applying this annotation system provides the field with a standard nomenclature and resource for future studies of VNC anatomy, connectivity, and development. This is a monumental effort that will fundamentally transform the field of Drosophila neuroscience and provide a roadmap for similar connectomic studies in other organisms.

Weaknesses:<br /> Despite the significant merit of these contributions, the manuscript is challenging to read and comprehend. In some places, it seems to be attempting to comprehensively document everything the authors found in this immense dataset. In other places, there are gaps in scholarship and analysis. As it is currently constructed, I worry that the manuscript will intimidate general readers looking for an entry point to the system, and ostracize specialized readers who are unable to use the paper as a comprehensive reference due to its confusing organization.

The bulk of the 559 pages of the submitted paper is taken up by a set of dashboard figures for each of ~40 hemilineages. Formatting the paper as an eLife publication will certainly help condense these supplemental figures into a more manageable format, but 68 primary figures will remain, and many of these also lack quality and clarity. Without articulating a clear function for each plot, it is hard to know what the authors missed or chose not to show. As an example, many of the axis labels indicate the hemilineage of a group of neurons, but are ordered haphazardly and so small as to be illegible; if the hemilineage name is too small, and in a bespoke order for that data, then is the reader meant to ignore the specific hemilineage labels?

The text has similar problems of emphasis. It is often meandering and repetitive. Overlapping information is found in multiple places, which causes the paper to be much longer than it needs to be. For example, the concept of hemilineages is introduced three times before the subtitle "Introduction to hemilineage-based organisation". When cell typing is introduced, it is unclear how this relates to serial motif, hemilineage, etc; "Secondary hemilineages" follow the Cell typing title. Like the overwhelming number of graphical elements, this gives the impression that little attention has been paid to curating and editing the text. It is unclear whether the authors intend for the paper to be read linearly or used as a reference. In addition, descriptions of the naming system are often followed by extensive caveats and exceptions, giving the impression that the system is not airtight and possibly fluid. At many points, the text vacillates between careful consideration of the dataset's limitations and overly grandiose claims. These presentation flaws overshadow the paper's fundamental contribution of describing a reasonable and useful cell-typing system and placing intrinsic neurons within this framework.

References to past Drosophila literature are inconsistent and references to work from other insects are generally not included; for example, the extensive past work on leg sensory neurons in locusts, cockroaches, and stick insects. Such omissions are understandable in a situation where brevity is paramount. However, this paper adopts a comprehensive and authoritative tone that gives the reader an impression of completeness that does not hold up under careful scrutiny.

The paper accompanies the release of the MANC dataset (EM images, segmentation, annotations) through a web browser-based tool: clio.janelia.org. The paper would be improved by distilling it down to its core elements, and then encouraging readers to explore the dataset through this interactive interface. Streamlining the paper by removing extraneous and incomplete analyses would provide the reader with a conceptual or practical framework on which to base their own queries of the connectome.

Reviewer #2 (Public Review):

In this research, Zhang et al. have pioneered the creation of an advanced organoid culture designed to emulate the intricate characteristics of endometrial tissue during the crucial Window of Implantation (WOI) phase. Their method involves the incorporation of three distinct hormones into the organoid culture, coupled with additives that replicate the dynamics of the menstrual cycle. Through a series of assays, they underscore the striking parallels between the endometrial tissue present during the WOI and their crafted organoids. Through a comparative analysis involving historical endometrial tissue data and control organoids, they establish a system that exhibits a capacity to simulate the intricate nuances of the WOI.

The authors made a commendable effort to address the majority of the statements. Developing an endometrial organoid culture methodology that mimics the window of implantation is a game-changer for studying the implantation process. However, the authors should strive to enhance the results to demonstrate how different WOI organoids are from SEC organoids, ensuring whether they are worth using in implantation studies, or a proper demonstration using implantation experiments.

Reviewer #2 (Public Review):

The current manuscript undoubtedly demonstrates that JAG1 can induced osteogenesis via non-canonical signaling. In fact, using the mouse-calvarial critical defect model, the authors have clearly shown the anabolic regenerative effect of JAG1 in via non-canonical pathways. Exploring the molecular mechanisms, the authors have shown that non-canonically JAG1 is regulating multiple pathways including STAT5, AKT, P38, JNK, NF-ĸB, and p70 S6K, which together possibly culminate to the activation of p70 S6K. In summary these findings have significant implications in designing new approaches for bone regenerative research.

Reviewer #3 (Public Review):<br /> This study seeks to investigate one aspect of disparity in academia: how gender balance in a discipline is valued in terms of evaluated research quality score and funding success. This is important in understanding disparities within academia.<br /> This study uses publicly available data to investigate covariation between gender balance in an academic discipline and:<br /> individual research quality scores of New Zealand academics as evaluated by one of 14 broader subject panels.<br /> [ii] funding success in Australia, Canada, Europe, UK.

The authors have addressed the concerns I had about the original version

Reviewer #2 (Public Review):

Summary:

This manuscript from Lee-Odegard et al reports proteomic profiling of exercise plasma in humans, leading to the discovery of CD300LG as a secreted exercise-inducible plasma protein. Correlational studies show associations of CD300LG with glycemic traits. Lastly, the authors query available public data from CD300LG-KO mice to establish a causal role for CD300LG as a potential link between exercise and glucose metabolism. However, the strengths of this manuscript were balanced by the moderate to major weaknesses. Therefore in my opinion, while this is an interesting study, the conclusions remain preliminary and are not fully supported by the experiments shown so far.

Strengths:

(1) Data from a well-phenotyped human cohort showing exercise-inducible increases in CD300LG.

(2) Associations between CD300LG and glucose and other cardiometabolic traits in humans, that have not previously been reported.

(3) Correlation to CD300LG mRNA levels in adipose provides additional evidence for exercise-inducible increases in CD300LG.

Weaknesses:

(1) CD300LG is by sequence a single-pass transmembrane protein that is exclusively localized to the plasma membrane. How CD300LG can be secreted remains a mystery. More evidence should be provided to understand the molecular nature of circulating CD300LG. Is it full-length? Is there a cleaved fragment? Where is the epitope where the o-link is binding to CD300LG? Does transfection of CD300LG to cells in vitro result in secreted CD300LG?

(2) There is a growing recognition of specificity issues with both the O-link and somalogic platforms. Therefore it is critical that the authors use antibodies, targeted mass spectrometry, or some other methods to validate that CD300LG really is increased instead of just relying on the O-link data.

(3) It is insufficient simply to query the IMPC phenotyping data for CD300LG; the authors should obtain the animals and reproduce or determine the glucose phenotypes in their own hands. In addition, this would allow the investigators to answer key questions like the phenotype of these animals after a GTT, whether glucose production or glucose uptake is affected, whether insulin secretion in response to glucose is normal, effects of high-fat diet, and other standard mouse metabolic phenotyping assays.

(4) I was unable to find the time point at which plasma was collected at the 12-week time point. Was it immediately after the last bout of exercise (an acute response) or after some time after the training protocol (trained state)?

Reviewer #2 (Public Review):

Summary:

In this manuscript, van der Kroeg et al have developed a method for creating 3D cortical organoids using iPSC-derived neural progenitor cells in 384-well plates, thus scaling down the neural organoids to adherent culture and a smaller format that is amenable to high throughput cultivation. These adherent cortical organoids, measuring 3 x 3 x 0.2 mm, self-organize over eight weeks and include multiple neuronal subtypes, astrocytes, and oligodendrocyte lineage cells.

Strengths:

(1) The organoids can be cultured for up to 10 months, exhibiting mature dendritic spines, axonal myelination, and robust neuronal activity.

(2) Unlike free-floating organoids, these do not develop necrotic cores, making them ideal for high-throughput drug discovery, neurotoxicological screening, and brain disorder studies.

(3) The method addresses the technical challenge of achieving higher-order neural complexity with reduced heterogeneity and the issue of necrosis in larger organoids. The method presents a technical advance in organoid culture.

(4) The method has been demonstrated with multiple cell lines which is a strength.

(5) The manuscript provides high-quality immunostaining for multiple markers.

Weaknesses:

(1) Direct head-to-head comparison with standard organoid culture seems to be missing and may be valuable for benchmarking, ie what can be done with the new method that cannot be done with standard culture and vice versa, ie what are the aspects in which new method could be inferior to the standard.

(2) It would be important to further benchmark the throughput, ie what is the success rate in filling and successfully growing the organoids in the entire 384 well plate?

(3) For each NPC line an optimal seeding density was estimated based on the proliferation rate of that NPC line and via visual observation after 6 weeks of culture. It would be important to delineate this protocol in more robust terms, in order to enable reproducibility with different cell lines and amongst the labs.

DOI: 10.1038/s42003-020-01170-2

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bpowell22

SciCrunch record: RRID:SCR_006457

What is this?

Reviewer #2 (Public Review):

Li et al present a method to extract "behaviorally relevant" signals from neural activity. The method is meant to solve a problem which likely has high utility for neuroscience researchers. There are numerous existing methods to achieve this goal some of which the authors compare their method to-thankfully, the revised version includes one of the major previous omissions (TNDM). However, I still believe that d-VAE is a promising approach that has its own advantages. Still, I have issues with the paper as-is. The authors have made relatively few modifications to the text based on my previous comments, and the responses have largely just dismissed my feedback and restated claims from the paper. Nearly all of my previous comments remain relevant for this revised manuscript. As such, they have done little to assuage my concerns, the most important of which I will restate here using the labels/notation (Q1, Q2, etc) from the reviewer response.

(Q1) I still remain unconvinced that the core findings of the paper are "unexpected". In the response to my previous Specific Comment #1, they say "We use the term 'unexpected' due to the disparity between our findings and the prior understanding concerning neural encoding and decoding." However, they provide no citations or grounding for why they make those claims. What prior understanding makes it unexpected that encoding is more complex than decoding given the entropy, sparseness, and high dimensionality of neural signals (the "encoding") compared to the smoothness and low dimensionality of typical behavioural signals (the "decoding")?

(Q2) I still take issue with the premise that signals in the brain are "irrelevant" simply because they do not correlate with a fixed temporal lag with a particular behavioural feature hand-chosen by the experimenter. In the response to my previous review, the authors say "we employ terms like 'behaviorally-relevant' and 'behaviorally-irrelevant' only regarding behavioral variables of interest measured within a given task, such as arm kinematics during a motor control task.". This is just a restatement of their definition, not a response to my concern, and does not address my concern that the method requires a fixed temporal lag and continual decoding/encoding. My example of reward signals remains. There is a huge body of literature dating back to the 70s on the linear relationships between neural and activity and arm kinematics; in a sense, the authors have chosen the "variable of interest" that proves their point. This all ties back to the previous comment: this is mostly expected, not unexpected, when relating apparently-stochastic, discrete action potential events to smoothly varying limb kinematics.

(Q5) The authors seem to have missed the spirit of my critique: to say "linear readout is performed in motor cortex" is an over-interpretation of what their model can show.

(Q7) Agreeing with my critique is not sufficient; please provide the data or simulations that provides the context for the reference in the fano factor. I believe my critique is still valid.

(Q8) Thank you for comparing to TNDM, it's a useful benchmark.

Reviewer #2 (Public Review):

Weng and colleagues investigated the relationship between sustained attention and substance use in a large cohort across three longitudinal visits (ages 14, 19, and 23). They employed a stop signal task to assess sustained attention and utilized the Timeline Followback self-report questionnaire to measure substance use. They assessed the linear relationship between sustained attention-associated functional connections and substance use at an earlier visit (age 14 or 19). Subsequently, they utilized this relationship along with the functional connection profile at a later age (age 19 or 23) to predict substance use at those respective ages. The authors found that connections in association with reduced sustained attention predicted subsequent increases in substance use, a conclusion validated in an external dataset. Altogether, the authors suggest that sustained attention could serve as a robust biomarker for predicting future substance use.

This study by Weng and colleagues focused on an important topic of substance use prediction in adolescence/early adulthood.

Reviewer #2 (Public Review):

Schommartz et al. present a manuscript characterizing neural signatures of reinstatement during cued retrieval of middle-aged children compared to adults. The authors utilize a paradigm where participants learn the spatial location of semantically related item-scene memoranda which they retrieve after short or long delays. The paradigm is especially strong as the authors include novel memoranda at each delayed time point to make comparisons across new and old learning. In brief, the authors find that children show more forgetting than adults, and adults show greater engagement of cortical networks after longer delays as well as stronger item-specific reinstatement. Interestingly, children show more category-based reinstatement, however, evidence supports that this marker may be maladaptive for retrieving episodic details. The question is extremely timely both given the boom in neurocognitive research on the neural development of memory, and the dearth of research on consolidation in this age group. Also, the results provide novel insights into why consolidation processes may be disrupted in children.

Reviewer #2 (Public Review):

Summary:

To investigate the impact of chemical ischemia induced by blocking mitochondrial function and glycolysis, the authors measured extracellular field potentials, performed whole-cell patch-clamp recordings, and measured glutamate release with optical techniques. They found that shorter two-minutes-lasting blockade of energy production initially blocked synaptic transmission but subsequently caused a potentiation of synaptic transmission due to increased glutamate release. In contrast, longer five-minutes-lasting blockage of energy production caused a sustained decrease of synaptic transmission. A correlation between the increase of extracellular potassium concentration and the response upon chemical ischemia indicates that the severity of the ischemia determines whether synapses potentiate or depress upon chemical ischemia. A subsequent mechanistic analysis revealed that the speed of uptake of glutamate is unchanged. An increase in the duration of the fiber volley reflecting the extracellular voltage of the action potentials of the axon bundle was interpreted as an action potential broadening, which could provide mechanistic explanation. In summary, the data convincingly demonstrate that synaptic potentiation induced by chemical ischemia is caused by increased glutamate release.

Strengths:

The manuscript is well written, and the experiments are carefully designed. The results are exciting, novel, and important for the field. The main strength of the manuscript is the combination of electrophysiological recordings and optical glutamate imaging. The main conclusion of increased glutamate release was furthermore supported with an independent approach relying on a low-affinity competitive antagonist of glutamate receptors. The data are of exceptional quality. Several important controls were carefully performed, such as the stability of the recordings and the size of the extracellular space. The number of experiments are sufficient for the conclusions. The careful data analysis justifies the classification of two types of responses, namely synaptic potentiation and depression after chemical ischemia. The data are carefully discussed and the conclusions are justified.

Weaknesses:

The weaknesses are minor. The authors measured the fiber volley, which reflects the extracellular voltage of the compound action potential of the fiber bundle. The half-duration of the fiber volley was increased. These results are consistent with action potential broadening in the axons but the action potential broadening was not experimentally demonstrated. However, these results are carefully discussed.

Reviewer #2 (Public Review):

Summary:

The study by Kremling et al. describes a study of the nsp16-nsp10 methyl transferase from SARS CoV-2 protein which is aimed at identifying inhibitors by x-ray crystallography-based compound screening.<br /> A set of 234 compounds were screened resulting in a set of adenosine-containing compounds or analogues thereof that bind in the SAM site of nsp16-nsp10. The compound selection was mainly based on similarity to SAM and docking of commercially available libraries. The resulting structures are of good quality and clearly show the binding mode of the compounds. It is not surprising to find that these compounds bind in the SAM pocket since they are structurally very similar to portions of SAM. Nevertheless, the result is novel and may be inspirational for the future design of inhibitors. Following up on the crystallographic screen the identified compounds were tested for antiviral activity and binding to np16-nsp10. In addition, an analysis of similar binding sites was presented.

Strengths:

The crystallography is solid and the structures are of good quality. The compound binding constitutes a novel finding.

Weaknesses:

The major weakness is the mismatch between antiviral activity and binding to the target protein. Only one of the compounds could be demonstrated to bind to the nsp16-nsp10 protein. By performing a displacement experiment using ITC Sangivamycin is concluded to bind with a Kd > 1mM. However, the same compound displays antiviral activity with an EC50 of 0.01 microM. Even though the authors do not make specific claims that the antiviral effect is due to inhibition of nsp16-nsp10, it is implicit. If the data is included, it should state specifically that the effect is not likely due to nsp16-nsp10 inhibition.

The structure of the paper and the language needs quite a lot of work to bring it to the expected quality.

Technical point:

Refinement of crystallographic occupancies to single digit percentage is not normally supported by electron density.

Reviewer #2 (Public Review):

Summary:

The flexibility of the ligand binding domain (LBD) of NRs allows various modes of ligand binding leading to various cellular outcomes. In the case of PPARγ, it's known that two ligands can co-bind to the receptor. However, whether a covalent inhibitor functions by blocking the binding of a non-covalent ligand, or co-bind in a manner that weakens the binding of a non-covalent ligand remains unclear. In this study, the authors first used TR-FRET and NMR to demonstrate that covalent inhibitors (such as GW9662 and T0070907) weaken but do not prevent non-covalent synthetic ligands from binding, likely via an allosteric mechanism. The AF-2 helix can exchange between active and repressive conformations, and covalent inhibitors shift the conformation toward a transcriptionally repressive one to reduce the orthosteric binding of the non-covalent ligands. By co-crystal studies, the authors further reveal the structural details of various non-covalent ligand binding mechanisms in a ligand-specific manner (e.g., an alternate binding site, or a new orthosteric binding mode by alerting covalent ligand binding pose).

Strengths:

The biochemical and biophysical evidence presented is strong and convincing.

Weaknesses:

However, the co-crystal studies were performed by soaking non-covalent ligands to LBD pre-crystalized with a covalent inhibitor. Since the covalent inhibitors would shift the LBD toward transcriptionally repressive conformation which reduces orthosteric binding of non-covalent ligands, if the sequence was reversed (i.e., soaking a covalent inhibitor to LBD pre-crystalized with a non-covalent ligand), would a similar conclusion be drawn? Additional discussion will broaden the implications of the conclusion.

Reviewer #2 (Public Review):

Summary:

Pech et al selected 5 Parkinson's disease-causing genes, and generated multiple Drosophila lines by replacing the Drosophila lrrk, rab39, auxilin (aux), synaptojanin (synj), and Pink1 genes with wild-type and pathogenic mutant human or Drosophila cDNA sequences. First, the authors performed a panel of assays to characterize the phenotypes of the models mentioned above. Next, by using single-cell RNA-seq and comparing fly data with human postmortem tissue data, the authors identified multiple cell clusters being commonly dysregulated in these models, highlighting the olfactory projection neurons. Next, by using selective expression of Ca2+-sensor GCaMP3 in the OPN, the authors confirmed the synaptic impairment in these models, which was further strengthened by olfactory performance defects.

Strengths:

The authors overall investigated the functionality of PD-related mutations at endogenous levels and found a very interesting shared pathway through single-cell analysis, more importantly, they performed nice follow-up work using multiple assays.

Weaknesses:

While the authors state this is a new collection of five familial PD knock-in models, the AuxR927G model has been published and carefully characterized in Jacquemyn et al., 2023. ERG has been performed for Aux R927G in Jacquemyn et al., 2023, but the findings are different from what's shown in Figure 1b and Supplementary Figure 1d, which the authors should try to explain. Moreover, according to the authors, the hPINK1control was the expression of human PINK1 with UAS-hPINK1 and nsyb-Gal4 due to technical obstacles.  Having PINK1 WT being an overexpression model, makes it difficult to explain PINK1 mutant phenotypes. It will be strengthened if the authors use UAS-hPINK1 and nsyb-Gal4 (or maybe ubiquitous Gal4) to rescue hPink1L347P and hPink1P399L phenotypes. In addition, although the authors picked these models targeting different biology/ pathways, however, Aux and Synj both act in related steps of Clathrin-mediated endocytosis, with LRRK2 being their accessory regulatory proteins. Therefore, is the data set more favorable in identifying synaptic-related defects?

GH146-GAL4+ PNs are derived from three neuroblast lineages, producing both cholinergic and GABAergic inhibitory PNs (Li et al, 2017). Therefore, OPN neurons have more than "cholinergic projection neurons". How do we know from single-cell data that cholinergic neurons were more vulnerable across 5 models?

In Figure 1b, the authors assumed that locomotion defects were caused by dopaminergic neuron dysfunction. However, to better support it, the author should perform rescue experiments using dopaminergic neuron-specific Gal4 drivers. Otherwise, the authors may consider staining DA neurons and performing cell counting. Furthermore, the authors stated in the discussion, that "We now place cholinergic failure firmly ahead of dopaminergic system failure in flies", which feels rushed and insufficient to draw such a conclusion, especially given no experimental evidence was provided, particularly related to DA neuron dysfunction, in this manuscript.

It is interesting to see that different familial PD mutations converge onto synapses. The authors have suggested that different mechanisms may be involved directly through regulating synaptic functions, or indirectly through mitochondria or transport. It will be improved if the authors extend their analysis on Figure 3, and better utilize their single-cell data to dissect the mechanisms. For example, for all the candidates listed in Figure 3C, are they all altered in the same direction across 5 models?

While this approach is carefully performed, the authors should state in the discussions the strengths and the caveats of the current strategy. For example, what kind of knowledge have we gained by introducing these mutations at an endogenous locus? Are there any caveats of having scRNAseq at day 5 only but being compared with postmortem human disease tissue?

Reviewer #2 (Public Review):

Summary:

Hiramatsu et al. investigated how cognate neurotransmitter receptors with antagonizing downstream effects localize within neurons when co-expressed. They focus on mapping the localization of the dopaminergic Dop1R1 and Dop2R receptors, which correspond to the mammalian D1- and D2-like dopamine receptors, which have opposing effects on intracellular cAMP levels, in neurons of the Drosophila mushroom body (MB). To visualize specific receptors in single neuron types within the crowded MB neuropil, the authors use existing dopamine receptor alleles tagged with 7 copies of split GFP to target reconstitution of GFP tags only in the neurons of interest as a read-out of receptor localization. The authors show that both Dop1R1 and Dop2R, with differing degrees, are enriched in axonal compartments of both the Kenyon Cells cholinergic presynaptic inputs and in different dopamine neurons (DANs), which project axons to the MB. Co-localization studies of dopamine receptors with the presynaptic marker Brp suggest that Dop1R1 and, to a larger extent Dop2R, localize in the proximity of release sites. This localization pattern in DANs suggests that Dop1R1 and Dop2R work in dual-feedback regulation as autoreceptors. Finally, they provide evidence that the balance of Dop1R1 and Dop2R in the axons of two different DAN populations is differentially modulated by starvation and that this regulation plays a role in regulating appetitive behaviors.

Strengths:

The authors use reconstitution of GFP fluorescence of split GFP tags knocked into the endogenous locus at the C-terminus of the dopamine receptors as a readout of dopamine receptor localization. This elegant approach preserves the endogenous transcriptional and post-transcriptional regulation of the receptor, which is essential for studies of protein localization.

The study focuses on mapping the localization of dopamine receptors in neurons of the mushroom body. This is an excellent choice of system to address the question posed in this study, as the neurons are well-studied, and their connections are carefully reconstructed in the mushroom body connectome. Furthermore, the role of this circuit in different behaviors and associative memory permits the linking of patterns of receptor localization to circuit function and resulting behavior. Because of these features, the authors can provide evidence that two antagonizing dopamine receptors can act as autoreceptors within the axonal compartment of MB innervating DANs. The differential regulation of the balance of the two receptors under starvation in two distinct DAN innervations provides evidence of the role that regulation of this balance can play in circuit function and behavioral output.

Weaknesses:

The approach of using endogenously tagged alleles to study localization is a strength of this study, but the authors do not provide sufficient evidence that the insertion of 7 copies of split GFP to the C terminus of the dopamine receptors does not interfere with the endogenous localization pattern or function. Both sets of tagged alleles (1X Venus and 7X split GFP tagged) were previously reported (Kondo et al., 2020), but only the 1X Venus tagged alleles were further functionally validated in assays of olfactory appetitive memory. Despite the smaller size of the 7X split-GFP array tag knocked into the same location as the 1X venus tag, the reconstitution of 7 copies of GFP at the C terminus of the dopamine receptor, might substantially increase the molecular bulk at this site, potentially impeding the function of the receptor more significantly than the smaller, single Venus tag. The data presented by Kondo et al. 2020, is insufficient to conclude that the two alleles are equivalent.

The authors' conclusion that the receptors localize to presynaptic sites is weak. The analysis of the colocalization of the active zone marker Brp whole-brain staining with dopamine receptors labeled in specific neurons is insufficient to conclude that the receptors are localized at presynaptic sites. Given the highly crowded neuropil environment, the data cannot differentiate between the receptor localization postsynaptic to a dopamine release site or at a presynaptic site within the same neuron. The known distribution of presynaptic sites within the neurons analyzed in the study provides evidence that the receptors are enriched in axonal compartments, but co-labeling of presynaptic sites and receptors in the same neuron or super-resolution methods are needed to provide evidence of receptor localization at active zones. The data presented in Figures 5K-5L provides compelling evidence that the receptors localize to neuronal varicosities in DANs where the receptors could play a role as autoreceptors.

Given the highly crowded environment of the mushroom body neuropil, the analysis of dopamine receptor localization in Kenyon cells is not conclusive. The data is sufficient to conclude that the receptors are preferentially localizing to the axonal compartment of Kenyon cells, but co-localization with brain-wide Brp active zone immunostaining is not sufficient to determine if the receptor localizes juxtaposed to dopaminergic release sites, in proximity of release sites in Kenyon cells, or both.

Reviewer #2 (Public Review):

Summary:

Yang and colleagues used a Hidden Markov Model (HMM) on whole-night fMRI to isolate sleep and wake brain states in a data-driven fashion. They identify more brain states (21) than the five sleep/wake stages described in conventional PSG-based sleep staging, show that the identified brain states are stable across nights, and characterize the brain states in terms of which networks they primarily engage.

Strengths:

This work's primary strengths are its dataset of two nights of whole-night concurrent EEG-fMRI (including REM sleep), and its sound methodology.

Weaknesses:

The study's weaknesses are its small sample size and the limited attempts at relating the identified fMRI brain states back to EEG.

General appraisal:

The paper's conclusions are generally well-supported, but some additional analyses and discussions could improve the work.

The authors' main focus lies in identifying fMRI-based brain states, and they succeed at demonstrating both the presence and robustness of these states in terms of cross-night stability. Additional characterization of brain states in terms of which networks these brain states primarily engage adds additional insights.

A somewhat missed opportunity is the absence of more analyses relating the HMM states back to EEG. It would be very helpful to the sleep field to see how EEG spectra of, say, different N2-related HMM states compare. Similarly, it is presently unclear whether anything noticeable happens within the EEG time course at the moment of an HMM class switch (particularly when the PSG stage remains stable). While the authors did look at slow wave density and various physiological signals in different HMM states, a characterization of the EEG itself in terms of spectral features is missing. Such analyses might have shown that fMRI-based brain states map onto familiar EEG substates, or reveal novel EEG changes that have so far gone unnoticed.

It is unclear how the presently identified HMM brain states relate to the previously identified NREM and wake states by Stevner et al. (2019), who used a roughly similar approach. This is important, as similar brain states across studies would suggest reproducibility, whereas large discrepancies could indicate a large dependence on particular methods and/or the sample (also see later point regarding generalizability).

More justice could be done to previous EEG-based efforts moving beyond conventional AASM-defined sleep/wake states. Various EEG studies performed data-driven clustering of brain states, typically indicating more than 5 traditional brain states (e.g., Koch et al. 2014, Christensen et al. 2019, Decat. et al 2022). Beyond that, countless subdivisions of classical sleep stages have been proposed (e.g., phasic/tonic REM, N2 with/without spindles, N3 with global/local slow waves, cyclic alternating patterns, and many more). While these aren't incorporated into standard sleep stage classification, the current manuscript could be misinterpreted to suggest that improved/data-driven classifications cannot be achieved from EEG, which is incorrect.

More discussion of the limitations of the current sample and generalizability would be helpful. A sample of N=12 is no doubt impressive for two nights of concurrent whole-night EEG-fMRI. Still, any data-driven approach can only capture the brain states that are present in the sample, and 12 individuals are unlikely to express all brain states present in the population of young healthy individuals. Add to that all the potentially different or altered brain states that come with healthy ageing, other demographic variables, and numerous clinical disorders. How do the authors expect their results to change with larger samples and/or varying these factors? Perhaps most importantly, I think it's important to mention that the particular number of identified brain states (here 21, and e.g. 19 in Stevner) is not set in stone and will likely vary as a function of many sample- and methods-related factors.

Reviewer #2 (Public Review):

Summary:<br /> The authors proposed a neural network model to explore the spatial representations of the hippocampal CA1 and entorhinal cortex (EC) and the remapping of these representations when multiple environments are learned. The model consists of a recurrent network and output units (a decoder) mimicking the EC and CA1, respectively. The major results of this study are: the EC network generates cells with their receptive fields tuned to a border of the arena; decoder develops neuron clusters arranged in a hexagonal lattice. Thus, the model accounts for entrohinal border cells and CA1 place cells. The authors also suggested the remapping of place cells occurs between different environments through state transitions corresponding to unstable dynamical modes in the recurrent network.

Strengths:<br /> The authors found a spatial arrangement of receptive fields similar to their model's prediction in experimental data recorded from CA1. Thus, the model proposes a plausible mechanisms to generate hippocampal spatial representations without relying on grid cells. This result is consistent with the observation that grid cells are unnecessary to generate CA1 place cells.

The suggestion about the remapping mechanism shows an interesting theoretical possibility.

Weaknesses:<br /> The explicit mechanisms of generating border cells and place cells and those underlying remapping were not clarified at a satisfactory level.

The model cannot generate entorhinal grid cells. Therefore, how the proposed model is integrated into the entire picture of the hippocampal mechanism of meｍory processing remains elusive.

Reviewer #2 (Public Review):

Summary:

The authors show that a combination of arginine methyltransferase inhibitors synergize with PARP inhibitors to kill ovarian and triple-negative cancer cell lines in vitro and in vivo using preclinical mouse models.

PARP inhibitors have been the common targeted-therapy options to treat high-grade serous ovarian cancer (HGSOC) and triple-negative breast cancer (TNBC). PRMTs are oncological therapeutic targets and specific inhibitors have been developed. However, due to the insufficiency of PRMTi or PARPi single treatment for HGSOC and TNBC, designing novel combinations of existing inhibitors is necessary. In previous studies, the authors and others developed an "induced PARPi sensitivity by epigenetic modulation" strategy to target resistant tumors. In this study, the authors presented a triple combination of PRMT1i, PRMT5i and PARPi that synergistically kills TNBC cells. A drug screen and RNA-seq analysis were performed to indicate cancer cell growth dependency of PRMT1 and PRMT5, and their CRISPR/Cas9 knockout sensitizes cancer cells to PARPi treatment. It was shown that the cells accumulate DNA damage and have increased caspase 3/7 activity. RNA-seq analysis identified BRCAness genes, and the authors closely studied a top hit ERCC1 as a downregulated DNA damage protein in PRMT inhibitor treatments. ERCC1 is known to be synthetic lethal with PARP inhibitors. Thus, the authors add back ERCC1 and reduce the effects of PRMT inhibitors suggesting PRMT inhibitors mediate, in part, their effect via ERCC1 downregulation. The combination therapy (PRMT/PARP) is validated in 2D cultures of cell lines (OVCAR3, 8 and MDA-MB-231) and has shown to be effective in nude mice with MDA-MB-231 xenograph models.

Strengths and weaknesses:

Overall, the data is well-presented. The experiments are well-performed, convincing, and have the appropriate controls (using inhibitors and genetic deletions) and statistics.

They identify the DNA damage protein ERCC1 to be reduced in expression with PRMT inhibitors. As ERCC1 is known to be synthetic lethal with PARPi, this provides a mechanism for the synergy. They use cell lines only for their study in 2D as well as xenograph models.

Reviewer #2 (Public Review):

Summary:

The manuscript by Herneisen et al. examines the Toxoplasma SPARK kinase orthologous to mammalian PDK1 kinase. The extracellular signals trigger cascades of the second messengers and play a central role in the apicomplexan parasites' survival. In Toxoplasma, these cascades regulate active replication of the tachyzoites, which manifests as acute toxoplasmosis, or the development into drug-resilient bradyzoites characteristic of the chronic stage of the disease. This study focuses on the poorly understood signaling mechanisms acting upstream of such second messenger kinases as PKA and PKG. The authors showed that similar to PDK1, Toxoplasma SPARK likely regulates several AGC kinases.

Strengths:

The study demonstrated a strong association of the SPARK kinase with the SPARKL factor and an uncharacterized AGC kinase. Using a set of standard assays, the authors determined the SPARK /SPARLS role in parasite egress, invasion, and bradyzoite differentiation.

Weaknesses:

Although the revised manuscript has significantly improved, the primary concern of incomplete data analysis still needs to be addressed.

Reviewer #2 (Public Review):

This manuscript reports several interesting observations that invite follow-up. The notion that hubs, and perhaps condensates that may (or may not embrace them) are functionally and physiologically important is an open issue at this time. The authors note that TFIIIC helps to prune extraneous connections from hubs, but do not comment that the connections that are maintained are also reinforced. At the same time only modest changes in gene expression associated with expanded or decreased connections and changes in bound proteins. One interesting possibility might be that standard methods for assessing expression miss changes global or background transcription. It seems that the TFIIIC-MYCN-ER connection has features that would help to suppress such background. The results invite a more global consideration of TFIIIC than as primarily RNAPIII/small RNA transcription factor and of MYCN as an E-box dependent transcription factor. The results use sate of the art methods to develop interesting new ideas that have the potential to instruct further studies that may reveal new mechanisms of action for TFIIIC and MYCN.

The work is however subject to a couple of caveats. First, the authors should be more cautious when drawing firm conclusions about the dynamics and kinetics of transcription from the static snapshots obtained from most genomic methods. For example, please take a look at Figure 1F of "Transcription elongation defects link oncogenicSF3B1 mutations to targetable alterations in chromatin landscape" by Buddu et al, https://doi.org/10.1016/j.molcel.2024.02.032. Here, an increase in RNAPSer2P is seen in gene bodies and a bit at the TES- superficially inviting the conclusion that expression is increased (a similar erroneous conclusion has been claimed in other genomic studies), but the increase is in fact, not due to increased transcription, rather to impaired elongation-this conclusion required performing TT-Seq which allowed inferences to be made about elongation rates. Acknowledging this qualification would help advise the reader.

The authors also need to discuss directly what differences between the MYC predominant SH-EP cells and the MYCN-predominant SH-EP-MYCNER+tamoxifen are qualitative versus quantitative. MYCNER indeed associates much more with chromatin than did MYC, but there seems to be a lot more MYCER than there was MYC prior to the addition of tamoxifen. (The true control for this would be to prepare SH-EP-MYCER cells expressed from the same promoter as was MYCNER. Some discussion of qualitative versus quantitative differences should be acknowledged.

Strengths:

Use of a variety of methods to assess the genomic response to increased MYCN in the presence or absence of TFIIIC. Clearly establishes in vitro and in vivo the TFIIIC-MYCN complex

Weaknesses:

Dynamic inferences are made without kinetic experiments.

Reviewer #2 (Public Review):

Summary:

The study is devoted to the deep investigation of the spermatogonial stem cell (SSC) niche in trans women after gender-affirming hormone therapy (GAHT). Both cellular structure and functionality of the niche were studied. The authors evidently demonstrated that all cellular components of SSC niche were affected by hormone therapy. Interestingly, the signs of "rejuvenation" within the niche were also observed indicating the possible reverse to the immature condition.

Strengths:

The obtained findings are important for the better understanding of hormonal regulation of testis and SSC niche and provide some clues for using the biomaterials from these specific and even unique donors for biomedical research.

Weaknesses:

This study has some limitations. Many studies can't be done using the testes cells of trans women, since their cells are significantly different from adult man cells and less from prepubertal and pubertal cells. The authors themselves identify some of the limitations: this material is suitable only for studying prepubertal processes in the testis. However, the authors also report large variability in data due to different hormonal therapy regimens and, apparently, age. Accordingly, not all material obtained from trans women can also be used for studies of prepubertal processes.

Reviewer #2 (Public Review):

Summary:

Golamalamdari, van Schaik, Wang, Kumar Zhang, Zhang, and colleagues study interactions between the speckle, nucleolus, and lamina in multiple cell types (K562, H1, HCT116, and HFF). Their datasets define how interactions between the genome and the different nuclear landmarks relate to each other and change across cell types. They also identify how these relationships change in K562 cells in which LBR and LMNA are knocked out.

Strengths:

Overall, there are a number of datasets that are provided, and several "integrative" analyses are performed. This is a major strength of the paper, and I imagine the datasets will be of use to the community to further probed and the relationships elucidated here further studied. An especially interesting result was that specific genomic regions (relative to their association with the speckle, lamina, and other molecular characteristics) segregate relative to the equatorial plane of the cell.

Weaknesses:

The experiments are largely descriptive, and it is difficult to draw many cause-and-effect relationships. Similarly, the paper would be very much strengthened if the authors provided additional summary statements and interpretation of their results (especially for those not as familiar with 3D genome organization). The study would benefit from a clear and specific hypothesis.

Reviewer #2 (Public Review):

Summary:

The manuscript from Belato et al. used advanced NMR approaches and a mutagenesis campaign to probe the conformational dynamics of the recognition lobe (Rec) of the CRISPR Cas9 enzyme from G. stearothermophilus (GeoCas9). Using truncated and full-length constructs they assess the impacts of two different point mutations have on the redistribution and timescale of these motions and assess gRNA recognition and specificity. Single point mutations in the Rec domain in a Cas9 from a related species had profound impacts on- and off-target DNA editing, therefore the authors reasoned analogous mutations in GeoCas9 would have similar effects. However, despite a redistribution of local motions and changes in global stability, their chosen mutations had little impact on DNA editing in the context of the full-length enzyme. Their studies highlight the species-specific complexity of interdomain communication and allosteric mechanisms used by these multi-domain endonucleases. Despite these negative results, their study is highly rigorous, and their approach will broadly support understanding how the activity and specificity of these enzymes can be engineered to tune activity and limit off-target cleavage by these enzymes.

Strengths:

(1) Atomistic investigation of the conformational dynamics of the GeoCas9 gRNA recognition lobe (GeoRec), probing dynamics on a broad range of timescales from ps to ms using advanced NMR approaches will be broadly interesting to both the structural biology and CRISPR engineering communities.

(2) Highly rigorous biophysical studies that push the boundaries of current techniques, provide insight into local dynamics of the GeoRec domain that serve to propagate allosteric information and potentially regulate enzymatic activity.

(3) The study highlights the complexities of understanding interdomain communication in Cas9 enzymes since analogous mutations in different species have different effects on target recognition and cleavage.

(4) The type of structural and dynamic insights derived from this study design could serve as foundational information to guide a rational design strategy aimed at improving the selectivity and reducing the off-target effects of Cas9 enzymes.

Weaknesses:

(1) Despite the rigor of the experiments, the mutations chosen by the authors do not have a profound effect on the overall substrate affinity or activity of GeoCas9 rendering little mechanistic insight into allosteric communication in this particular Cas9. However, the double mutant K267E/R332A has a more pronounced effect on the cleavage of WT and mismatched (at nucleotides 19 and 20) DNA substrates while minimally affecting the cleavage of mismatched (at nucleotides 5 and 6), suggesting more could be learned about the allosteric mechanism from the detailed characterization of this mutant.

(2) Follow-up experiments with other residues that were identified as being highly dynamic might affect substrate recognition and cleavage activity in different ways providing additional insight.

(3) Details regarding the authors' experimental approach are incomplete such as a description of the model used to fit the CD data, a detailed explanation of the global fitting of the relaxation dispersion data describing how the best-fit model was selected, and the description of the ModelFree fitting of fast timescale dynamics is incomplete.

Reviewer #3 (Public Review):

Summary:

Deng et al. assess neonatal cord blood methylation profiles and the association with (self-reported) maternal smoking in multiple populations, including two European (CHILD, FAMILY) and one South Asian (START), via two approaches: 1) they perform an independent epigenome-wide association study (EWAS) and meta-analysis across the CHILD and FAMILY cohort, during which they also benchmark previously reported maternal-smoking associated sites, and 2) they generate new composite methylation risk scores for maternal smoking, and assess their performance and association with phenotypic characteristics in the three populations, in addition to previously described maternal smoking methylation risk scores.

Strengths and weaknesses:

Their meta-analysis across multiple cohorts and comparison with previous findings represents a strength. In particular the inclusion of a South Asian birth cohort is commendable as it may help to bolster generalizability. However, their conclusions are limited by several important weaknesses:

(1) the low number of (self-reported) maternal smokers in particular their South Asian population, resulting in an inability to conduct benchmarking of maternal smoking sites in this cohort. As such, the inclusion of the START cohort in certain figures is not warranted (e.g., Figure 3) and the overall statement that smoking-associated MRS are portable across populations are not fully supported;<br /> (2) different methylation profiling tools were used: START and CHILD methylation profiles were generated using the more comprehensive 450K array while the FAMILY cohort blood samples were profiled using a targeted array covering only 3,000, as opposed to 450,000 sites, resulting in different coverage of certain sites which affects downstream analyses and MRS, and importantly, omission of potentially relevant sites as the array was designed in 2016 and substantial additional work into epigenetic traits has been conducted since then;<br /> (3) the authors train methylation risk scores (MRS) in CHILD or FAMILY populations based on sites that are associated with maternal smoking in both cohorts and internally validate them in the other cohort, respectively. As START cohort due to insufficient numbers of self-reported maternal smokers, the authors cannot fully independently validated their MRS, thus limiting the strength of their results.

Overall strength of evidence and conclusions:

Despite these limitations, the study overall does explore the feasibility of using neonatal cord blood for the assessment of maternal smoking. However, their conclusion on generalizability of the maternal smoking risk score is currently not supported by their data as they were not able to validate their score in a sufficiently large number of maternal smokers and never smokers of South Asian populations.

While their generalizability remains limited due to small sample numbers and previous studies with methylation risk scores exist, their findings may nonetheless provide the basis for future work into prenatal exposures which will be of interest to the research community. In particular their finding that the maternal smoking-associated MRS was associated with small birth sizes and weights across birth cohorts, including the South Asian birth cohort that had very few self-reported smokers, is interesting and the author suggest these findings could be associated with factors other than smoking alone (e.g., pollution), which warrant further investigation and would be highly novel.<br /> Future exploration should also include a strong focus on more diverse health outcomes, including respiratory conditions that may have long-lasting health consequences.

Reviewer #2 (Public Review):

The authors investigated the conformational dynamics and energetics of the SthK Clinker/CNBD fragment using both steady-state and time-resolved transition metal ion Förster resonance energy transfer (tmFRET) experiments. To do so, they engineered donor-acceptor pairs at specific sites of the CNBD (C-helix and β-roll) by incorporating a fluorescent noncanonical amino acid donor and metal ion acceptors. In particular, the authors employed two cysteine-reactive metal chelators (TETAC and phenM). This allowed them to coordinate three transition metals (Cu2+, Fe2+, and Ru2+) to measure both short (10-20 Å, Cu2+) and long distances (25-50 Å, Fe2+, and Ru2+). By measuring tmFRET with fluorescence lifetimes, the authors determined intramolecular distance distributions in the absence and presence of the full agonist cAMP or the partial agonist cGMP. The probability distributions between conformational states without and with ligands were used to calculate the changes in free energy (ΔG) and differences in free energy change (ΔΔG) in the context of a simple four-state model.

Overall, the work is conducted in a rigorous manner, and it is well-written. I greatly enjoyed reading it.

Nonetheless, I do not see the novelty that the authors claim.

In terms of methodology, this work provides further support to steady-state and time-resolved tmFRET approaches previously developed by the authors of the present work to probe conformational rearrangements by using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor (Zagotta et al., eLIfe 2021; Gordon et al., Biophysical Journal 2024; Zagotta et al., Biophysical Journal 2024).

Regarding cyclic nucleotide-binding domain (CNBD)-containing ion channels, I disagree with the authors when they state that "the precise allosteric mechanism governing channel activation upon ligand binding, particularly the energetic changes within domains, remains poorly understood". On the contrary, I would say that the literature on this subject is rather vast and based on a significantly large variety of methodologies. This is a not exhaustive list of papers: Zagotta et al., Nature 2003; Craven et al., GJP, 2004; Craven et al., JBC, 2008; Taraska et al., Nature Methods, 2009; Puljung et al., JBC, 2013; Saponaro et al., PNAS 2014; Goldschen-Ohm et al., eLife, 2016; Bankston et al., JBC, 2017; Hummert et al., PLoS Comput Biol., 2018; Porro et al., eLife, 2019; Ng et al., JGP, 2019; Porro et al., JGP, 2020; Evans et al., PNAS, 2020; Pfleger et al., Biophys J. 2021; Saponaro et al., Mol Cell, 2021; Dai et al., Nat Commun. 2021; Kondapuram et al., Commun Biol. 2022. These studies were conducted either on the isolated Clinker/CNBD fragments or on the entire full-length proteins. As is evident from the above list, the authors of the present work have significantly contributed to the understanding of the allosteric mechanism governing the ligand-induced activation of CNBD-containing channels, including a detailed description of the energetic changes induced by ligand binding. Particularly relevant are their works based on DEER spectroscopy. In DeBerg et al., JBC 2016, the authors described, in atomic detail, the conformational changes induced by different cyclic nucleotides on the HCN CNBD fragment and derived energetics associated with ligand binding to the CNBD (ΔΔG). In Collauto et al., Phys Chem Chem Phys. 2017, they further detailed the ligand-CNBD conformational changes by combining DEER spectroscopy with microfluidic rapid freeze quench to resolve these processes and obtain both equilibrium constants and reaction rates, thus demonstrating that DEER can quantitatively resolve both the thermodynamics and the kinetics of ligand binding and the associated conformational changes.

Suggestions:

- In light of the above, I suggest the authors better clarify the contribution/novelty that the present work provides to the state-of-the-art methodology employed (steady-state and time-resolved tmFRET) and of CNBD-containing ion channels. In particular, it would be nice to have a comparison with the conformational dynamics and energetics reported in the previous works of the authors based on DEER spectroscopy (DeBerg et al., JBC 2016, Collauto et al., Phys Chem Chem Phys. 2017 and Evans et al., PNAS, 2020) and with Goldschen-Ohm et al., eLife, 2016, where single-molecule events (FRET-based) of cAMP binding to HCN CNBD were measured and kinetic rate constants were models in the context of a simple four-state model, reminiscent of the model employed in the present work.

- Even considering the bacterial SthK channel, cryo-EM has significantly advanced the atomistic understanding of its ligand-dependent regulation (Rheinberger et al., eLife, 2018). More recently, the authors of the present work have elegantly employed DEER on full-length SthK protein to reveal ligand-dependent conformational rearrangements in the Clinker region (Evans et al., PNAS, 2020). In light of the above, what is the contribution/novelty that the present work provides to the SthK biophysics?

- The authors decided to use the Clinker/CNBD fragment of SthK. On the basis of the above-cited work (Evans et al., PNAS, 2020) the authors should clarify why they have decided to work on the isolated Clinker/CNBD fragment and not on the full-length protein. I assume that the use of the C-licker/CNBD fragment was necessary to isolate tetramers with only one labelled subunit (fSEC and MP were used to confirm this) to avoid inter-subunit crass-talk. However, I am not clear if this is correct.

- What is the advantage of using the Clinker/CNBD fragment of a bacterial protein and not one of HCN channels, as already successfully employed by the authors (see above citations)?

Reviewer #2 (Public Review):

Summary:

Wine et al. describe a framework to view the estimation of gene-context interaction analysis through the lens of bias-variance tradeoff. They show that, depending on trait variance and context-specific effect sizes, effect estimates may be estimated more accurately in context-combined analysis rather than in context-specific analysis. They proceed by investigating, primarily via simulations, implications for the study or utilization of gene-context interaction, for testing and prediction, in traits with polygenic architecture. First, the authors describe an assessment of the identification of context-specificity (or context differences) focusing on "top hits" from association analyses. Next, they describe an assessment of polygenic scores (PGSs) that account for context-specific effect sizes, showing, in simulations, that often the PGSs that do not attempt to estimate context-specific effect sizes have superior prediction performance. An exception is a PGS approach that utilizes information across contexts.

Strengths:

The bias-variance tradeoff framing of GxE is useful, interesting, and rigorous. The PGS analysis under pervasive amplification is also interesting and demonstrates the bias-variance tradeoff.

Weaknesses:

The weakness of this paper is that the first part -- the bias-variance tradeoff analysis -- is not tightly connected to, i.e. not sufficiently informing, the later parts, that focus on polygenic architecture. For example, the analysis of "top hits" focuses on the question of testing, rather than estimation, and testing was not discussed within the bias-variance tradeoff framework. Similarly, while the PGS analysis does demonstrate (well) the bias-variance tradeoff, the reader is left to wonder whether a bias-variance deviation rule (discussed in the first part of the manuscript) should or could be utilized for PGS construction.

Reviewer #2 (Public Review):<br /> The authors investigate the disparity between spatial extant and temporal variance of electrophysiological-fMRI correlations in a rodent model. They found high correspondence in spatial extent but a disparity in temporal variance. From this, they propose a model of an electrophysiologically-invisible signal affecting temporal variance.

I remain skeptical about the "electrophysiologically invisible signal" model but the authors have done a much better job of both explaining it and hedging it in this version. Readers can decide for themselves.

The revision submitted by the authors substantially improves writing and methods.

Reviewer #2 (Public Review):

Summary:

In this study, Chia-Lo Ho et al. study the impact of CD5high CD8 T cells in the pathophysiology of type 1 diabetes (T1D) in NOD mice. The authors used high expression of CD5 as a surrogate of high TCR signaling and self-reactivity and compared the phenotype, transcriptome, TCR usage, function, and pathogenic properties of CD5high vs. CD5low CD8 T cells extracted from the so-called naive T cell pool. The study shows that CD5high CD8 T cells resemble memory T cells poised for a stronger response to TCR stimulation and that they exacerbate disease upon transfer in RAG-deficient NOD mice. The authors attempt to link these features to the thymic selection events of these CD5high CD8 T cells. Importantly, forced overexpression of the phosphatase PTPN22 in T cells attenuated TCR signaling and reduced pathogenicity of polyclonal CD8 T cells but not highly autoreactive 8.3-TCR CD8 T cells.

Strengths:

The study is nicely performed and the manuscript is clear and well-written. Interpretation of the data is careful and fair. The data are novel and likely important. However, some issues would need to be clarified through either text changes or the addition of new data.

Weaknesses:

The definition of naïve T cells based solely on CD44low and CD62Lhigh staining may be oversimplistic. Indeed, even within this definition, naïve CD5high CD8 T cells express much higher levels of CD44 than CD5low CD8 T cells.

Reviewer #2 (Public Review):

Summary:

This study comprehensively presents data from single nuclei sequencing of Heigai pig skeletal muscle in response to conjugated linoleic acid supplementation. The authors identify changes in myofiber type and adipocyte subpopulations induced by linoleic acid at depth previously unobserved. The authors show that linoleic acid supplementation decreased the total myofiber count, specifically reducing type II muscle fiber types (IIB), myotendinous junctions, and neuromuscular junctions, whereas type I muscle fibers are increased. Moreover, the authors identify changes in adipocyte pools, specifically in a population marked by SCD1/DGAT2. To validate the skeletal muscle remodeling in response to linoleic acid supplementation, the authors compare transcriptomics data from Laiwu pigs, a model of high intramuscular fat, to Heigai pigs. The results verify changes in adipocyte subpopulations when pigs have higher intramuscular fat, either genetically or diet-induced. Targeted examination using cell-cell communication network analysis revealed associations with high intramuscular fat with fibro-adipogenic progenitors (FAPs).  The authors then conclude that conjugated linoleic acid induces FAPs towards adipogenic commitment. Specifically, they show that linoleic acid stimulates FAPs to become SCD1/DGAT2+ adipocytes via JNK signaling. The authors conclude that their findings demonstrate the effects of conjugated linoleic acid on skeletal muscle fat formation in pigs, which could serve as a model for studying human skeletal muscle diseases.

Strengths:

The comprehensive data analysis provides information on conjugated linoleic acid effects on pig skeletal muscle and organ function. The notion that linoleic acid induces skeletal muscle composition and fat accumulation is considered a strength and demonstrates the effect of dietary interactions on organ remodeling. This could have implications for the pig farming industry to promote muscle marbling. Additionally, these data may inform the remodeling of human skeletal muscle under dietary behaviors, such as elimination and supplementation diets and chronic overnutrition of nutrient-poor diets. However, the biggest strength resides in thorough data collection at the single nuclei level, which was extrapolated to other types of Chinese pigs.

Weaknesses:

While the authors generated a sizeable comprehensive dataset, cellular and molecular validation needed to be improved. For example, the single nuclei data suggest changes in myofiber type after linoleic acid supplementation, yet these data are not validated by other methodologies. Similarly, the authors suggest that linoleic acid alters adipocyte populations, FAPs, and preadipocytes; however, no cellular and molecular analysis was performed to reveal if these trajectories indeed apply. Attempts to identify JNK signaling pathways appear superficial and do not delve deeper into mechanistic action or transcriptional regulation. Notably, a variety of single cell studies have been performed on mouse/human skeletal muscle and adipose tissues. Yet, the authors need to discuss how the populations they have identified support the existing literature on cell-type populations in skeletal muscle. Moreover, the authors nicely incorporate the two pig models into their results, but the authors only examine one muscle group. It would be interesting if other muscle groups respond similarly or differently in response to linoleic acid supplementation. Further, it was unclear whether Heigai and Laiwu pigs were both fed conjugated linoleic acid or whether the comparison between Heigai-fed linoleic acid and Laiwu pigs (as a model of high intramuscular fat). With this in mind, the authors do not discuss how their results could be implicated in human and pig nutrition, such as desirability and cost-effectiveness for pig farmers and human diets high in linoleic acid. Notably, while single nuclei data is comprehensive, there needs to be a statement on data deposition and code availability, allowing others access to these datasets. Moreover, the experimental designs do not denote the conjugated linoleic acid supplementation duration. Several immunostainings performed could be quantified to validate statements. This reviewer also found the Nile Red staining hard to interpret visually and did not appear to support the conclusions convincingly. Within Figure 7, several letters (assuming they represent statistical significance) are present on the graphs but are not denoted within the figure legend.

Reviewer #2 (Public Review):

Summary:

The authors used a large cohort of patients with metastatic lung cancer pre- and 1-3 weeks post-immunotherapy. The goal was to investigate whether immunotherapy results in changes in CHIP clones (using targeted sequencing and whole exome sequencing) as well as to investigate whether patients with CHIP changed their response to immunotherapy (single-cell RNA sequencing).

Strengths:

This represents a large cohort of patients, and comprehensive assays - including targeted sequencing, whole exome sequencing, and single-cell RNA sequencing.

Weaknesses:

Findings are not necessarily unexpected. With regards to clonal dynamics, it would be very unlikely to see any changes within a few weeks' time frame. Longer follow-up to assess clonal dynamics would realistically be necessary.

Reviewer #2 (Public Review):

Summary:

In this paper, the function of trpγ in lipid metabolism was investigated. The authors found that lipid accumulation levels were increased in trpγ mutants and remained high during starvation; the increased TAG levels in trpγ mutants were restored by the expression of active AMPK in DH44 neurons and oral administration of the anti-diabetic drug metformin. Furthermore, oral administration of lipase, TAG, and free fatty acids effectively restored the survival of trpγ mutants under starvation conditions. These results indicate that TRPv plays an important role in the maintenance of systemic lipid levels through the proper expression of lipase. Furthermore, authors have shown that this function is mediated by DH44R2. This study provides an interesting finding in that the neuropeptide DH44 released from the brain regulates lipid metabolism through a brain-gut axis, acting on the receptor DH44R2 presumably expressed in gut cells.

Strengths:

Using Drosophila genetics, careful analysis of which cells express trpγ regulates lipid metabolism is performed in this study. The study supports its conclusions from various angles, including not only TAG levels, but also fat droplet staining and survival rate under starved conditions, and oral administration of substances involved in lipid metabolism.

Weaknesses:

Lipid metabolism in the gut of DH44R2-expressing cells should be investigated for a better understanding of the mechanism. Fat accumulation in the gut is not mechanistically linked with fat accumulation in the fat body. The function of lipase in the gut (esp. R2 region) should be addressed, e.g. by manipulating gut-lipases such as magro or Lip3 in the gut in the contest of trpγ mutant. Also, it is not clarified which cell types in the gut DH44R2 is expressed. The study also mentioned only in the text that bmm expression in the gut cannot restore lipid droplet enlargement in the fat body, but this result might be presented as a figure.

Reviewer #2 (Public Review):

Summary:

The study investigates the molecular mechanisms underlying chronic pain-related memory impairment by focusing on S1P/S1PR1 signaling in the dentate gyrus (DG) of the hippocampus. Through behavioural tests (Y-maze and Morris water maze) and RNA-seq analysis, the researchers segregated chronic pain mice into memory impairment-susceptible and -unsusceptible subpopulations. They discovered that S1P/S1PR1 signaling is crucial for determining susceptibility to memory impairment, with decreased S1PR1 expression linked to structural plasticity changes and memory deficits.

Knockdown of S1PR1 in the DG induced a susceptible phenotype, while overexpression or pharmacological activation of S1PR1 promoted resistance to memory impairment and restored normal synaptic structure. The study identifies actin cytoskeleton-related pathways, including ITGA2 and its downstream Rac1/Cdc42 signaling, as key mediators of S1PR1's effects, offering new insights and potential therapeutic targets for chronic pain-related cognitive dysfunction.

This manuscript consists of a comprehensive investigation and significant findings. The study provides novel insights into the molecular mechanisms of chronic pain-related memory impairment, highlighting the critical role of S1P/S1PR1 signaling in the hippocampal dentate gyrus. The clear identification of S1P/S1PR1 as a potential therapeutic target offers promising avenues for future research and treatment strategies. The manuscript is well-structured, methodologically sound, and presents valuable contributions to the field.

Strengths:

(1) The manuscript is well-structured and written in clear, concise language. The flow of information is logical and easy to follow.

(2) The segregation of mice into memory impairment-susceptible and -unsusceptible subpopulations is innovative and well-justified. The statistical analyses are robust and appropriate for the data.

(3) The detailed examination of S1PR1 expression and its impact on synaptic plasticity and actin cytoskeleton reorganization is impressive. The findings are significant and contribute to the understanding of chronic pain-related memory impairment.

Weaknesses:

(1) Results: While the results are comprehensive, some sections are data-heavy and could be more reader-friendly with summarized key points before diving into detailed data.

(2) Discussion: There is a need for a more balanced discussion regarding the limitations of the study. For example, addressing potential biases in the animal model or limitations in the generalizability of the findings to humans would strengthen the discussion. Also, providing specific suggestions for follow-up studies would be beneficial.

(3) Conclusion: The conclusion, while concise, could better highlight the study's broader impact on the field and potential clinical implications.

Reviewer #2 (Public Review):

Summary:

The study investigates the brain's functional connectivity (FC) dynamics across different timescales using simultaneous recordings of intracranial EEG/source-localized EEG and fMRI. The primary research goal was to determine which of three convergence/divergence scenarios is the most likely to occur.

The results indicate that despite similar FC patterns found in different data modalities, the time points were not aligned, indicating spatial convergence but temporal divergence.

The researchers also found that FC patterns in different frequencies do not overlap significantly, emphasizing the multi-frequency nature of brain connectivity. Such asynchronous activity across frequency bands supports the idea of multiple connectivity states that operate independently and are organized into a multiplex system.

Strengths:

The data supporting the authors' claims are convincing and come from simultaneous recordings of fMRI and iEEG/EEG, which has been recently developed and adapted.

The analysis methods are solid and involve a novel approach to analyzing the co-occurrence of FC patterns across modalities (cross-modal recurrence plot, CRP) and robust statistics, including replication of the main results using multiple operationalizations of the functional connectome (e.g., amplitude, orthogonalized, and phase-based coupling).

In addition, the authors provided a detailed interpretation of the results, placing them in the context of recent advances and understanding of the relationships between functional connectivity and cognitive states.

Weaknesses:

Despite the impressive work, the paper still lacks some analyses to make it complete.

Firstly, the effect of the window size is unclear, especially in the case of different frequencies where the number of cycles that fall in a window will vary drastically. A typical oscillation lasts just a few cycles (see Myrov et al., 2024), and brain states are usually short-lived because of meta-stability (see Roberts et al., 2019).

Secondly, the authors didn't examine frequencies lower than 1Hz despite similarities between fMRI and infra-slow oscillations found in prior literature (see Palva et al., 2014; Zhang et al., 2023).

On a minor note, the phase-locking value (PLV) is positively biased for EEG data (see Palva et al., 2018) and a different metric for phase coupling could be a more appropriate choice (e.g., iPLV/wPLI, see Vinck et al., 2011). The repository with the code is also unavailable.

Reviewer #2 (Public Review):

Summary:

The authors evaluate spectral changes in electroencephalography (EEG) data as a function of the congruency of audio and visual information associated with biological motion (BM) or non-biological motion. The results show supra-additive power gains in the neural response to gait dynamics, with trials in which audio and visual information were presented simultaneously producing higher average amplitude than the combined average power for auditory and visual conditions alone. Further analyses suggest that such supra-additivity is specific to BM and emerges from temporoparietal areas. The authors also find that the BM-specific supra-additivity is negatively correlated with autism traits.

Strengths:

The manuscript is well-written, with a concise and clear writing style. The visual presentation is largely clear. The study involves multiple experiments with different participant groups. Each experiment involves specific considered changes to the experimental paradigm that both replicate the previous experiment's finding yet extend it in a relevant manner.

Weaknesses:

The manuscript interprets the neural findings using mechanistic and cognitive claims that are not justified by the presented analyses and results.

First, entrainment and cortical tracking are both invoked in this manuscript, sometimes interchangeably so, but it is becoming the standard of the field to recognize their separate evidential requirements. Namely, step and gate cycles are striking perceptual or cognitive events that are expected to produce event-related potentials (ERPs). The regular presentation of these events in the paradigm will naturally evoke a series of ERPs that leave a trace in the power spectrum at stimulation rates even if no oscillations are at play. Thus, the findings should not be interpreted from an entrainment framework except if it is contextualized as speculation, or if additional analyses or experiments are carried out to support the assumption that oscillations are present. Even if oscillations are shown to be present, it is then a further question whether the oscillations are causally relevant toward the integration of biological motion and for the orchestration of cognitive processes.

Second, if only a cortical tracking account is adopted, it is not clear why the demonstration of supra-additivity in spectral amplitude is cognitively or behaviorally relevant. Namely, the fact that frequency-specific neural responses to the [audio & visual] condition are stronger than those to [audio] and [visual] combined does not mean this has implications for behavioral performance. While the correlation to autism traits could suggest some relation to behavior and is interesting in its own right, this correlation is a highly indirect way of assessing behavioral relevance. It would be helpful to test the relevance of supra-additive cortical tracking on a behavioral task directly related to the processing of biological motion to justify the claim that inputs are being integrated with the service of behavior. Under either framework, cortical tracking or entrainment, the causal relevance of neural findings toward cognition is lacking.

Overall, I believe this study finds neural correlates of biological motion, and it is possible that such neural correlates relate to behaviorally relevant neural mechanisms, but based on the current task and associated analyses this has not been shown.

DOI: 10.1007/s00330-024-10886-2

Resource: Parkinson's Progression Markers Initiative (RRID:SCR_006431)

Curator: @dhovakimyan1

SciCrunch record: RRID:SCR_006431

What is this?

DOI: 10.1007/s12264-020-00518-2

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

Curator: @bpowell22

SciCrunch record: RRID:BDSC_458

What is this?

Reviewer #2 (Public Review):

MotorNet aims to provide a unified interface where the trained RNN controller exists within the same TensorFlow environment as the end effectors being controlled. This architecture provides a much simpler interface for the researcher to develop and iterate through computational hypotheses. In addition, the authors have built a set of biomechanically realistic end effectors (e.g., a 2 joint arm model with realistic muscles) within TensorFlow that are fully differentiable.

MotorNet will prove a highly useful starting point for researchers interested in exploring the challenges of controlling movement with realistic muscle and joint dynamics. The architecture features a conveniently modular design and the inclusion of simpler arm models provides an approachable learning curve. Other state-of-the-art simulation engines offer realistic models of muscles and multi-joint arms and afford more complex object manipulation and contact dynamics than MotorNet. However, MotorNet's approach allows for direct optimization of the controller network via gradient descent rather than reinforcement learning, which is a compromise currently required when other simulation engines (as these engines' code cannot be differentiated through).

The paper has been reorganized to provide clearer signposts to guide the reader. Importantly, the software has been rewritten atop PyTorch which is increasingly popular in ML and computational neuroscience research.

Reviewer #3 (Public Review):

Summary:

In this paper Hajra et al have attempted to identify the role of Sirt1 and Sirt3 in regulating metabolic reprogramming and macrophage host defense. They have performed gene knock down experiments in RAW macrophage cell line to show that depletion of Sirt1 or Sirt3 enhances the ability of macrophages to eliminate Salmonella Typhimurium. However, in mice inhibition of Sirt1 resulted in dissemination of the bacteria but the bacterial burden was still reduced in macrophages. They suggest that the effect they have observed is due to increased inflammation and ROS production by macrophages. They also try to establish a weak link with metabolism. They present data to show that the switch in metabolism from glycolysis to fatty acid oxidation is regulated by acetylation of Hif1a, and PDHA1.

Strengths:

The strength of the manuscript is that the role of Sirtuins in host-pathogen interactions has not been previously explored in-depth making the study interesting. It is also interesting to see that depletion of either Sirt1 or Sirt3 results in a similar outcome.

Weaknesses:

The major weakness of the paper is the low quality of data, making it harder to substantiate the claims. Also, there are too many pathways and mechanisms being investigated. It would have been better if the authors had focussed on either Sirt1 or Sirt3 and elucidated how it reprograms metabolism to eventually modulate host response against Salmonella Typhimurium. Experimental evidence is also lacking to prove the proposed mechanisms. For instance they show correlative data that knock down of Sirt1 mediated shift in metabolism is due to HIF1a acetylation but this needs to be proven with further experiments.

Reviewer #2 (Public Review):

Summary:

In this study, Hauser et al investigate the role of amphibian (Xenopus laevis) mast cells in cutaneous immune responses to the ecologically important pathogen Batrachochytrium dendrobatidis (Bd) using novel methods of in vitro differentiation of bone marrow-derived mast cells and in vivo expansion of skin mast cell populations. They find that bone marrow-derived myeloid precursors cultured in the presence of recombinant X. laevis Stem Cell Factor (rSCF) differentiate into cells that display hallmark characteristics of mast cells. They inject their novel (r)SCF reagent in the skin of X. laevis and find that this stimulates expansion of cutaneous mast cell populations in vivo. They then apply this model of cutaneous mast cell expansion in the setting of Bd infection and find that mast cell expansion attenuates skin burden of Bd zoospores and pathologic features including epithelial thickness and improves protective mucus production and transcriptional markers of barrier function. Utilizing their prior expertise with expanding neutrophil populations in X. laevis, the authors compare mast cell expansion using (r)SCF to neutrophil expansion using recombinant colony stimulating factor 3 (rCSF3) and find that neutrophil expansion in Bd infection leads to greater burden of zoospores and worse skin pathology. Combining these two observations, they demonstrate that mast cell expansion using rSCF attenuates cutaneous neutrophilic infiltration. They further show that mast cell expansion correlates to cutaneous IL-4 expression, and that treatment with exogenous rIL-4 reduces neutrophilic infiltration and restores markers of epithelial health, offering a mechanism by which mast cell expansion protects from Bd infection.

Strengths:

The authors report a novel method of expanding amphibian mast cells utilizing their custom-made rSCF reagent. They rigorously characterize expanded mast cells in vitro and in vivo using histologic, morphologic, transcriptional, and functional assays. This establishes solid footing with which to then study the role of rSCF-stimulated mast cell expansion in the Bd infection model. This appears to be the first demonstration of exogenous use of rSCF in amphibians to expand mast cell populations and may set a foundation for future mechanistic studies of mast cells in the X. laevis model organism. Building on prior work, they are able to contrast mast cell expansion with their neutrophil expansion model, allowing them to infer a mechanistic link between mast cell expansion and IL-4 production and subsequent suppression of neutrophil infiltration and cutaneous dysbiosis.

Weaknesses:

The main weaknesses derive from technical limitations inherent to the Xenopus model at this time. For example, in mice a mechanistic study would be expected to use IL-4 knockouts, preferably mast cell-specific, to prove the link between mast cell expansion and IL-4 production being necessary and sufficient to suppress neutrophils. However, the novel reagents in this manuscript present a compelling technical advance and a step forward in the tools available to study amphibian biology.

In addition to their discussion, one open question from the revised manuscript is how a single treatment with rSCF leads to a peak in mast cell numbers and then decline to baseline in mock-infected frogs, while Bd infection either sustains rSCF-boosted mast cells or leads to steady mast cell increase over time in control-treated frogs. Whether this is mediated by endogenous SCF or some other factor remains unexplored.

Reviewer #2 (Public Review):

In this study, the authors aim to understand how neurons in the anterior insular cortex (insula) modulate fear behaviors. They report that the activity of a subpopulation of insula neurons is positively correlated with freezing behaviors, while the activity of another subpopulation of neurons is negatively correlated to the same freezing episodes. They then used optogenetics and showed that activation of anterior insula excitatory neurons during tones predicting a footshock increases the amount of freezing outside the tone presentation, while optogenetic inhibition had no effect. Finally, they found that two neuronal projections of the anterior insula, one to the amygdala and another to the medial thalamus, are increasing and decreasing freezing behaviors respectively.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

After revision, the bioinformatics section of the methods is still jumbled and may indicate issues in the pipeline. Important parameters are not included to replicate analyses. Merging the forward and reverse reads may represent a problem for denoising. Chimera detection was performed prior to denoising.

Potential denoising issues for NovaSeq data was not addressed in the response. The authors did not clarify if multiple testing correction was applied; however, it may be assumed not as written. The raw sequencing data made available through the SRA accession (if for the correct project) indicates it was a MiSeq platform; however, the sample names do not appear to link up to this experimental design and metadata not sufficient to replicate analyses.

Reviewer #2 (Public Review):

Deciphering the metabolic alterations characterizing the prediabetes-diabetes spectrum could provide early time windows for targeted preventive measures to extend precision medicine while avoiding disproportionate healthcare costs. The authors identified a panel of 9 circulating metabolites combined with basic clinical variables that significantly improved the prediction from prediabetes to diabetes. These findings provided insights into the integration of these metabolites into clinical and public health practice. However, the interpretation of these findings should take account of the following limitations.

First, the causal relationship between identified metabolites and diabetes or prediabetes deserves to be further examined particularly when the prediabetic status was partially defined. Some metabolites might be the results of prediabetes rather than the casual factors for progression to diabetes.

Second, the blood samples were taken at random (not all in a non-fasting state) and so the findings were subjected to greater variability. This should be discussed in the limitations.

Third, the strength of NMR in metabolic profiling compared to other techniques (i.e., mass spectrometry [MS], another commonly used metabolic profiling method) could be added in the Discussion section.

Fourth, the applied platform focuses mostly on lipid species which may be a limitation as well.

Fifth, it is a very large group with pre-diabetes, but the results only apply to prediabetes and not to the general population. This should be clear, although the authors have also validated the predictive value of these metabolites in the general population.

Reviewer #2 (Public Review):

Summary:

This study explores the fundamental neuroscience question of the stability of neuronal representation. The concept of 'representational-drift' has been put forward after observations made using 2-photon imaging of neuronal activity over many days revealed that neurons contribute in a time-limited manner to population representation of stimuli or experiences. The authors contribute to the still contested concept of 'drifts' by measuring representation across days using electrophysiology and thus with sufficient temporal resolution to characterize the receptive fields of neurons in timescales relevant to the stimuli used. The data obtained from chronic recordings over days combined with nonlinear stimulus-response estimation allows the authors to conclude that both the spectrotemporal receptive fields as well as contextual gain fields dependent on combination sensitivity to complex stimuli were stable over time. This suggests that when a neuron is responsive to experimental parameters across long periods of time (days), its sensory receptive field is stable.

Strengths:

The strength of this study lies in the capacity to draw novel conclusions on auditory cortex representation based on the experimentally difficult combination of stable recordings of neuronal activity, behavior, and pupil over days and state-of-the-art analysis of receptive fields.

Weaknesses:

It would have been desirable, but too ambitious in the current setting, to be able to assess what proportion if any of the neurons drop out or in to draw a closer parallel with the 2-photon studies.

Reviewer #2 (Public Review):

Summary:

This study is an investigation of galanin and galanin receptor signaling on whole-brain activity in the context of recurrent seizure activity or under homeostatic basal conditions. The authors primarily use calcium imaging to observe whole-brain neuronal activity accompanied by galanin qPCR to determine how manipulations of galanin or the galr1a receptor affect the activity of the whole-brain under non-ictal or seizure event conditions. The authors' Eaat2a-/- model (introduced in their Glia 2022 paper, PMID 34716961) that shows recurrent seizure activity alongside suppression of neuronal activity and locomotion in the time periods lacking seizures is used in this paper in comparison to the well-known pentylenetetrazole (PTZ) pharmacological model of epilepsy in zebrafish. Given the literature cited in their Introduction, the authors reasonably hypothesize that galanin will exert a net inhibitory effect on brain activity in models of epilepsy and at homeostatic baseline, but were surprised to find that this hypothesis was only moderately supported in their Eaat2a-/- model. In contrast, under PTZ challenge, fish with galanin overexpression showed increased seizure number and reduced duration while fish with galanin KO showed reduced seizure number and increased duration. These results would have been greatly enriched by the inclusion of behavioral analyses of seizure activity and locomotion (similar to the authors' 2022 Glia paper and/or PMIDs 15730879, 24002024). In addition, the authors have not accounted for sex as a biological variable, though they did note that sex sorting zebrafish larvae precludes sex selection at the younger ages used. It would be helpful to include smaller experiments taken from pilot experiments in older, sex-balanced groups of the relevant zebrafish to increase confidence in the findings' robustness across sexes. A possible major caveat is that all of the various genetic manipulations are non-conditional as performed, meaning that developmental impacts of galanin overexpression or galanin or galr1a knockout on the observed results have not been controlled for and may have had a confounding influence on the authors' findings. Overall, this study is important and solid (yet limited), and carries clear value for understanding the multifaceted functions that neuronal galanin can have under homeostatic and disease conditions.

Strengths:

- The authors convincingly show that galanin is upregulated across multiple contexts that feature seizure activity or hyperexcitability in zebrafish, and appears to reduce neuronal activity overall, with key identified exceptions (PTZ model).

- The authors use both genetic and pharmacological models to answer their question, and through this diverse approach, find serendipitous results that suggest novel underexplored functions of galanin and its receptors in basal and disease conditions. Their question is well-informed by the cited literature, though the authors should cite and consider their findings in the context of Mazarati et al., 1998 (PMID:982276). The authors' Discussion places their findings in context, allowing for multiple interpretations and suggesting some convincing explanations.

- Sample sizes are robust and the methods used are well-characterized, with a few exceptions (as the paper is currently written).

- Use of a glutamatergic signaling-based genetic model of epilepsy (Eaat2a-/-) is likely the most appropriate selection to test how galanin signaling can alter seizure activity, as galanin is known to reduce glutamatergic release as an inhibitory mechanism in rodent hippocampal neurons via GalR1a (alongside GIRK activation effects). Given that PTZ instead acts through GABAergic signaling pathways, it is reasonable and useful to note that their glutamate-based genetic model showed different effects than did their GABAergic-based model of seizure activity.

Weaknesses:

- The authors do not include behavioral assessments of seizure or locomotor activity that would be expected in this paper given their characterizations of their Eaat2a-/- model in the Glia 2022 paper that showed these behavioral data for this zebrafish model. These data would inform the reader of the behavioral phenotypes to expect under the various conditions and would likely further support the authors' findings if obtained and reported.

- No assessment of sex as a biological variable is included, though it is understood that these specific studied ages of the larvae may preclude sex sorting for experimental balancing as stated by the authors.

- The reported results may have been influenced by the loss or overexpression of galanin or loss of galr1a during developmental stages. The authors did attempt to use the hsp70l system to overexpress galanin, but noted that the heat shock induction step led to reduced brain activity on its own (Supplementary Figure 1). Their hsp70l:gal model shows galanin overexpression anyways (8x fold) regardless of heat induction, so this model is still useful as a way to overexpress galanin, but it should be noted that this galanin overexpression is not restricted to post-developmental timepoints and is present during development.

Reviewer #2 (Public Review):

Summary:

In the study titled "Functional genomics reveals the mechanism of hypoxic adaptation in nontuberculous mycobacteria" by Tateishi et al., the authors have used TnSeq to identify the common essential and growth-defect-associated genes that represent the genomic diversity of clinical M. intracellulare strains in comparison to the reference type strain. By estimating the frequency of Tn insertion, the authors speculate that genes involved in gluconeogenesis, the type VII secretion system, and cysteine desulfurase are relatively critical in the clinical MAC-PD strains than in the type strain, both for the extracellular survival and in a mouse lung infection model.

Based on their analysis, the authors proposed to identify the mechanism of hypoxic adaptation in nontuberculous mycobacteria (NTM) which offer promising drug targets in the strains causing clinical Mycobacterium avium-intracellulare complex pulmonary disease (MAC-PD).

Strengths:

A major strength of the manuscript is the performance of the exhaustive set of TnSeq experiments with multiple strains of M. intracellulare during in vitro growth and animal infection.

Weaknesses:

(1) The study suffers from the authors' preconceived bias toward a small subset of genes involved in hypoxic pellicle formation in ATCC13950.

(2) An important set of data with the ATCC13950 reference strain is missing in the mouse infection study. In the absence of this, it is difficult to establish whether the identified genes are critical for infection/intracellular proliferation, specifically in the clinical isolates that are relatively more adapted for hypoxia.

(3) Statistical enrichment analysis of gene sets by GSEA wrongly involves genes required for hypoxic pellicle formation in ATCC13950 together with the gene sets found essential in the clinical MAC-PD strains, to claim that a significant % of genes belong to hypoxia-adaptation pathways. It could be factually incorrect because a majority of these might overlap with those found critical for the in vitro survival of MAC-PD strains (and may not be related to hypoxia).

(4) Validation of mouse infection experiments with individual mutants is missing.

(5) Phenotypes with TnSeq and CRISPRi-based KD exhibit poor correlation with misleading justifications by the authors.

In summary, this study is unable to provide mechanistic insights into why and how different MAC-PD mutant strains exhibit differential survival (in vitro and in animals) and adaptation to hypoxia. It remains to understand why the clinical strains show better adaptation to hypoxia and what is the impact of other stresses on their growth rates.

Reviewer #2 (Public Review):

The manuscript entitled "Intestinal microbiome dysbiosis increases Mycobacteria pulmonary colonization in mice by regulating the Nos2-associated pathways" by Han et al reported that using clindamycin, an antibiotic to selectively disorder anaerobic Bacteriodetes, intestinal microbiome dysbiosis resulted in Mycobacterium smegmatis (MS) colonization in the mice lungs. The authors found that clindamycin induced damage of the enterocytes and gut permeability and also enhanced the fermentation of cecum contents, which finally increased MS colonization in the mice's lungs. The study showed that gut microbiota dysbiosis up-regulated the Nos2 gene-associated pathways, leading to increased nitric oxide (NO) levels and decreased reactive oxygen species (ROS) and β-defensin 1 (Defb1) levels. These changes in the host's immune response created an antimicrobial and anti-inflammatory environment that favored MS colonization in the lungs. The findings suggest that gut microbiota dysbiosis can modulate the host's immune response and increase susceptibility to pulmonary infections by altering the expression of key genes and pathways involved in innate immunity. The authors reasonably provided experimental data and subsequent gene profiles to support their conclusion. Although the overall outcomes are convincing, there are several issues that need to be addressed:

(1) In Figure S1, the reviewer suggests checking the image sizes of the pathological sections of intestinal tissue from the control group and the CL-treatment group. When compared to the same intestinal tissue images in Figure S4, they do not appear to be consistently magnified at 40x. The numerical scale bars should be presented instead of just magnification such as "40x".

(2) In Figure 4d, the ratio of Firmicutes in the CL-FMT group decreased compared to the CON-FMT group, whereas the CL-treatment group showed an increase in Firmicutes compared to the Control group in Figure 3b. The author should explain this discrepancy and discuss its potential implications on the study's findings.

(3) In Figure 6, did the authors have a specific reason for selecting Nos2 but not Tnf for further investigation? The expression level of the Tnf gene appears to be the most significant in both RT-qPCR and RNA-sequencing results in Figure 5f. Tnf is an important cytokine involved in immune responses to bacterial infections, so it is also a factor that can influence NO, ROS, and Defb1 levels.

Reviewer #2 (Public Review):

The manuscript by Carbo et al. reports a novel role for the MltG homolog AgmT in gliding motility in M. xanthus. The authors conclusively show that AgmT is a cell wall lytic enzyme (likely a lytic transglycosylase), its lytic activity is required for gliding motility, and that its activity is required for proper binding of a component of the motility apparatus to the cell wall. The data are generally well-controlled. The marked strength of the manuscript includes the detailed characterization of AgmT as a cell wall lytic enzyme, and the careful dissection of its role in motility. Using multiple lines of evidence, the authors conclusively show that AgmT does not directly associate with the motility complexes, but that instead its absence (or the overexpression of its active site mutant) results in the failure of focal adhesion complexes to properly interact with the cell wall.