Reviewer #3 (Public review):

The manuscript by Fargeot and colleagues assesses the relative effects of species and genetic diversity on ecosystem functioning. This study is very well written and examines the interesting question of whether within-species or among-species diversity correlates with ecosystem functioning, and whether these effects are consistent across trophic levels. The main findings are that genetic diversity appears to have a stronger positive effect on function than species diversity (which appears negative). These results are interesting and have value.

However, I do have some concerns that could influence the interpretation.

(1) Scale: the different measures of diversity and function for the different trophic levels are measured over very different spatial scales, for example, trees along 200 m transects and 15 cm traps. It is not clear whether trees 200 m away are having an effect on small-scale function.

(2) Size of diversity gradients: More information is needed on the actual diversity gradients. One of the issues with surveys of natural systems is that they are of species that have already gone through selection filters from a regional pool, and theoretically, if the environments are similar, you should get similar sets of species, without monocultures. So, if the species diversity gradients range from say, 6 to 8 species, but genetic diversity gradients span an order of magnitude more, you can explain much more variance with genetic diversity. Related to this, species diversity effects on function are often asymptotic at high diversity and so if you are only sampling at the high diversity range, we should expect a strong effect.

(3) Ecosystem functions: The functions are largely biomass estimates (expect decomposition), and I fail to see how the biomass of a single species can be construed as an ecosystem function. Aren't you just estimating a selection effect in this case?

Note that the article claims to be one of the only studies to look at function across trophic levels, but there are several others out there, for example:

Li, F., Altermatt, F., Yang, J., An, S., Li, A., & Zhang, X. (2020). Human activities' fingerprint on multitrophic biodiversity and ecosystem functions across a major river catchment in China. Global change biology, 26(12), 6867-6879.

Luo, Y. H., Cadotte, M. W., Liu, J., Burgess, K. S., Tan, S. L., Ye, L. J., ... & Gao, L. M. (2022). Multitrophic diversity and biotic associations influence subalpine forest ecosystem multifunctionality. Ecology, 103(9), e3745.

Moi, D. A., Romero, G. Q., Antiqueira, P. A., Mormul, R. P., Teixeira de Mello, F., & Bonecker, C. C. (2021). Multitrophic richness enhances ecosystem multifunctionality of tropical shallow lakes. Functional Ecology, 35(4), 942-954.

Wan, B., Liu, T., Gong, X., Zhang, Y., Li, C., Chen, X., ... & Liu, M. (2022). Energy flux across multitrophic levels drives ecosystem multifunctionality: Evidence from nematode food webs. Soil Biology and Biochemistry, 169, 108656.

And the case was made strongly by:

Seibold, S., Cadotte, M. W., MacIvor, J. S., Thorn, S., & Müller, J. (2018). The necessity of multitrophic approaches in community ecology. Trends in ecology & evolution, 33(10), 754-764.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #3 (Public review):

Summary:

The systematic way in which path selection is parametrically investigated is the main contribution.

Strengths:

The authors have developed an impressive workflow to study gait and gaze in natural terrain. They are able to determine footholds and gaze points in the 3D world, and explore different path selections in the terrain.

Weaknesses:

The finding that walkers prefer less tortuous, demanding paths is hardly surprising, and from the data it is still not clear what actual visual features are used to choose among alternative routes or what the nature of the decision process is. The authors discuss energetic cost and other "factors" that might influence path selection, but as yet there is no way to express these ideas rigorously in such complex natural settings.

Reviewer #3 (Public review):

This paper analyses self-citation rates in the field of Neuroscience, comprising in this case, Neurology, Neuroscience and Psychiatry. Based on data from Scopus, the authors identify self-citations, that is, whether references from a paper by some authors cite work that is written by one of the same authors. They separately analyse this in terms of first-author self-citations and last-author self-citations. The analysis is well-executed and the analysis and results are written down clearly. The interpretation of some of the results might prove more challenging. That is, it is not always clear what is being estimated.

This issue of interpretability was already raised in my review of the previous revision, where I argued that the authors should take a more explicit causal framework. The authors have now revised some of the language in this revision, in order to downplay causal language. Although this is perfectly fine, this misses the broader point, namely that it is not clear what is being estimated. Perhaps it is best to refer to Lundberg et al. (2021) and ask the authors to clarify "What is your Estimand?" In my view, the theoretical estimands the authors are interested in are causal in nature. Perhaps the authors would argue that their estimands are descriptive. In either case, it would be good if the authors could clarify that theoretical estimand.

Finally, in my previous review, I raised the issue of when self-citations become "problematic". The authors have addressed this issue satisfactorily, I believe, and now formulate their conclusions more carefully.

Lundberg, I., Johnson, R., & Stewart, B. M. (2021). What Is Your Estimand? Defining the Target Quantity Connects Statistical Evidence to Theory. American Sociological Review, 86(3), 532-565. https://doi.org/10.1177/00031224211004187

Reviewer #3 (Public Review):

In this manuscript by Berrocal and coworkers, the authors do a deep dive into the transcriptional regulation of the eve gene in both an endogenous and ectopic background. The idea is that by looking at eve expression under non-native conditions, one might infer how enhancers control transcriptional bursting. The main conclusion is that eve enhancers have not evolved to have specific behaviors in the eve stripes, but rather the same rates in the telegraph model are utilized as control rates even under ectopic or 'de novo' conditions. For example, they achieve ectopic expression (outside of the canonical eve stripes) through a BAC construct where the binding sites for the TF Giant are disrupted along with one of the eve enhancers. Perhaps the most general conclusion is that burst duration is largely constant throughout at ~ 1 - 2 min. This conclusion is consistent with work in human cell lines that enhancers mostly control frequency and that burst duration is largely conserved across genes, pointing to an underlying mechanistic basis that has yet to be determined.

Reviewer #3 (Public review):

Summary:

Mitochondrial injury activates eiF2α kinases - PERK, GCN2, HRI, and PKR - which collectively regulate the Integrated Stress Response (ISR) to preserve mitochondrial function and integrity. Previous work has demonstrated that stress-induced and pharmacologic stress-independent ISR activation promotes adaptive mitochondrial elongation via the PERK and GCN2 kinases, respectively. Here, the authors demonstrate that pharmacologic ISR inducers of HRI and GCN2 enhance mitochondrial elongation and suppress mitochondrial fragmentation in two disease models, illustrating the therapeutic potential of pharmacologic ISR activators. Specifically, the authors first used an innovative ISR translational reporter to screen for nucleoside mimetic compounds that induce ISR signaling and identified two compounds, 0357 and 3610, that preferentially activate HRI. Using a mitochondrial-targeted GFP MEF cell line, the authors next determined that these compounds (as well as the GCN2 activator, halofuginone) enhance mitochondrial elongation in an ISR-dependent manner. Moreover, pretreatment of MEFs with these ISR kinase activators suppressed pathological mitochondrial fragmentation caused by a calcium ionophore. Finally, pharmacologic HRI and GCN2 activation were found to preserve mitochondrial morphology in human fibroblasts expressing a pathologic variant in MFN2, a defect that leads to mitochondrial fragmentation and is a cause of Charcot Marie Tooth Type 2A disease.

Strengths:

This well-written manuscript has several notable strengths, including the demonstration of the potential therapeutic benefit of ISR modulation. New chemical entities with which to further interrogate this stress response pathway are also reported. In addition, the authors used an elegant screen to isolate compounds that selectively activate the ISR and identify which of the four kinases was responsible for activation. Special attention was also paid to a thorough evaluation of the effect of their compounds on other stress response pathways (i.e. the UPR, and heat and oxidative stress responses), thereby minimizing the potential for off-target effects. The implementation of automated image analysis rather than manual scoring to quantify mitochondrial elongation is not only practical but also adds to the scientific rigor, as does the complementary use of both the calcium ionophore and MFN2 models to enhance confidence and the broad therapeutic potential for pharmacology ISR manipulation.

Weaknesses:

The only minor concerns are with regard to effects on cell health and the timing of pharmacological administration.

Reviewer #3 (Public review):

Summary:

Dong et al tackle the mechanism leading to polarized migration of tracheal progenitors during Drosophila metamorphosis. This work fits in the stem cell research field and its crucial role in growth and regeneration. While it has been previously reported by others that tracheal progenitors migrate in response to FGF and Insulin signals emanating from the fat body in order to regenerate tracheal branches, the authors identified an additional mechanism involved in the communication of the fat body and tracheal progenitors.

Strengths:

The data presented were obtained using a wide range of complementary techniques combining genetics, molecular biology, quantitative, and live imaging techniques. The authors provide convincing evidence that the fat body, found in close proximity to the trachea, secrete vesicles containing the Upd2 cytokine that reach tracheal progenitors leading to JAK-STAT pathway activation, which is required for their polarized migration. In addition, the authors show that genes regulating planar cell polarity are also involved in this inter-organ communication.

Weaknesses:

(1) Affecting this inter-organ communication leads to a quite discrete phenotype where polarized migration of tracheal progenitors is partially compromised. The study lacks data showing the consequences of this phenotype on the final trachea morphology, function, and/or regeneration capacities at later pupal and adult stages. This could potentially increase the significance of the findings.

(2) The conclusions of this paper are mostly well supported by data, but some aspects of data acquisition and analysis need to be clarified and corrected, such as recurrent errors in plotting of tracheal progenitor migration distance that mislead the reader regarding the severity of the phenotype.

(3) The number of tracheal progenitors should be assessed since they seem to be found in excess in some genetic conditions that affect their behavior. A change in progenitor number could lead to crowding, thus affecting their localization rather than migration capacities, thereby changing the proposed interpretation. In addition, the authors show data suggesting a reduced progenitor migration speed when the fat body is affected, which would also be consistent with a crowding of progenitors.

(4) The authors claim that tracheal progenitors display a polarized distribution of PCP proteins that is controlled by JAK-STAT signaling. However, this conclusion is made from a single experiment that is not quantified and for which there is no explanation of how the plot profile measurements were performed. It also seems that this experiment was done only once. Altogether, this is insufficient to support the claim. Finally, a quantification of the number of posterior edges presenting filopodia rather than the number of filopodia at the anterior and posterior leading edges would be more appropriate.

(5) The authors demonstrate that Upd2 is transported through vesicles from the fat body to the tracheal progenitors where they propose they are internalized. Since the Upd2 receptor Dome ligand binding sites are exposed to the extracellular environment, it is difficult to envision in the proposed model how Upd2 would be released from vesicles to bind Dome extracellularly and activate the JAK-STAT pathway. Moreover, data regarding the mechanism of the vesicular transport of Upd2 are not fully convincing since the PLA experiments between Upd2 and Rab5, Rab7, and Lbm are not supported by proper positive and negative controls and co-immunoprecipitation data in the main figure do not always correlate to the raw data.

Reviewer #3 (Public review):

Summary:

In this study, Sanchez-Leon et al. combined extracellular recordings of Purkinje cell activity in awake and anesthetized mice with juxtacellular recordings and Purkinje cell staining to link Purkinje cell orientation to their stimulation response. The authors find a relationship between neuron orientation and firing rate, dependent on stimulation type (anodal/cathodal). They also show the effects of stimulation intensity and rebound effects.

Strengths:

Overall, the work is methodologically sound and the manuscript is well written. The authors have taken great care to explain their rationale and methodological choices.

Weaknesses:

My only reservation is the lack of reporting of the precise test statistics, p-values, and multiple comparison corrections. The work would benefit from adding this and other information.

Major Comments:

(1) The authors should report the exact test statistics. These are missing for all comparisons and hinder the reader from understanding what exactly was tested for each of the experiments. For example, having the exact test statistics would help better understand the non-significant differences in Figure 1h where there is at least a numeric difference in CS firing rate during tDCS.

(2) Did the authors apply any corrections for multiple comparisons? Generally, it would be helpful if they could clarify the statistical analysis (which values were subjected to the tests, how many tests were performed for each question, etc.).

(3) The relationship shown in Figure 2g seems to be influenced by the two outliers. Have the authors confirmed the results using a robust linear regression method?

(4) The authors conclude that tDCS modulates vermal PCs more than Crus I/II PCs - but they don't seem to test this statistically. It would be helpful to submit the firing rate change values to an actual statistical test to conclude this directly from the data

Reviewer #3 (Public review):

Experimentally, this study provides sufficient data to support the authors' conclusion that MinD dimerization but not ATPase activity is both necessary and sufficient for concentrating it and its binding partner, the division inhibitor MinC, at cell poles. Biochemical data appears to be rigorously acquired and includes proper controls. Although cytological data are consistent with the authors' model, quantitative information on MinD localization in a statistically relevant set of cells is missing (e.g. Figure 2B). 

The study's other major conclusion, as outlined in their discussion, that a reaction-diffusion model explains MinD localization in wild-type cells, is unsubstantiated. If they would like to make this a major conclusion of the final manuscript, they will need to include modeling that takes into account biochemical and cytological data. 

From a presentation perspective, the manuscript is challenging to read and will require substantial rewriting and revision prior to publication.

Reviewer #3 (Public review):

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

Comments on revised version:

The authors did a thorough revision of the manuscript which now is very clear. All the missing information has been added and the technical issue clarified. I think that it is a very good and important paper.

Reviewer #3 (Public review):

Summary:

In the current manuscript, Piccin et al. identify a role for CRF type 1 receptors in morphine-induced social deficits using a 3-chamber social interaction task in mice. They demonstrate that pre-treatment with a CRFR1 antagonist blocks morphine-induced social deficits in male, but not female, mice, and this is associated with the CRF R1 antagonist blocking morphine-induced increases in PVN neuronal excitability in male but not female mice. They followed up by using a transgenic mouse CRFR1 knockout mouse line. CRFR1 genetic deletion also blocked morphine-induced social deficits, similar to the pharmacological approach, in male mice. This was also associated with morphine-induced increases in PVN neuronal excitability being blocked in CRFR1 knockout mice. Interestingly they found that the pharmacological antagonism of the CRFR1 specifically blocked morphine-induced increases in oxytocin/AVP neurons in the PVN in male mice.

Strengths:

The authors used both male and female mice where possible and the studies were fairly well controlled. The authors provided sufficient methodological detail and detailed statistical information. They also examined measures of locomotion in all of the behavioral tasks to separate changes in sociability from overall changes in locomotion. The experiments were well thought out and well controlled. The use of both the pharmacological and genetic approaches provides converging lines of evidence for the role of CRFR1 in morphine-induced social deficits. Additionally, they have identified the PVN as a potential site of action for these CRFR1 effects.

Weaknesses:

While the authors included both sexes they analyzed them independently. This was done for simplicity's sake as they have multiple measures but there are several measures where the number of factors is reduced and the inclusion of sex as a factor would be possible. Additionally, single doses of both the CRFR1 antagonist and morphine are used within an experiment without justification for the doses. In fact, a lower dose of morphine was needed for the genetic CRFR1 mouse line. This would suggest that the dose of morphine being used is likely causing some aversion that may be more present in the females, as they have lower overall time in the ROI areas of both the object and the mouse following morphine exposure. As for the discussion, the authors do not sufficiently address why CRFR1 has an effect in males but not females and what might be driving that difference, or why male and female mice have different distribution of PVN cell types during the recordings. Additionally, the authors attribute their effect to CRF and CRFR1 within the PVN but do not consider the role of extrahypothalamic CRF and CRFR1. While the PVN does contain the largest density of CRF neurons there are other CRF neurons, notably in the central amygdala and BNST, that have been shown to play important roles in the impact of stress on drug-related behavior. This also holds true for the expression of CRFR1 in other regions of the brain, including the VTA, which is important for drug-related behavior and social behavior. The treatments used in the current manuscript were systemic or brain-wide deletion of CRFR1. Therefore, the authors should consider that the effects could be outside the PVN.

Reviewer #3 (Public Review):

Summary:

Kearns et al. explored a computational approach DASAr to identify stable peptide epitopes on SARS-CoV-2 proteins. They find that the computational approach has a high success rate at identifying stable and soluble peptides that may reserve the native conformation. The approach identified multiple peptides in Spike, Nucleoprotein, Membrane, and Envelope proteins of SARS-CoV-2. Most surprisingly, a high prevalence of IgM response is to recognize a newly exposed Membrane epitope, M1. Anti-M1 IgM titer is associated with a protective anti-Spike titer, severe disease and long COVID. The data also indicate that anti-M1 IgM may arise from T cell-independent B cell activation.

Strengths:

The computational approach can be widely applied to study antibody epitopes in many pathogens. The observations from this study provide clues to further understanding the role of anti-M1 response and the mechanisms of anti-M1 IgM response to SARS-CoV-2 associated diseases.

Weaknesses:

A subset of the conclusions of this paper are well supported by data, but some statements and analyses need to be clarified, revised, and extended.

Reviewer #3 (Public Review):

Vazquez-Fernandez et al. present a comprehensive and detailed analysis of the S. cerevisiae APC/C complex, providing new insights into its structure and function. The authors determined the medium-resolution structures of three recombinant S. cerevisiae APC/C complexes, including unphosphorylated apo-APC/C (4.9 Å), the ternary APC/CCDH1-substrate complex (APC/CCDH1:Hsl1 , 4.0 Å), and phosphorylated apo-APC/C (4.4 Å). Prior structures of human, E. cuniculi, S. cerevisiae, and S. pombe APC/C subunits, as well as AlphaFold2 predictions were used to guide model building. Although the determined structures are not sufficient to fully explain the molecular mechanism of APC/C activation and regulation in S. cerevisiae, they provide valuable insights into the similarities and differences with the human complex, shedding light on the conserved and divergent features of APC/C function.

The manuscript synthesizes the structural analysis of the APC/C complex in S. cerevisiae, with literature into a cohesive and clear picture of the complex's structure and function. It is well-written and clear, making the complex biology of the APC/C complex accessible to a wide range of readers. The complex forms a triangular shape, with a central cavity surrounded by two modules: the TPR lobe and the platform module. The TPR lobe consists of three TPR proteins (APC3, APC6, and APC8), which stack on top of each other to form a quasi-symmetric structure. The platform module is composed of the large APC1 subunit, together with APC4 and APC5. The authors also analyzed the structure of several smaller subunits that are involved in regulating the activity of the APC/C complex and showed their structural similarities to and discrepancies from their human counterparts. These subunits, including CDC26/APC12, SWM1/APC13, APC9, and MND2/APC15, form extended, irregular structures that simultaneously contact multiple large globular APC/C subunits.

While the authors report the similarity between the overall structure of S. cerevisiae and human APC/C complexes, they also found two unexpected differences. First, in the S. cerevisiae apo-complex, the E2 binding site on APC11RING is accessible, whereas, in humans, it requires CDH1 binding. Second, a structural element similar to the human APC1 auto-inhibitory segment is missing in S. cerevisiae. In humans, the phosphorylation-dependent displacement of this segment allows CDC20 binding to APC/C. In S. cerevisiae, the binding requires phosphorylation however the structures reported here are suggestive that this could involve a different (presently unknown) mechanism. These structural insights highlight the importance of understanding the species-specific features of APC/C function.

Strengths:

The manuscript does a great job of revealing new structures.

Opportunity for increasing impact: It would have been nice if some functional differences were demonstrated, for example regarding the mechanism of CDC20 binding, and the comparison between apo-APC/C and ternary APC/CCDH1:Hsl1 does not explain the molecular activation mechanism of S. cerevisiae APC/C. Nonetheless, the authors nicely integrate their data with well-established literature on the similarities and differences between yeast and human systems.

Reviewer #3 (Public review):

Summary:

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

Comment on revised version:

The reviewer has addressed all my concerns and I have no further comments.

Reviewer #3 (Public review):

Summary:

This paper identifies GTSE1 as a potential substrate of cyclin D1-CDK4/6 and shows that GTSE1 correlates with cancer prognosis, probably through an effect on cell proliferation. The main problem is that the phosphorylation analysis relies on the over-expression of cyclin D1. It is unclear if the endogenous cyclin D1 is responsible for any phosphorylation of GTSE1 in vivo, and what, if anything, this moderate amount of GTSE1 phosphorylation does to drive proliferation.

Strengths:

There are few bonafide cyclin D1-Cdk4/6 substrates identified to be important in vivo so GTSE1 represents a potentially important finding for the field. Currently, the only cyclin D1 substrates involved in proliferation are the Rb family proteins.

Weaknesses:

The main weakness is that it is unclear if the endogenous cyclin D1 is responsible for phosphorylating GTSE1 in the G1 phase. For example, in Figure 2G there doesn't seem to be a higher band in the phos-tag gel in the early time points for the parental cells. This experiment could be redone with the addition of palbociclib to the parental to see if there is a reduction in GTSE1 phosphorylation and an increase in the amount in the G1 phase as predicted by the authors' model.

The experiments involving palbociclib do not disentangle cell cycle effects. Adding Cdk4 inhibitors will progressively arrest more and more cells in the G1 phase and so there will be a reduction not just in Cdk4 activity but also in Cdk2 and Cdk1 activity. More experiments, like the serum starvation/release in Figure 2G, with synchronized populations of cells would be needed to disentangle the cell cycle effects of palbociclib treatment.

It is unclear if GTSE1 drives the G1/S transition. Presumably, this is part of the authors' model and should be tested.

The proliferation assays need to be more quantitative. Figure 4B should be plotted on a log scale so that the slope can be used to infer the proliferation rate of an exponentially increasing population of cells. Figure 4c should be done with more replicates and error analysis since the effects shown in the lower right-hand panel are modest.

Reviewer #3 (Public review):

Summary:

In this study, the authors aim to uncover how 3D tongue direction is represented in the Motor (M1o) and Somatosensory (S1o) cortex. In non-human primates implanted with chronic electrode arrays, they use X-ray-based imaging to track the kinematics of the tongue and jaw as the animal is either chewing food or licking from a spout. They then correlate the tongue kinematics with the recorded neural activity. Using linear regressions, they characterize the tuning properties and distributions of the recorded population during feeding and licking. Then, they recharacterize the tuning properties after bilateral lidocaine injections in the two sensory branches of the trigeminal nerve. They report that their nerve block causes a reorganization of the tuning properties. Overall, this paper concludes that M1o and S1o both contain representations of the tongue direction, but their numbers, their tuning properties, and susceptibility to perturbed sensory input are different.

Strengths:

The major strengths of this paper are in the state-of-the-art experimental methods employed to collect the electrophysiological and kinematic data.

Weaknesses:

However, this paper has a number of weaknesses in the analysis of this data.

It is unclear how reliable the neural responses are to the stimuli. The trial-by-trial variability of the neural firing rates is not reported. Thus, it is unclear if the methods used for establishing that a neuron is modulated and tuned to a direction are susceptible to spurious correlations. The authors do not use shuffling or bootstrapping tests to determine the robustness of their fits or determining the 'preferred direction' of the neurons. This weakness colors the rest of the paper.

The authors compare the tuning properties during feeding to those during licking but only focus on the tongue-tip. However, the two behaviors are different also in their engagement of the jaw muscles. Thus many of the differences observed between the two 'tasks' might have very little to do with an alternation in the properties of the neural code - and more to do with the differences in the movements involved. Many of the neurons are likely correlated with both Jaw movements and tongue movements - this complicates the interpretations and raises the possibility that the differences in tuning properties across tasks are trivial.

The population analyses for decoding are rudimentary and provide very coarse estimates (left, center, or right), it is also unclear what the major takeaways from the population decoding analyses are. The reduced classification accuracy could very well be a consequence of linear models being unable to account for the complexity of feeding movements, while the licking movements are 'simpler' and thus are better accounted for.

The nature of the nerve block and what sensory pathways are being affected is unclear - the trigeminal nerve contains many different sensory afferents - is there a characterization of how effectively the nerve impulses are being blocked? Have the authors confirmed or characterized the strength of their inactivation or block, I was unable to find any electrophysiological evidence characterizing the perturbation.

Overall, while this paper provides a descriptive account of the observed neural correlations and their alteration by perturbation, a synthesis of the observed changes and some insight into neural processing of tongue kinematics would strengthen this paper.

https://www.franklin-christoph.com/

Among various fountain pens, notebooks, and other papers, they sell archival quality 3 x 5 inch index cards suited to fountain pen use.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

(1) The characters (NAD+ and Zn2+-independent deacetylase activity in an unknown domain) shown in this study are very unexpected. To convince readers, MSMS data must be necessary to accurately detect (de)acetylation at the target site in the deacetylase activity assay. The authors showed the MSMS data in assays with acetylated BSA, but other assays only rely on western blot.

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

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

Reviewer #3 (Public review):

Summary:

The authors have compared different groups of AUD patients at different levels and have examined metabolomics.

Strengths:

A well-written and comprehensive study.

Reviewer #3 (Public review):

This revised study described changes in membrane excitability and Na+ and K+ current amplitudes of sympathetic motor neurons in culture. The findings indicate that neurons isolated from aged animals show increased membrane excitability manifested as increased firing rates in response to electrical stimulation and changes in related membrane properties including depolarized resting membrane potential, increased rheobase, and spontaneous firing. By contrast, neuron cultures from young mice show little to no spontaneous firing and relatively low firing rates in response to current injection. These changes in excitability correlate with reductions in the magnitude of KCNQ currents in neurons cultured from aged mice compared to neurons from cultured from young mice. The authors conclude that aging promotes hyperexcitability of sympathetic motor neurons through changes in KCNQ channels.

Reviewer #3 (Public review):

Summary:

The authors studied the function of Hsp110 co-chaperones (e.g. yeast Sse1) in Hsp70-dependent protein disaggregation reactions. The study builds on former work by the authors (Wyszkowski et al., 2021, PNAS), analyzing the binding of Hsp70 and J-domain protein (JDP) cochaperones to protein aggregates using bio-layer interferometry (BLI). It was shown before by other groups that Hsp110 enhances Hsp70 disaggregation activity. The mechanism of Hsp110-stimulated disaggregation activity, however, remained poorly defined. Here, the authors show that yeast Hsp110 increases Hsp70 recruitment to the surface of protein aggregates. The effect is largely dependent on J-domain protein (JDP) identity and particularly pronounced for class B JDPs (e.g. yeast Sis1), which are also more effective in disaggregation reactions. The authors also confirm former results, showing inhibition by increased Hsp110 levels and provide novel evidence that the inhibitory effect is caused by competition between Hsp110 and JDPs for Hsp70 binding.

Strengths:

The work represents a very thoroughly executed study, which provides novel insights into the mechanism of Hsp70-mediated protein disaggregation. Key findings established for yeast chaperones are also documented for human counterparts. The observation that Hsp110 might displace JDPs from Hsp70 during the disaggregation reaction is very appealing. It will now become important to validate this initial finding and dissect how it propels the disaggregation reaction.

Weaknesses:

How exactly the interplay between JDPs and Hsp110 orchestrates protein disaggregation remains largely speculative and further analysis is required for a deeper mechanistic understanding.

Reviewer #4 (Public review):

Summary:

Li and Chouhan et al. follow up on a previous publication describing the role of anterior-posterior (ap) and medial (m) ɑ′/β′ Kenyon cells in mediating sleep-dependent and sleep-independent memory consolidation, respectively, based on feeding state in Drosophila melanogaster. The authors sequenced bulk RNA of ap ɑ′/β′ Kenyon cells 1h after flies were either trained-fed, trained-starved or untrained-fed and find a small number of genes (59) differentially expressed (3 upregulated, 56 downregulated) between trained-fed and trained-starved conditions. Many of these genes encode proteins involved in the regulation of gene expression. The authors then screened these differentially expressed genes for sleep phenotypes by expressing RNAi hairpins constitutively in ap ɑ′/β′ Kenyon cells and measuring sleep patterns. Two hits were selected for further analysis: Polr1F, which promoted sleep, and Regnase-1, which reduced sleep. The pan-neuronal expression of Polr1F and Regnase-1 RNAi constructs was then temporally restricted to adult flies using the GeneSwitch system. Polr1F sleep phenotypes were still observed, while Regnase-1 sleep phenotypes were not, indicating developmental defects. Appetitive memory was then assessed in flies with constitutive knockdown of Polr1F and Regnase-1 in ap ɑ′/β′ Kenyon cells. Polr1F knockdown did not affect sleep-dependent or sleep-independent memory, while Regnase-1 knockdown disrupted sleep-dependent memory, sleep-independent memory, as well as learning. Polr1F knockdown increased pre-ribosomal RNA transcripts in the brain, as measured by qPCR, in line with its predicted role as part of the RNA polymerase I complex. A puromycin incorporation assay to fluorescently label newly synthesized proteins also indicated higher levels of bulk translation upon Polr1F knockdown. Regnase-1 knockdown did not lead to observable changes in measurements of bulk translation.

Strengths:

The proposed involvement of RNA processing genes in regulating sleep and memory processes is interesting, and relatively unexplored. The methods are satisfactory.

Weaknesses:

The main weakness of the paper is in the overinterpretation of their results, particularly relating to the proposed link between sleep and memory consolidation, as stated in the title. Constitutive Polr1F knockdown in ap ɑ′/β′ Kenyon cells had no effect on appetitive long-term memory, while constitutive Regnase-1 knockdown affected both learning and memory. Since the effects of constitutive Regnase-1 knockdown on sleep could be attributed to developmental defects, it is quite plausible that these same developmental defects are what drive the observed learning and memory phenotypes. In this case, an alternative explanation of the authors' findings is that constitutive Regnase-1 knockdown disrupts the entire functioning of ap ɑ′/β′ Kenyon cells, and as a consequence behaviors involving these neurons (i.e. learning, memory and sleep) are disrupted. It will be important to provide further evidence of the function of RNA processing genes in memory in order to substantiate the memory link proposed by the authors.

Reviewer #3 (Public review):

Summary:

Rollenhagen et al. offer a detailed description of layer 1 of the human neocortex. They use electron microscopy to assess the morphological parameters of presynaptic terminals, active zones, vesicle density/distribution, mitochondrial morphology, and astrocytic coverage. The data is collected from tissue from four patients undergoing epilepsy surgery. As the epileptic focus was localized in all patients to the hippocampus, the tissue examined in this manuscript is considered non-epileptic (access) tissue.

Strengths:

The quality of the electron microscopic images is very high, and the data is analyzed carefully. Data from human tissue is always precious and the authors here provide a detailed analysis using adequate approaches, and the data is clearly presented.

Weaknesses:

The study provides only morphological details, these can be useful in the future when combined with functional assessments or computational approaches. The authors emphasize the importance of their findings on astrocytic coverage and suggest important implications for glutamate spillover. However, the percentage of synapses that form tripartite synapses has not been quantified, the authors' functional claims are based solely on volumetric fraction measurements.

The distinction between excitatory and inhibitory synapses is not clear, they should be analyzed separately.

The text connects functional and morphological characteristics in a very direct way. For example, connecting plasticity to any measurement the authors present would be rather difficult without any additional functional experiments. References to various vesicle pools based on the location of the vesicles are also more complex than suggested in the manuscript. The text should better reflect the limitations of the conclusions that can be drawn from the authors' data.

Reviewer #3 (Public review):

Summary:

This work demonstrates that people's imprecision in numeric perception varies with the stimulus context and task goal. By measuring imprecision across different widths of uniform prior distributions in estimation and discrimination tasks, the authors find that imprecision changes sublinearly with prior width, challenging previous range normalization models. They further show that these changes align with the efficient encoding model, where decision-makers balance expected rewards and encoding costs optimally.

Strengths:

The experimental design is straightforward, controlling the mean of the number distribution while varying the prior width. By assessing estimation errors and discrimination accuracy, the authors effectively highlight how imprecision adjusts across conditions.

The model's predictions align well with the data, with the exponential terms (1/2 and 3/4) of imprecision changes matching the empirical results impressively.

Weaknesses:

Some details in the model section are unclear. Specifically, I'm puzzled by the Wiener process assumption where r∣x∼N(m(x)T,s^2T). Does this imply that both the representation of number x and the noise are nearly zero at the beginning, increasing as observation time progresses? This seems counterintuitive, and a clearer explanation would be helpful.

The authors explore range normalization models with Gaussian representation, but another common approach is the logarithmic representation (Barretto-García et al., 2023; Khaw et al., 2021). Could the logarithmic representation similarly lead to sublinearity in noise and distribution width?

Additionally, Heng et al. (2020) found that subjects did not alter their encoding strategy across different task goals, which seems inconsistent with the fully adaptive representation proposed here. I didn't find the analysis of participants' temporal dynamics of adaptation. The behavioral results in the manuscript seem to imply that the subjects adopted different coding schemes in a very short period of time. Yet in previous studies of adaptation, experimental results seem to be more supportive of a partial adaptive behavior (Bujold et al., 2021; Heng et al., 2020), which might balance experimental and real-world prior distributions. Analyzing temporal dynamics might provide more insight. Noting that the authors informed subjects about the shape of the prior distribution before the experiment, do the results in this manuscript suggest a top-down rapid modulation of number representation?

Barretto-García, M., De Hollander, G., Grueschow, M., Polanía, R., Woodford, M., & Ruff, C. C. (2023). Individual risk attitudes arise from noise in neurocognitive magnitude representations. Nature Human Behaviour, 7(9), 1551-1567. https://doi.org/10.1038/s41562-023-01643-4

Bujold, P. M., Ferrari-Toniolo, S., & Schultz, W. (2021). Adaptation of utility functions to reward distribution in rhesus monkeys. Cognition, 214, 104764. https://doi.org/10.1016/j.cognition.2021.104764

Heng, J. A., Woodford, M., & Polania, R. (2020). Efficient sampling and noisy decisions. eLife, 9, e54962. https://doi.org/10.7554/eLife.54962

Khaw, M. W., Li, Z., & Woodford, M. (2021). Cognitive Imprecision and Small-Stakes Risk Aversion. The Review of Economic Studies, 88(4), 1979-2013. https://doi.org/10.1093/restud/rdaa044

Reviewer #3 (Public review):

Summary:

The activity of serotonin (5-HT) releasing neurons as well as 5-HT levels in brain structures targeted by serotonergic axons are known to fluctuate substantially across the animal's sleep/wake cycle, with high 5-HT levels during wakefulness (WAKE), intermediate levels during non-REM sleep (NREM) and very low levels during REM sleep. Recent studies have shown that during NREM, the activity of 5-HT neurons in raphe nuclei oscillates at very low frequencies (0.01 - 0.05 Hz) and this ultraslow oscillation is negatively coupled to broadband EEG power. However, how exactly this 5-HT oscillation affects neural activity in downstream structures is unclear.

The present study addresses this gap by replicating the observation of the ultraslow oscillation in the 5-HT system, and further observing that hippocampal sharp wave-ripples (SWRs), biomarkers of offline memory processing, occur preferentially in barrages on the falling phase of the 5-HT oscillation during both wakefulness and NREM sleep. In contrast, the raising phase of the 5-HT oscillation is associated with microarousals during NREM and increased muscular activity during WAKE. Finally, the raising 5-HT phase was also found to be associated with increased synchrony between the hippocampus and neocortex. Overall, the study constitutes a valuable contribution to the field by reporting a close association between raising 5-HT and arousal, as well as between falling 5-HT and offline memory processes.

Strengths:

The study makes compelling use of the state-of-the-art methodology to address its aims: the genetically encoded 5-HT sensor used in the study is ideal for capturing the ultraslow 5-HT dynamics and the novel detection method for SWRs outperforms current state-of-the-art algorithms and will be useful to many scientists in the field. Explicit validation of both of these methods is a particular strength of this study.

The analytical methods used in the article are appropriate and are convincingly applied, the use of a general linear mixed model for statistical analysis is a particularly welcome choice as it guards against pseudoreplication while preserving statistical power.

Overall, the manuscript makes a strong case for distinct sub-states across WAKE and NREM, associated with different phases of the 5-HT oscillation.

Weaknesses:

All of the evidence presented in the study is correlational. While the study mostly avoids claims of causality, it would still benefit from establishing whether the 5-HT oscillation has a direct role in the modulation of SWR rate via e.g. optogenetic activation/inactivation of 5-HT axons. As it stands, the possibility that 5-HT levels and SWRs are modulated by the same upstream mechanism cannot be excluded.

Reviewer #3 (Public review):

Summary:

In this study, Stein and colleagues use a clever masking/attentional blink paradigm using Kanisza stimuli, coupled with EEG decoding and the NMDA antagonist memantine, to isolate putative neural markers of feedforward, lateral, and feedback processing.

In two elegant experiments, they show that memantine selective influences EEG decoding of only illusory Kanisza surfaces (but not contour continuation or raw contrast), only when unmasked, only when attention is available (not when "blinked"), and only when task-relevant.

This neatly implicates NMDA receptors in the feedback mechanisms that are believed to be involved in inferring illusory Kanisza surfaces, and builds a difficult bridge between the large body of human perceptual experiments and pharmacological and neurophysiological work in animals.

Strengths:

Three key strengths of the paper are<br /> (1) The elegant and thorough experimental design, which includes internal replication of some key findings.<br /> (2) The clear pattern of results across the full set of experiments.<br /> (3) The clear writing and presentation of results.

The paper effectively reports a 4-way interaction, with memantine only influencing decoding of surfaces (1) that are unmasked (2), with attention available (3) and task-relevant (4). Nevertheless, the results are very clear, with a clear separation between null effects on other conditions and quite a strong (and thus highly selective) effect on this one intersection of conditions. This makes the pattern of findings very convincing.

Weaknesses:

Overall this is an impressive and important paper. However, to my mind, there are two minor weaknesses.

First, despite its clear pattern of neural effects, there is no corresponding perceptual effect. Although the manipulation fits neatly within the conceptual framework, and there are many reasons for not finding such an effect (floor and ceiling effects, narrow perceptual tasks, etc), this does leave open the possibility that the observation is entirely epiphenomenal, and that the mechanisms being recorded here are not actually causally involved in perception per se.

Second, although it is clear that there is an effect on decoding in this particular condition, what that means is not entirely clear - particularly since performance improves, rather than decreases. It should be noted here that improvements in decoding performance do not necessarily need to map onto functional improvements, and we should all be careful to remain agnostic about what is driving classifier performance. Here too, the effect of memantine on decoding might be epiphenomenal - unrelated to the information carried in the neural population, but somehow changing the balance of how that is electrically aggregated on the surface of the skull. *Something* is changing, but that might be a neurochemical or electrical side-effect unrelated to actual processing (particularly since no corresponding behavioural impact is observed.)

Reviewer #3 (Public review):

Summary:

This study provides evidence for translational changes in inhibitory spinal dorsal horn neurons following chronic nerve injury. Gene expression changes have been widely studied in the context of pain induction and provided key insights into the adaptation of the nervous system in the early phases of chronic pain. Whereas this is interesting biologically, most patients will arrive in the clinic beyond the acute phase of their injury, thus limiting the translational relevance of these studies. Recent studies have extended this work to highlight the difference between acute and chronic pain states, potentially explaining the cascading factors leading to chronic pain, and hopefully how to prevent this in vulnerable populations. The present study suggests that translational changes within spinal inhibitory populations could underlie long-term chronic pain, leading to decreased inhibition and heightened pain thresholds.

Strengths:

The approaches used and the broad outcomes of the manuscript are interesting and could be an exciting development in the field. The authors are using approaches more common in molecular biology and extending these into neuroscientific research, getting into the detail of how pathology could impact gene expression differentially across the course of an injury. This could open up new areas of research to selectively target not only defined populations but additionally help alleviate pain symptoms once an injury has already reached the maintenance phase. There is an opportunity to delve into what must be a very large data set and learn more about what genes are differentially translated and how this could affect circuit function.

Weaknesses:

Whereas the authors approach a key question in pain chronicity, the manuscript falls a little short of providing any conclusive data.

The manuscript was in some areas very difficult to follow. Terminology was not always consistent or clear, and the flow of the manuscript could use some attention to highlight key areas. Whereas the overall message is clear in the summary, this would not necessarily be the case when reading the manuscript alone.

The study claims to show that translational control mechanisms in the spinal cord play a role in mediating neuropathic pain hypersensitivity, but the studies presented do not fully support this statement. The authors instead provide some correlation between translation and behavioural reflex excitability (namely vfh and Hargreaves).

It is difficult to fully interpret the work, as there are a number of inconsistencies, namely the range of timings pre- and post-injury, lack of controls for manipulations, the use of shmiRNA versus lineage deletions, and lack of detailed somatosensory testing. It is not completely clear how this work could be translatable as is, without a deeper understanding of how translational control affects circuit function and whether all of this is necessarily bad for the system, or whether this is a positive homeostatic adaptation to the hyperexcitability of the circuit following injury.

A large portion of the work is focussed on showing an inhibitory-selective change in translation following chronic nerve injury. The evidence for this is however lacking. Statistics to show that translational effects are restricted to inhibitory subpopulations are inadequate. The author's choice of transgenic lines is not clear and seems to rely on availability rather than hypothesis.

Reviewer #3 (Public review):

The authors delved into an important aspect of abortifacient diseases of livestock in Tanzania. The thoughts of the authors on the topic and its significance have been clarified. The number of wards in the study area, statistical selection of wards, type of questionnaire ie open or close ended. and statistical analyses of outcomes have been clearly elucidated in the manuscript. The exclusion criteria for two wards out of the fifteen wards mentioned in the text are clearly stated. Observations were from pastoral, agro-pastoral and small holder agro ecological farmers. Sample numbers or questionnaires attributed to the above farming systems correlate findings with management systems. The impacts of the research investigation output are clearly visible as to warrant intervention methods. The identified pathogens from laboratory investigation, particularly with the use of culture and PCR, as well as the zoonotic pathogens encountered are stated in the manuscript and the supplementary files.

In conclusion, based on the intent of the authors and content of this research, and the weight of the research topic, the seeming weaknesses in the critical data analysis observed have been clarified, to demonstrate cause, effect and impact.

The authors have carried out the necessary corrections.

The findings do imply that identification of some of the abortifacient of livestock in Tanzania will necessitate important interventions in the control of the diseases in the study area

Reviewer #3 (Public review):

The study provides a detailed analysis of the chromosomal rearrangements related to the deletions of histidine-rich protein 2 (pfhrp2) and pfhrp3 genes in P. falciparum that have clinical significance since malaria rapid diagnostic tests detect these parasite proteins. A large number of publicly available short sequence reads for whole-genome of the parasite were analyzed and data on coverage and on discordant mapping allowed to identify deletions, duplications and chromosomal rearrangements related to pfhrp3 deletions. Long-read sequences showed support for the presence of a normal chromosome 11 and a hybrid 13-11 chromosome lacking pfhrp3 in some of the pfhrp3-deleted parasites. The findings support that these translocations have repeatedly occurred in natural populations. The authors discuss the implications of these findings and how they support or not previous hypothesis on the emergence of these deletions and the possible selective pressures involved.

The genomic regions where these genes are located are challenging to study since they are highly repetitive and paralogous and the use of long read sequencing allowed to span the duplicated regions, giving support to the identification of the hybrid 13-11 chromosome.

All publicly available whole-genome sequences of the malaria parasite from around the world were analysed which allowed an overview of the worldwide variability, even though this analysis is biased by the availability of sequences, as the authors recognize.

Despite the reduced sample size, the detailed analysis of haplotypes and identification of location of breakpoints gives support to a single origin event for the 13-5++ parasites.

The analysis of haplotype variation across the duplicated chromosome-11 segment identified breakpoints at varied locations that support multiple translocation events in natural populations. The authors suggest these translocations may be occurring at high frequency in meiosis in natural populations but strongly selected against in most circumstances, which remains to be tested.

In this new version, the authors have addressed the points raised previously and adequately discuss the limitations of the study.

Reviewer #3 (Public review):

Summary:

The authors applied an innovative approach (CO-Detection by indEXing - CODEX) together with sophisticated computational analyses to image pancreas tissues from rare organ donors with type 1 diabetes. They aimed to assess key features of inflammation in both islet and extra-islet tissue areas; they reported that the extra-islet space of lobules with extensive islet infiltration differs from the extra-islet space of less infiltrated areas within the same tissue section. The study also identifies four sub-states of inflamed islets characterized by the activation profiles of CD8+T cells enriched in islets relative to the surrounding tissue. Lymphoid structures are identified in the pancreas tissue away from islets, and these were enriched in CD45RA+ T cells - a population also enriched in one of the inflamed islet sub-states. Together, these data help define the coordination between islets and the extra-islet pancreas in the pathogenesis of human T1D.

Strengths:

The analysis of tissue from well-characterized organ donors, provided by the Network for the Pancreatic Organ Donor with Diabetes, adds strength to the validity of the findings.

By using their innovative imaging/computation approaches, key known features of islet autoimmunity were confirmed, providing validation of the methodology.

The detection of IDO+ vasculature in inflamed islets - but not in normal islets or islets that have lost insulin-expression links this expression to the islet inflammation, and it is a novel observation. IDO expression in the vasculature may be induced by inflammation and may be lost as disease progresses, and it may provide a potential therapeutic avenue.

The high-dimensional spatial phenotyping of CD8+T cells in T1D islets confirmed that most T cells were antigen-experienced. Some additional subsets were noted: a small population of T cells expressing CD45RA and CD69, possibly naive or TEMRA cells, and cells expressing Lag-3, Granzyme-B, and ICOS.

While much attention has been devoted to the study of the insulitis lesion in T1D, our current knowledge is quite limited; the description of four sub-clusters characterized by the activation profile of the islet-infiltrating CD8+T cells is novel. Their presence in all T1D donors indicates that the disease process is asynchronous and is not at the same stage across all islets. Although this concept is not novel, this appears to be the most advanced characterization of insulitis stages.

When examining together both the exocrine and islet areas, which is rarely done, authors report that pancreatic lobules affected by insulitis are characterized by distinct tissue markers. Their data support the concept that disease progression may require crosstalk between cells in the islet and extra-islet compartments. Lobules enriched in β-cell-depleted islets were also enriched in nerves, vasculature, and Granzyme-B+/CD3- cells, which may be natural killer cells.

Lastly, authors report that immature tertiary lymphoid structures (TLS) exist both near and away from islets, where CD45RA+ CD8+T cells aggregate, and also observed an inflamed islet-subcluster characterized by an abundance of CD45RA+/CD8+ T cells. These TLS may represent a point of entry for T cells and this study further supports their role in islet autoimmunity.

Weaknesses:

As the authors themselves acknowledge, the major limitation is that the number of donors examined is limited as those satisfying study criteria are rare. Thus, it is not possible to examine disease heterogeneity and the impact of age at diagnosis. Of 8 T1D donors examined, 4 would be considered newly diagnosed (less than 3 months from onset) and 4 had longer disease durations (2, 2, 5, and 6 years). It was unclear if disease duration impacted the results in this small cohort. In the introduction, the authors discuss that most of the pancreata from nPOD donors with T1D lack insulitis. This is correct, yet it is a function of time from diagnosis. Donors with shorter duration will be more likely to have insulitis. A related point is that the proportion of islets with insulitis is low even near diagnosis, Finally, only one donor was examined that while not diagnosed with T1D, was likely in the preclinical disease stage and had autoantibodies and insulitis. This is a critically important disease stage where the methodology developed by the investigators could be applied in future efforts.

While this was not the focus of this investigation, it appears that the approach was very much immune-focused and there could be value in examining islet cells in greater depth using the methodology the authors developed.

Additional comments:

Overall, the authors were able to study pancreas tissues from T1D donors and perform sophisticated imaging and computational analysis that reproduce and importantly extend our understanding of inflammation in T1D. Despite the limitations associated with the small sample size, the results appear robust, and the claims well-supported.

The study expands the conceptual framework of inflammation and islet autoimmunity, especially by the definition of different clusters (stages) of insulitis and by the characterization of immune cells in and outside the islets.

DOI: 10.1038/s41467-023-42626-3

Resource: RRID:BDSC_26314

Curator: @maulamb

SciCrunch record: RRID:BDSC_26314

What is this?

Reviewer #3 (Public review):

Summary:

The authors developed and optimized the methods for detecting G4s and R-loops independent of BG4 and S9.6 antibody, and mapped genomic native G4s and R-loops by HepG4-seq and HBD-seq, revealing that co-localized G4s and R-loops participate in regulating transcription and affecting the self-renewal and differentiation capabilities of mESCs.

Strengths:

By utilizing the peroxidase activity of G4-hemin complex and combining proximity labeling technology, the authors developed HepG4-seq (high throughput sequencing of hemin-induced proximal labelled G4s) , which can detect the dynamics of G4s in vivo. Meanwhile, the "GST-His6-2xHBD"-mediated CUT&Tag protocol (Wang et al., 2021) was optimized by replacing fusion protein and tag, the optimized HBD-seq avoids the generation of GST fusion protein aggregates and can reflect the genome-wide distribution of R-loops in vivo.

The authors employed HepG4-seq and HBD-seq to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). The data indicate that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes.

Combined with Dhx9 ChIP-seq and co-localized G4s and R-loops data in wild-type and dhx9KO mESCs, the authors found that the helicase Dhx9, a major regulator of co-localized G4s and R-loops, affects the self-renewal and differentiation capacities of mESCs.

In conclusion, the authors provide an approach to study the interplay between G4s and R-loops, shedding light on the important roles of co-localized G4s and R-loops in development and disease by regulating the transcription of related genes.

Weaknesses:

As we know, there are at least two structure data of S9.6 antibody very recently, and the questions about the specificity of the S9.6 antibody on RNA:DNA hybrids should be finished. The authors referred (Hartono et al., 2018; Konig et al., 2017; Phillips et al., 2013) need to be updated, and the author's bias against S9.6 antibodies needs also to be changed. In contrast to S9.6 CUT&Tag and other inactive ribonucleotide H1-based methods including MapR (inactive ribonucleotide H1-mediated CUT&Run) (Yan et al., 2019)and GST-2xHBD CUT&Tag (Wang et al., 2021), HBD-seq did not perform satisfactorily and its binding specificity was questionable.

Although HepG4-seq is an effective G4s detection technique, and the authors have also verified its reliability to some extent, given the strong link between ROS homeostasis and G4s formation, hemin's affinity for different types of G4s and their differences in peroxidase activities, whether HepG4-seq reflects the dynamics of G4s in vivo more accurately than existing detection techniques still needs to be more carefully corroborated.

The authors focus on the interaction of non-B DNA structures G4s and R-loops and their roles in development and disease by regulating the transcription of related genes. Compared to the complex regulatory network of G4s and R-loops, the authors provide limited mechanistic insight into the major regulator of co-localized G4s and R-loops, helicase Dhx9. However, the authors propose that "A degron system-mediated simultaneous and/or stepwise degradation system of multiple regulators will help us elucidate the interplaying effects between G4s and R-loops." is attractive. The main innovations of this article are the proposal of new antibody-independent methods for detecting G4s and the optimization of the GST-2xHBD CUT&Tag (Wang et al., 2021) method for detecting R-loops. Unfortunately, however, the reliability and accuracy of these methods are still debatable, and the reference value of the G4s and R-loops datasets based on these methods is relatively limited.

Reviewer #3 (Public review):

Summary:

This manuscript reveals an important mechanism of KCNQ1/IKs channel gating such that the open state of the pore is unstable and undergoes intermittent closed and open conformations. PUFA enhances the maximum open probability of IKs by binding to a crevice adjacent to the pore and stabilize the open conformation. This mechanism is supported by convincing single channel recordings that show empty and open channel traces and the ratio of such traces is affected by PUFA. In addition, mutations of the pore residues alter PUFA effects, convincingly supporting that PUFA alters the interactions among these pore residues.

Strengths:

The data are of high quality and the description is clear.

Reviewer #3 (Public review):

Summary:

The primary aim of this manuscript was to investigate how context, defined from visual object information in multimodal movies, impacts the neural representation of concrete and abstract conceptual knowledge. The authors first conduct a series of analyses to identify context independent regional response to concrete and abstract concepts in order to compare these results with the networks observed in prior research using non-naturalistic paradigms. The authors then conduct analyses to investigate whether regional response to abstract and concrete concepts changes when the concepts are either contextually situated or displaced. A concept is considered displaced if the visual information immediately preceding the word is weakly associated with the word whereas a concept is situated if the association is high. The results suggest that, when ignoring context, abstract and concrete concepts engage different brain regions with overlap in core language areas. When context is accounted for, however, similar brain regions are activated for processing concrete and situated abstract concepts and for processing abstract and displaced concrete concepts. The authors suggest that contextual information dynamically changes the brain regions that support the representation of abstract and concrete conceptual knowledge.

Strengths:

There is significant interest in understanding both the acquisition and neural representation of abstract and concrete concepts, and most of the work in this area has used highly constrained, decontextualized experimental stimuli and paradigms to do so. This manuscript addresses this limitation by using multimodal narratives which allows for an investigation of how context-sensitive the regional response to abstract and concrete concepts is. The authors characterize the regional response in a comprehensive way.

Weaknesses:

The edits made to the manuscript in response to the reviewer comments have clarified and strengthened the methodological concerns flagged by all reviewers, giving me greater confidence that the authors are capturing what they aimed to and are making appropriate inferences given the results.

Reviewer #3 (Public Review):

This study presented a valuable inventory of scoring a neuropsychological test, ROCFT, with constructing an artificial intelligence model.

Comments on latest version:

The authors made the system with fundamental AI that is sufficient for clinical use for humans. In human neuropsychology, the test that generates the score is fundamental and relatively easy. Neuropsychologists apply patients to many tests; therefore, the present system is one of them, where we cannot tell the total neurofunction of a patient. The evidence for scoring is thought to be compelling quality, enough for clinical use now and we progress to evaluate other more complicated human neuropsychological functions. For example, persons with dementia change their performance easily when they feel other emotions (worry, boredom, etc. ) and notice other stimulation (announcements in the hospital, a walking nurse by chance, etc.). The score of ROCF is definitely changing, compelling the effort of AI scoring. We should grasp this behavior of humans with diverse tests totally. Therefore, scoring AI with compelling quality is a fundamental step for the next, evaluation against the changeable and ambiguous neurobehavior of humans.

Reviewer #3 (Public review):

Summary:

In this study, the authors studied the effects of traumatic brain injury created by LFPI procedure on the CA1 at the network level. The major findings in this study seem to be that the TBI reduces theta and gamma powers in CA1, reduces phase-amplitude coupling in between theta and gamma bands as well as disrupts the gamma entrainment of interneurons. I think the authors have made some important discoveries that could help advance the understanding of TBI effects at the physiological level, however, more investigations into deciphering the relationship of the behavioral and brain states to the observed effects would help clarify the interpretations for the readers.

Strengths:

The authors in this study were able to combine behavioral verification of the TBI model with the laminar electrophysiological recordings of the CA1 region to bring forward network-level anomalies such as the temporal coordination of network-level oscillations as well as in the firing of the interneurons. Indeed, it seems that the findings may serve future studies to functionally better understand and/or refine the therapies for the TBI.

Weaknesses:

Discoveries made in the paper and their broad interpretations can be helped with further characterization and comparison among the brain and behavioral states both during immobility and movement. The impact of brain injury in several parts of the brain can alter brain-wide LFP and/or behavior. The altered behavior and/or LFP patterns might then lead to reduced spiking and unreliable LFP oscillations in the hippocampus. Hence, claims made in the abstract such as "These results reveal deficits in information encoding and retrieval schemes essential to cognition that likely underlie TBI-associated learning and memory impairments, and elucidate potential targets for future neuromodulation therapies" do not have enough evidence to test whether the disruptions were information encoding and retrieval related or due to sensory-motor and/or behavioral deficits that could also occur during TBI.

Movement velocity is already known to be correlated to the entrainment of spikes with the theta rhythm and also in some cases with the gamma oscillations. So, it is important to disentangle the differences in behavioral variables and the observed effects. As an example, the author's claims of disrupted temporal coding (as shown in the graphical abstract) might have suffered from these confounds. The observed results of reduced entrainment might, on one hand, be due to the decreased LFP power (induced by injury in different brain areas) resulting in altered behavior and/or the unreliable oscillations of the LFP bands such as theta and gamma, rather than memory encoding and retrieval related disruption of spikes synchrony to the rhythms, while on the other hand, they may simply be due to reduced excitability in the neurons particularly in the behavioral and brain state in which the effects were observed, rather than disrupted temporal code. Hence, further investigations into dissociating these factors could help readers mechanistically understand the interesting results observed by the authors.

Reviewer #3 (Public review):

Summary:

The authors described cilia deficits, phospho-Rab12 accumulation, dystrophic axons in cholinergic neurons, and loss of the cholinergic neurons in the mouse brains of G2019S-LRRK2 knock-in mice, a preclinical animal model for Parkinson's disease. They showed that the above changes associated with cholinergic neurons are age-dependent and region-specific. The observation is interesting considering the neuron-type-specific effect of the LRRK2-G2019S in mice.

Strengths:

The observations are important and show neuron type-specific effects of the PD mutation of LRRK2 relevant to PD pathologies.

Weaknesses:

The authors may over-interpret the data, and the study may lack mechanistic investigation.

Reviewer #3 (Public review):

Summary:

The authors study the temporal summation of caged EPSPs in dendrite-targeting hippocampal CA1 interneurons. There are some descriptive data presented, indicating non-linear summation, which seems to be larger in dendrites of NDNF expressing neurogliaform cells versus OLM cells. However, the underlying mechanisms are largely unclear.

Strengths:

Focal 2-photon uncaging of glutamate is a nice and detailed method to study temporal summation of small potentials in dendritic segments.

Weaknesses:

(1) NMDA-receptor signaling in NDNF-IN. The authors nicely show that temporal summation in dendrites of NDNF-INs is to a certain extent non-linear. However, this non-linearity varies massively from cell to cell (or dendrite to dendrite) from 0% up to 400% (Figure S2). The reason for this variability is totally unclear. Pharmacology with AP5 hints towards a contribution of NMDA receptors. However, the authors claim that the non-linearity is not dependent on EPSP amplitude (Figure S2), which should be the case if NMDA-receptors are involved. Unfortunately, there are no voltage-clamp data of NMDA currents similar to the previous study. This would help to see whether NMDA-receptor contribution varies from synapse to synapse to generate the observed variability? Furthermore, the NMDA- and AMPA-currents would help to compare NDNF with the previously characterized PV cells and would help to contribute to our understanding of interneuron function.

(2) Sublinear summation in NDNF-INs. In the presence of AP5, the temporal summation of caged EPSPs is sublinear. That is potentially interesting. The authors claim that this might be dependent on the diameter of dendrites. Many voltage-gated channels can mediate such things as well. To conclude the contribution of dendritic diameter, it would be helpful to at least plot the extent of sublinearity in single NDNF dendrites versus the dendritic diameter. Otherwise, this statement should be deleted.

(3) Nonlinear EPSP summation in OLM-IN. The authors do similar experiments in dendrite-targeting OLM-INs and show that the non-linear summation is smaller than in NDNF cells. The reason for this remains unclear. The authors claim that this is due to the larger dendritic diameter in OLM cells. However, there is no analysis. The minimum would be to correlate non-linearity with dendritic diameter in OLM-cells. Very likely there is an important role of synapse density and glutamate receptor density, which was shown to be very low in proximal dendrites of OLM cells and strongly increase with distance (Guirado et al. 2014, Cerebral Cortex 24:3014-24, Gramuntell et al. 2021, Front Aging Neurosci 13:782737). Therefore, the authors should perform a set of experiments in more distal dendrites of OLM cells with diameters similar to the diameters of the NDNF cells. Even better would be if the authors would quantify synapse density by counting spines and show how this density compares with non-linearity in the analyzed NDNF and OLM dendrites.

(4) NMDA in OLM. Similar to the NDNF cells, the authors claim the involvement of NMDA receptors in OLM cells. Again there seems to be no dependence on EPSP amplitude, which is not understandable at this point (Figure S3). Even more remarkable is the fact that the authors claim that there is no dendritic calcium increase after activation of NMDA receptors. Similar to NDNF-cell analysis there are no NMDA currents in OLMs. Unfortunately, even no calcium imaging experiments were shown. Why? Are there calcium-impermeable NNDA receptors in OLM cells? To understand this phenomenon the minimum is to show some physiological signature of NMDA-receptors, for example, voltage-clamp currents. Furthermore, it would be helpful to systematically vary stimulus intensity to see some calcium signals with larger stimulation. In case there is still no calcium signal, it would be helpful to measure reversal potentials with different ion compositions to characterize the potentially 'Ca2+ impermeable' voltage-dependent NMDA receptors in OLM cells.

Reviewer #3 (Public review):

Summary:

The authors employ a series of well-conceived and well-executed experiments involving photometric imaging of the dentate gyrus and raphe nucleus, as well as cell-type specific genetic manipulations of serotonergic receptors that together serve to directly implicate serotonergic regulation of dentate gyrus (DG) granule (GC) and mossy cell (MC) activity in association with an infra slow oscillation (ISO) of neural activity has been previously linked to general cortical regulation during NREM sleep and microarousals.

Strengths:

There are a number of novel and important results, including the modulation of dentage granule cell activity by the infraslow oscillation during NREM sleep, the selective association of different subpopulations of granule cells to microarousals (MA), the anticorrelation of raphe activity with infraslow dentate activity.

The discussion includes a general survey of ISOs and recent work relating to their expression in other brain areas and other potential neuromodulatory system involvement, as well as possible connections with infraslow oscillations, micro-arousals, and sensory sensitivity.

Weaknesses:

(1) The behavioral results showing contextual memory impairment resulting from 5-HT1a knockdown are fine but are over-interpreted. The term memory consolidation is used several times, as well as references to sleep-dependence. This is not what was tested. The receptor was knocked down, and then 2 weeks later animals were found to have fear conditioning deficits. They can certainly describe this result as indicating a connection between 5-HT1a receptor function and memory performance, but the connection to sleep and consolidation would just be speculation. The fact that 5-HT1a knockdown also impacted DG ISOs does not establish dependency. Some examples of this are:

a. The final conclusion asserts "Together, our study highlights the role of neuromodulation in organizing neuronal activity during sleep and sleep-dependent brain functions, such as memory.". However, the reported memory effects (impairment of fear conditioning) were not shown to be explicitly sleep-dependent.

b. Earlier in the discussion it mentions "Finally, we showed that local genetic ablation of 5-HT1a receptors in GCs impaired the ISO and memory consolidation". The effect shown was on general memory performance - consolidation was not specifically implicated.

(2) The assertion on page 9 that the results demonstrate "that the 5-HT is directly acting in the DG to gate the oscillations" is a bit strong given the magnitude of effect shown in Figure 6D, and the absence of demonstration of negative effect on cortical areas that also show ISO activity and could impact DG activity (see requested cortical sigma power analysis).

(3) Recent work has shown that abnormal DG GC activity can result from the use of the specific Ca indicator being used (GCaMP6s). (Teng, S., Wang, W., Wen, J.J.J. et al. Expression of GCaMP6s in the dentate gyrus induces tonic-clonic seizures. Sci Rep 14, 8104 (2024). https://doi.org/10.1038/s41598-024-58819-9). The authors of that study found that the effect seemed to be specific to GCaMP6s and that GCaMP6f did not lead to abnormal excitability. Note this is of particular concern given similar infraslow variation of cortical excitability in epilepsy (cf Vanhatalo et al. PNAS 2004). While I don't think that the experiments need to be repeated with a different indicator to address this concern, you should be able to use the 2p GCaMP7 experiments that have already been done to provide additional validation by repeating the analyses done for the GCaMP6s photometry experiments. This should be done anyway to allow appropriate comparison of the 2p and photometry results.

(4) While the discussion mentions previous work that has linked ISOs during sleep with regulation of cortical oscillations in the sigma band, oddly no such analysis is performed in the current work even though it is presumably available and would be highly relevant to the interpretation of a number of primary results including the relationship between the ISOs and MAs observed in the DG and similar results reported in other areas, as well as the selective impact of DG 5-HT1a knockdown on DG ISOs. For example, in the initial results describing the cross-correlation of calcium activity and EMG/EEG with MA episodes (paragraph 1, page 4), similar results relating brief arousals to the infraslow fluctuation in sleep spindles (sigma band) have been reported also at .02 Hz associated with variation in sensory arousability (cf. Cardis et al., "Cortico-autonomic local arousals and heightened somatosensory arousability during NREMS of mice in neuropathic pain", eLife 2021). It would be important to know whether the current results show similar cortical sigma band correlations. Also, in the results on ISO attenuation following 5-HT1 knockdown on page 7 (Figure 6), how is cortical EEG affected? Is ISO still seen in EEG but attenuated in DG?

(5) The illustrations of the effect of 5-HT1a knockdown shown in Figure 6 are somewhat misleading. The examples in panels B and C show an effect that is much more dramatic than the overall effect shown in panel D. Panels B and C do not appear to be representative examples. Which of the sample points in panel D are illustrated in panels B and C? It is not appropriate to arbitrarily select two points from different animals for comparison, or worse, to take points from the extremes of the distributions. If the intent is to illustrate what the effect shown in D looks like in the raw data, then you need to select examples that reflect the means shown in panel D. It is also important to show the effect on cortical EEG, particularly in sigma band to see if the effects are restricted to the DG ISOs. It would also be helpful to show that MAs and their correlations as shown in Figure 1 or G as well as broader sleep architecture are not affected.

(6) On page 9 of the results it states that GCs and MCs are upregulated during NREM and their activity is abruptly terminated by MAs through a 5-HT mediated mechanism. I didn't see anything showing the 5-HT dependence of the MA activity correlation. The results indicate a reduction in ISO modulation of GC activity but not the MA-correlated activity. I would like to see the equivalent of Figure 1,2 G panels with the 5-HT1a manipulation.

Reviewer #3 (Public review):

The authors tested whether the number of stimulus-stimulus pairings alters whether preconditioned fear depends on online integration during the formation of the stimulus-outcome memory or during the probe test/mobilization phase, when the original stimulus, which was never paired with aversive events, elicits fear via chaining of stimulus-stimulus and stimulus-outcome memories. They found that sensory preconditioning was successful with either 8 or 32 stimulus-stimulus pairings. Perirhinal cortex NMDA receptor blockade during stimulus-outcome learning impaired preconditioning following 8 but not 32 pairings during preconditioning. Therefore, perirhinal cortex NMDA activity is required for online integration or mediated learning. Perirhinal-basolateral amygdala had nearly identical effects with the same interpretation: these areas communicate during stimulus-outcome learning, and this online communication is required for later expressing preconditioned fear. Disconnection prior to the probe test, when chaining might occur, had different effects: it impaired the expression of preconditioned fear in rats that received 32, but not 8, pairings during preconditioning. The study has several strengths and provides a thoughtful discussion of future experiments. The study is highly impactful and significant; the authors were successful in describing the behavioral and neurobiological mechanisms of mediated learning versus chaining in sensory preconditioning, which is often debated in the learning field. Therefore this study will have a significant impact on the behavioral neurobiology and learning fields.

Strengths:

Careful, rigorous experimental design and statistics.

The discussion leaves open questions that are very much worth exploring. For example - why did perirhinal-amygdala disconnection prior to the probe have no effect in the 8-pairing group, when bilateral perirhinal inactivation did (in Wong et al, 2019)? The authors propose that perirhinal cortex outputs bypass the amygdala during the probe test, which is an excellent hypothesis to test.

The authors provide evidence that both mediated learning and chaining occur.

Weaknesses:

This is inherent to all neural interference and behavioral experiments: biological/psychological functions do not typically operate binarily. There is no single clear number or parameter at which mediated learning or chaining happens, and both probably happen to some extent. Addressing this is even more difficult given behavioral variability across subjects, implant sites, etc. Thus, this is not so much a weakness particular to this study as much as an existential problem, which the authors were able to work around with careful experimental design and appropriate controls.

ვფიქრობა,ამის მიზეზი ადამიანების სტერეოტიპული აზროვნება და ვითარების გონივრულად შეფასების უცოდინრობაა. პიროვნების დისკრიმინაცია მისი სქესის მიხედვით და ამის საფუძველზე მისი მიღწევებისა და ცოდნის დაკნინება,ვფიქრობ,უცოდინრობის გამოვლინებაა მსგავსი ქმედებით ადამიანები ძირს უთხრიან ქალების კარიერულ წინსვლასა და წარმატებას

for - earth system justice - 3 aspects

earth system justice - 3 aspects - interspecies justice - intergenerational justice - intragenerational justice

Reviewer #3 (Public review):

This work offers a novel perspective to the question of how hippocampal networks can adaptively generate different spatial maps and replays of the corresponding place cells, without any such maps pre-existing in the network architecture or its inputs. And how can these place cells preplay their sequences even before the environment is experienced? Previous models required pre-existing spatial representations to be artificially introduced, limiting their adaptability to new environments. Others depended on synaptic plasticity rules which made remapping slower that what is seen in recordings. In contrast, this modeling study proposes that quickly-adaptive intrinsic spiking sequences (preplays) and spatially tuned spiking (place cells) can be generated in a network through randomly clustered recurrent connectivity. By simulating spatial exploration through border-cell-like synaptic inputs, the model generates place cells for different "environments" without the need to reconfigure its synaptic connectivity or introduce plasticity. By simulating sleep-like random synaptic inputs, the model generates sequential activations of cells, mimicking preplays. These "preplays" require small-world connectivity, so that cell clusters are activated in sequence. Using a set of electrophysiological recordings from CA1, the authors confirm that the modeled place cells and replays share many features with recorded ones.

Many features of the model are thoroughly examined, and conclusions are overall convincing (within the simple architecture of the model). Even though the modeled connectivity applies more closely to CA3, it remains unclear whether CA3 recapitulates the proposed small world architecture.

In any case, the proposal that a small-world-structured, clustered network can generate flexible place cells and replays without the need for pre-configured maps is novel and of potential interest to a wide computational and experimental community.

Reviewer #3 (Public review):

Summary:

This manuscript works with a hypothesis where the overall m6A methylation levels in cells are influenced by mRNA metabolism (sub-cellular localization and decay). The basic assumption is that m6A causes mRNA decay and this happens in the cytoplasm. They go on to experimentally test their model to confirm its predictions. This is confirmed by sub-cellular fractionation experiments which show high m6A levels in the nuclear RNA. Nuclear localized RNAs have higher methylation. Using a heat shock model, they demonstrate that RNAs with increased nuclear localization or transcription, are methylated at higher levels. Their overall argument is that changes in m6A levels are rather determined by passive processes that are influenced by RNA processing/metabolism. However, it should be considered that erasers have their roles under specific environments (early embryos or germline) and are not modelled by the cell culture systems used here.

Strengths:

This is a thought-provoking series of experiments that challenge the idea that active mechanisms of recruitment or erasure are major determinants for m6A distribution and levels.

Reviewer #3 (Public Review):

Summary:

This groundbreaking study provided the most advanced transgenic lineage tracing and advanced imaging techniques in deciphering dental/periodontal mesenchyme cells. In this study, authors utilized CRISPR/Cas9-mediated transgenic lineage tracing techniques to concurrently demonstrate the inclusive, exclusive, and hierarchical distributions of NFATc1+ and PDGFR-α+ cells and their lineage commitment in dental and periodontal mesenchyme.

Strengths:

In cooperating with tissue clearing-based advanced imaging and three-dimensional slices reconstruction, the distribution and hierarchical relationship of NFATc1+ and PDGFR-α+ cells and progeny cells plainly emerged, which undoubtedly broadens our understanding of their in vivo fate trajectories in craniomaxillofacial tissue. Also, the experiment design is comprehensive and well-executed, and the results are convincing and compelling.

Weaknesses:

Minor modifications could be made to the paper, including more details on the advantages of the methodology used by the authors in this study, compared to other studies.

DOI: 10.1038/s41420-023-01643-3

Resource: RRID:BDSC_43642

Curator: @maulamb

SciCrunch record: RRID:BDSC_43642

What is this?

Reviewer #3 (Public Review):

Summary:

In this manuscript, Barber and colleagues propose a dual role for the RNA-binding protein Mushashi-2 (Msi2) in lung adenocarcinoma initial transformation and subsequent tumor propagation. First, authors show that Msi2 is expressed in a subset of Club/BASC (37%) and AT2 (26%) cells in the normal lung and displayed a distinct transcriptional profile than non-expressing Msi2 cells. Furthermore, Msi2 is broadly expressed/activated in vivo in genetically induced lung adenocarcinoma tumors (Kras/p53 mouse model) and Msi2+ cells displayed a significantly higher ability to form tumor spheres in vitro. Authors demonstrated by in vivo and in vitro assays that Msi2 loss of function significantly impair tumor growth and progression in lung adenocarcinoma. Data showed that Msi2 function is conserved in human adenocarcinoma tumor growth in patient-derived xenograft. Lastly, novel genes regulated by Msi2 and involved in lung adenocarcinoma tumor growth were identified.

Strengths:

The authors provided convincing data for a key role of Msi2 in lung adenocarcinoma tumor progression and growth. Multiple evidences using Msi2 knock-out genetic mouse model and shRNA knock-down in tumor sphere formation assay are clearly demonstrated. The conservation and importance of Msi2 was further shown in human patient-derived xenograft. Although specific cell types (Club/BASC, AT2) were not isolated, authors further delved in the transcriptional difference between Msi2+ and Msi2- cells in the normal lung. Furthermore, novel genes and pathways regulated by Msi2 in lung adenocarcinoma were identified and tested for their ability to inhibit tumor growth in vitro. These 2 RNA-Seq datasets will be useful in the future and provide a basis to explore 1) potential propensity of a given cell to initiate oncogenic transformation, and 2) potential novel regulators of lung adenocarcinoma.

Weaknesses:

Although this work strongly demonstrated the importance of Msi2 in lung adenocarcinoma tumor progression and growth, the following points remain to be clarified or addressed.

- In Figure 1, characterization of Msi2 expression in the normal mouse lung was carried out by using a Msi2-GFP Knock-in reporter and analyzed by flow cytometry followed by cytospins and immunostaining. Additional characterization of Msi2 expression by co-immunostaining with well-known markers of airway and alveolar cell types in intact lung tissue will strengthen the existing data and provide more specific information about Msi2 expression and abundancy in relevant cell types. It will be also interesting to know whether Msi2 is expressed or not in other abundant lung cell types such as ciliated and AT1 cells.

- While this set of experiments provide strong evidence that Msi2 is required for tumor progression and growth in lung adenocarcinoma, it is unclear whether normal Msi2+ lung cells are more responsive to transformation or whether Msi2 is upregulated early during the process of tumorigenesis. Future lineage tracing experiments using Msi2-CreER and mouse models of chemically-induced lung carcinogenesis will provide additional data that will fully support this claim.

- In Figure 4F, Patient-derived xenograft (PDX) assays were conducted in 2 patients only and the percentage of cells infected by shRNA-Msi2 is low in both PDX (30% and 10% for patient 1 and 2 respectively). It is surprising that Msi2 downregulation in a small percentage of tumor cells has such a dramatic effect on tumor growth and expansion. Confirmation of this finding with additional patient samples would suggest an important non-cell autonomous role for Msi2 in lung adenocarcinoma.

Reviewer #4 (Public Review):

Summary:<br /> In the manuscript titled "Hemispheric Divergence of Interoceptive Processing Across Psychiatric Disorders", the authors analyzed a subset of data collected for a larger project investigating interoception in anorexia nervosa and generalized anxiety disorder (ClinicalTrials.gov Identifier: NCT02615119). This study utilized fMRI and various analyses with a special focus on the insula and its connectivity to map the neural commonalities and differences in both top-down and bottom-up interoceptive processing.

The primary aim was to compare whether these neural activations were quantitatively and qualitatively different in a sample of healthy controls (HC) versus patients diagnosed with anxiety, depression, and/or eating disorders (ADE).

The study initially recruited 70 patients with primary diagnoses of ADE and 57 HC. After applying exclusion criteria, the final sample consisted of 46 ADE patients and 46 matched HC. Participants underwent task-related and resting-state fMRI scan sessions.

Specifically, participants performed 2 tasks in fMRI: i) a bottom-up interoceptive (ISO) task involving intravenous infusions of isoproterenol (a peripherally-acting beta-adrenergic receptor agonist) administered in a double-blind, placebo-controlled fashion to alter cardiovascular activity where participants were asked about their visceral awareness; and ii) a top-down interoceptive attention (VIA) task where participants were asked to focus on their visceral sensations triggered by words indicating specific body parts (e.g., STOMACH, HEART, LUNGS) or to pay attention to color changes of the word TARGET during an exteroceptive control task.<br /> Main results show overlapping patterns of neural activation within the dysgranular mid-insula during top-down and bottom-up interoceptive processing with hemispheric differences. The patterns of dysgranular activation distinguished individuals with ADE compared to HC. Also differences in the activation of the anterior agranular insula during periods of interoceptive uncertainty differentiate ADE patients from HC.

Strengths:<br /> - This is a very nice study that aligns with modern Clinical Neuroscience approaches, as recommended by NIH policy (i.e. RDoC initiative), which puts emphasis describing clinical conditions via transdiagnostic dimensions measured on psychological processes, behaviors, and neural processes rather than merely identifying a series of symptoms.

I appreciated very much the different analyses that authors performed to characterize differences at the qualitative and quantitative regarding the insular activity and its connectivity during bottom-up and top-down interoceptive processes.

These findings may open avenues for new studies that will explain the mechanisms underlying these phenomena and provide useful insights for developing novel interventions.

Weaknesses:<br /> Weakness/Requests of additional clarifications<br /> (1) The sample<br /> (1.1) The authors describe the patient's group as having a primary diagnosis of anxiety, depression, and/or eating disorders. However, Table 1 shows that the majority had Anxiety disorders, some Major Depression (it is not clear which are the percentages of patients that at the time of the study had a concurred problem of major depression, please clarify), and very few had a diagnosis of Anorexia Nervosa. The leftward activation asymmetry and distinct activation patterns in the left dysgranular mid-insula across both the ISO and VIA tasks found on ADE did not correlate with symptoms measured by the SCOFF questionnaire, but correlated with anxiety and depressive symptoms. It would be nice if the authors can comment on these results in relation to eating disorders.

(1.2) Furthermore, the sample consisted of 5 males and 41 females in the HC group and 1 male and 45 females in the ADE group. In order to generalize these findings, the authors should acknowledge this gender imbalance and discuss whether they expect similar results in a predominantly male sample.

(2) The procedure<br /> While the fixed order of tasks reflects the primary emphasis on acquiring data from the infusion (ISO) task, this could introduce confounding order effects. The authors should acknowledge this as a limitation of this study.

(3) The rationale behind the study<br /> - The authors recognized that there was a broader aim behind this data collection. It would be important to clarify a little bit more how the differences in insular areas mapping both (or specifically) bottom-up and top-down interoceptive processes and insular connectivity, recorded in ADE patients compared to healthy controls (HC), contribute to psychiatric diagnoses (hypothesis 3).<br /> For example, they should explain the psychopathological dimensions common to the three patient groups. Are disturbances in bottom-up and top-down interoceptive processing common traits in these patients, reflected in the asymmetric interhemispheric dysgranular mid-insular activation? The link between these disturbances and anatomical evidence of convergence/divergence of top-down vs. bottom-up interoceptive processes should be clearly stated.

(4) Operationalization of Convergence / Divergence maps underlying top-down and bottom-up interoceptive processes in HC vs ADE patients<br /> It is not clear to me the concept of Convergence / Divergence maps underlying top-down and bottom-up interoceptive processes. The authors want to compare, in HCs and ADE patients, the neural structures that are co-activated (convergence maps) vs those that are uniquely involved (divergence maps) in top-down and bottom-up interoceptive processes in the two groups. Thus, I would expect that these two different analyses would have been performed on similar portions of data, instead different moments of the tasks (= different bottom-up / top-down interoceptive processes) have been analyzed.<br /> Specifically, the convergence maps have been identified by comparing active voxels recorded when participants were focusing on the heart and the lungs (compared to when they were focused on the exteroceptive features of the target) in the VIA task, and during infusions (Peak period) of 2mcg isoproterenol (compared to baseline) in the ISO task. The divergence maps have been identified by comparing voxels uniquely active during the anticipatory phases of both isoproterenol and saline infusions (compared to baseline) and during the peak period of saline dose of the ISO task with respect to when participants focused their attention on the heart and the lungs (compared to when they were focuses on the exteroceptive features) in the VIA task.<br /> I understand the idea of mapping interoceptive uncertainty, however I think that these two analyses do not show commonalities and differences in the neural structures involved in bottom up vs top down processes (in ADE vs HC), but also neural correlates underlying different types of interoceptive processes involving or nor top-down expectations.<br /> According to the authors, which is the most important neural marker that differentiates the ADE group: the difference in hemispheric activations within the left and right dysgranular insula or the less granular anterior insular activation during periods of interoceptive uncertainty? Also, do they reflect different transdiagnostic dimensions?

(5) Collected physiological measures<br /> The authors speak about cardiorespiratory interoceptive processes, but they only included cardiac measures. Including respiratory changes could provide a more comprehensive comparison between bottom-up signals and top-down attentional processes. Also, I guess that the "STOMACH" trials of the VIA task were not analyzed in this study since those are used in the bigger study and since no gastric measures were collected? Please clarify this point.

(6) ISO task instructions<br /> To better understand the task and participants' expectations, could the authors clarify the instructions given to participants regarding the isoproterenol and saline infusions. Did the participants have two types of expectations?

(7) Title of the study<br /> I understand that the term "divergence" in the title refers to the different hemispheric activations characterizing ADE patients compared to HC. However, it also suggests an analysis based on convergence/divergence maps, which might be ambiguous. Could the authors make some small modifications to the title to make it clearer?

(8) Caption of Figure 7<br /> The caption of Fig.7 notes that no difference in HR was found during the Saline infusion between the HC and ADE groups. However, it would be fair to mention the significant difference in dial ratings observed during the Saline infusion. How do the authors explain this difference?

Typos<br /> Figure 3 In Figure 3, "Hemispheric divergence", I think, should be corrected to "Hemispheric convergence."

I believe that by addressing these points, the manuscript will provide a clearer and more comprehensive understanding of the rationale, methods, and findings underlying this study.

Reviewer #3 (Public review):

Summary:

Redman and colleagues analyze grid cell data obtained from public databases. They show that there is significant variability in spacing and orientation within a module. They show that the difference in spacing and orientation for a pair of cells is larger than the one obtained for two independent maps of the same cell. They speculate that this variability could be useful to disambiguate the rat position if only information from a single module is used by a decoder.

Strengths:

The strengths of this work lie in its conciseness, clarity, and the potential significance of its findings for the grid cell community, which has largely overlooked this issue for the past two decades. Their hypothesis is well stated and the analyses are solid.

Weaknesses:

On the side of weaknesses, we identified two aspects of concern. First, alternative explanations for the main result exist that should be explored and ruled out. Second, the authors' speculation about the benefits of variability in angle and spacing for spatial coding is not particularly convincing, although this issue does not diminish the importance or impact of the results.

Major comments:

(1) One possible explanation of the dispersion in lambda (not in theta) could be variability in the typical width of the field. For a fixed spacing, wider fields might push the six fields around the center of the autocorrelogram toward the outside, depending on the details of how exactly the position of these fields is calculated. We recommend authors show that lambda does not correlate with field width, or at least that the variability explained by field width is smaller than the overall lambda variability.

(2) An alternative explanation could be related to what happens at the borders. The authors tackle this issue in Figure S2 but introduce a different way of measuring lambda based on three fields, which in our view is not optimal. We recommend showing that the dispersions in lambda and theta remain invariant as one removes the border-most part of the maps but estimating lambda through the autocorrelogram of the remaining part of the map. Of course, there is a limit to how much can be removed before measures of lambda and theta become very noisy.

(3) A third possibility is slightly more tricky. Some works (for example Kropff et al, 2015) have shown that fields anticipate the rat position, so every time the rat traverses them they appear slightly displaced opposite to the direction of movement. The amount of displacement depends on the velocity. Maps that we construct out of a whole session should be deformed in a perfectly symmetric way if rats traverse fields in all directions and speeds. However, if the cell is conjunctive, we would expect a deformation mainly along the cell's preferred head direction. Since conjunctive cells have all possible preferred directions, and many grid cells are not conjunctive at all, this phenomenon could create variability in theta and lambda that is not a legitimate one but rather associated with the way we pool data to construct maps. To rule away this possibility, we recommend the authors study the variability in theta and lambda of conjunctive vs non-conjunctive grid cells. If the authors suspect that this phenomenon could explain part of their results, they should also take into account the findings of Gerlei and colleagues (2020) from the Nolan lab, that add complexity to this issue.

(4) The results in Figure 6 are correct, but we are not convinced by the argument. The fact that grid cells fire in the same way in different parts of the environment and in different environments is what gives them their appeal as a platform for path integration since displacement can be calculated independently of the location of the animal. Losing this universal platform is, in our view, too much of a price to pay when the only gain is the possibility of decoding position from a single module (or non-adjacent modules) which, as the authors discuss, is probably never the case. Besides, similar disambiguation of positions within the environment would come for free by adding to the decoding algorithm spatial cells (non-hexagonal but spatially stable), which are ubiquitous across the entorhinal cortex. Thus, it seems to us that - at least along this line of argumentation - with variability the network is losing a lot but not gaining much.

(5) In Figure 4 one axis has markedly lower variability. Is this always the same axis? Can the authors comment more on this finding?

(6) The paper would gain in depth if maps coming out of different computational models could be analyzed in the same way.

(7) Similarly, it would be very interesting to expand the study with some other data to understand if between-cell delta_theta and delta_lambda are invariant across environments. In a related matter, is there a correlation between delta_theta (delta_lambda) for the first vs for the second half of the session? We expect there should be a significant correlation, it would be nice to show it.

Reviewer #3 (Public review):

Summary:

The authors present a study building on their previous work on activation of the general stress response phosphatase, RsbU, from Bacillus subtilis. Using computed structural models of the RsbU dimer the authors map previously identified activating mutations onto the structure and suggest further protein variants to test the role of the predicted linker helix and the interaction with RsbT on the activation of the phosphatase activity.

Using in vivo and in vitro activity assays, the authors demonstrate that linker variants can constitutively activate RsbU and increase the affinity of the protein for RsbT, thus showing a link between the structure of the linker region and RsbT binding.

Small angle X-ray scattering experiments on RsbU variants alone, and in complex with RsbT show structural changes consistent with a decreased flexibility of the RsbU protein, which is hypothesised to indicate a disorder-order transition in the linker when RsbT binds. This interpretation of the data is consistent with the biochemical data presented by the authors.

Further computed structure models are presented for other protein phosphates from different bacterial species and the authors propose a model for phosphatase activation by partner binding. They compare this to the activation mechanisms proposed for histidine kinase two-component systems and GGDEF proteins and suggest the individual domains could be swapped to give a toolkit of modular parts for bacterial signalling.

Strengths:

The key mutagenesis data is presented with two lines of evidence to demonstrate RsbU activation - in vivo sigma-b activation assays utilising a beta-galactosidase reporter and in vitro activity assays against the RsbV protein, which is the downstream target of RsbU. These data support the hypothesis for RsbT binding to the RsbU linker region as well as the dimerisation domain to activate the RsbU activity.

Weaknesses:

Small angle scattering curves are difficult to unambiguously interpret, but the authors present reasonable interpretations that fit with the biochemical data presented. These interpretations should be considered as good models for future testing with other methods - hydrogen/deuterium exchange mass spectrometry, would be a good additional method to use, as exchange rates in the linker region would be affected significantly by the disorder/order transition on RsbT binding.

The interpretation of the computed structure models should be toned down with the addition of a few caveats related to the bias in the models returned by AlphaFold2. For the full-length models of RsbU and other phosphatase proteins, the relationship of the domains to each other is likely to be the least reliable part of the models - this is apparent from the PAE plots shown in Supplementary Figure 8. Furthermore, the authors should show models coloured by pLDDT scores in an additional supplementary figure to help the reader interpret the confidence level of the predicted structures.

Reviewer #3 (Public Review):

Summary:

The interaction between FGF signaling and SHH-mediated GNP expansion in MB, particularly in the context of Sufu LoF, has just begun to be understood. The manuscript by Yabut et al. establishes a connection between ectopic FGF5 expression and GNP over-expansion in a late-stage embryonic Sufu LoF model. The data provided links region-specific interaction between aberrant FGF5 signaling with the SHH subtype of medulloblastoma. New data from Yabut et al. suggest that ectopic FGF5 expression correlates with GNP expansion near the secondary fissure in Sufu LoF cerebella. Furthermore, pharmacological blockade of FGF signaling inhibits GNP proliferation. Interestingly, the data indicate that the timing of conditional Sufu deletion (E13.5 using the hGFAP-Cre line) results in different outcomes compared to later deletion (using Math1-cre line, Jiwani et al., 2020). This study provides significant insights into the molecular mechanisms driving GNP expansion in SHH subgroup MB, particularly in the context of Sufu LoF. It highlights the potential of targeting FGF5 signaling as a therapeutic strategy. Additionally, the research offers a model for better understanding MB subtypes and developing targeted treatments.

Strengths:

One notable strength of this study is the extraction and analysis of ectopic FGF5 expression from a subset of MB patient tumor samples. This translational aspect of the study enhances its relevance to human disease. By correlating findings from mouse models with patient data, the authors strengthen the validity of their conclusions and highlight the potential clinical implications of targeting FGF5 in MB therapy.

The data convincingly show that FGFR signaling activation drives GNP proliferation in Sufu, conditional knockout models. This finding is supported by robust experimental evidence, including pharmacological blockade of FGF signaling, which effectively inhibits GNP proliferation. The clear demonstration of a functional link between FGFR signaling and GNP expansion underscores the potential of FGFR as a therapeutic target in SHH subgroup medulloblastoma.

Previous studies have demonstrated the inhibitory effect of FGF2 on tumor cell proliferation in certain MB types, such as the ptc mutant (Fogarty et al., 2006)(Yaguchi et al., 2009). Findings in this manuscript provide additional support suggesting multiple roles for FGF signaling in cerebellar patterning and development.

Weaknesses:

In the GEO dataset analysis, where FGF5 expression is extracted, the reporting of the P-value lacks detail on the statistical methods used, such as whether an ANOVA or t-test was employed. Providing comprehensive statistical methodologies is crucial for assessing the rigor and reproducibility of the results. The absence of this information raises concerns about the robustness of the statistical analysis.

Another concern is related to the controls used in the study. Cre recombinase induces double-strand DNA breaks within the loxP sites, and the control mice did not carry the Cre transgene (as stated in the Method section), while Sufu-cKO mice did. This discrepancy necessitates an additional control group to evaluate the effects of Cre-induced double-strand breaks on phosphorylated H2AX-DSB signaling. Including this control would strengthen the validity of the findings by ensuring that observed effects are not artifacts of Cre recombinase activity.

Although the use of the hGFAP-Cre line allows genetic access to the late embryonic stage, this also targets multiple celltypes, including both GNPs and cerebellar glial cells. However, the authors focus primarily on GNPs without fully addressing the potential contributions of neuron-glial interaction. This oversight could limit the understanding of the broader cellular context in which FGF signaling influences tumor development.

Reviewer #3 (Public review):

The DNA and RNA binding protein TDP-43 has been pathologically implicated in a number of neurodegenerative diseases including ALS, FTD, and AD. Normally residing in the nucleus, in TDP-43 proteinopathies, TDP-43 mislocalizes to the cytoplasm where it is found in cytoplasmic aggregates. It is thought that both loss of nuclear function and cytoplasmic gain of toxic function are contributors to disease pathogenesis in TDP-43 proteinopathies. Recent studies have demonstrated that depletion of nuclear TDP-43 leads to loss of its nuclear function characterized by changes in gene expression and splicing of target mRNAs. However, to date, most readouts of TDP-43 loss of function events are dependent upon PCR-based assays for single mRNA targets. Thus, reliable and robust assays for detection of global changes in TDP-43 splicing events are lacking. In this manuscript, Xie, Merjane, Bergmann and colleagues describe a biosensor that reports on TDP-43 splicing function in real time. Overall, this is a well described unique resource that would be of high interest and utility to a number of researchers. Nonetheless, a couple of points should be addressed by the authors to enhance the overall utility and applicability of this biosensor.

Generating new ideas and thinking in different ways or about different things is good for your brain.

Reviewer #3 (Public review):

Summary:

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

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

Strengths:

The main strength of the manuscript is the high-quality microscopy data. The authors use multiple markers and high-resolution timelapse imaging to track chloroplast dynamics under light limiting conditions.

Weaknesses:

The main weakness of the manuscript is the limited quantitative data. While it can be challenging to quantify dynamic intracellular events, quantification of these processes is important to appreciate the significance of these findings.

Reviewer #3 (Public review):

The manuscript by Geurts and colleagues studies the effects of methylphenidate on Pavlovian to instrumental transfer in humans and demonstrates that the effects of the drug depend on the baseline working memory capacity of the participants. The experiment used a well established cognitive task that allows to measure the effects of Pavlovian cues predicting monetary wins and losses on instrumental responding in two different contexts, namely approach and withdraw. By administering the drug after participants went through the instrumental and Pavlovian learning phases of the experiment, the authors limited the effects of the drug to the transfer phase in extinction. This allowed the authors to make inference about the invigorating effects of the cues independently from any learning bias. Moreover, the authors employed a within subject design to study the effect of the drug on 100 participants, which also allows to detect continuous between-subject relationships with covariates such as working memory capacity.

The study replicates previous findings using this task, namely that appetitive cues promote active responding, and aversive cues promote passive responding in an approach instrumental context, whereas the effect of the cues reverses in a withdraw instrumental context. The results of the methylphenidate manipulation show that the drug decreases the effects of the Pavlovian cues on instrumental responding in participants with low working memory capacity but increases the Pavlovian effects in participants with high working memory capacity. Importantly, in the latter group, methylphenidate increases the invigorating effect of appetitive Pavlovian cues on active approach and aversive Pavlovian cues on active withdrawal as well as the inhibitory effects of aversive Pavlovian cues on active approach and appetitive Pavlovian cues on active withdrawal. These results cannot be explained if catecholamines are just involved in Pavlovian biases by modulating behavioral invigoration driven by the anticipation of reward and punishment in the striatum, as this account can't account for the reversal of the effects of a valence cue on vigor depending on the instrumental context.

In general, I find the methods of this study very robust and the results very convincing and important. However, I have some concerns:

I am not convinced that the inclusion of impulsivity scores in the logistic mixed model to analyze the effects of methylphenidate on PIT is warranted. The authors do not show whether inclusion of this covariate is justified in terms of BIC. Moreover, they include this covariate but do not report the effects. Finally, it is possible that impulsivity is correlated with working memory capacity. In that case, multicollinearity may impact the estimation of the coefficient estimates and may inflate the p-values for the correlated covariates. Are the reported results robust when this factor is not included?

The authors state that working memory capacity is an established proxy for dopamine synthesis capacity and cite some studies supporting this view. However, the authors omit a recent reference by van den Bosch et al that provides evidence for the absence of links between striatal dopamine synthesis capacity and working memory capacity. The lack of a robust link between working memory capacity and dopamine synthesis capacity in the striatum strengthens the alternative explanations of the results suggested in the discussion.

Reviewer #3 (Public Review):

The Mutational Hazard Hypothesis (MHH) suggests that lineages with smaller effective population sizes should accumulate slightly deleterious transposable elements leading to larger genome sizes. Marino and colleagues tested the MHH using a set of 807 vertebrate, mollusc, and insect species. The authors mined repeats de novo and estimated dN/dS for each genome. Then, they used dN/dS and life history traits as reliable proxies for effective population size and tested for correlations between these proxies and repeat content while accounting for phylogenetic nonindependence. The results suggest that overall, lineages with lower effective population sizes do not exhibit increases in repeat content or genome size. This contrasts with expectations from the MHH. The authors speculate that changes in genome size may be driven by lineage-specific host-TE conflicts rather than effective population size.

The general conclusions of this paper are supported by a powerful dataset of phylogenetically diverse species. The use of C-values rather than assembly size for many species (when available) helps mitigate the challenges associated with the underrepresentation of repetitive regions in short-read-based genome assemblies. As expected, genome size and repeat content are highly correlated across species. Nonetheless, the authors report divergent relationships between genome size and dN/dS and TE content and dN/dS in multiple clades: Insecta, Actinopteri, Aves, and Mammalia. These discrepancies are interesting but could reflect biases associated with the authors' methodology for repeat detection and quantification rather than the true biology.

The authors used dnaPipeTE for repeat quantification. Although dnaPipeTE is a useful tool for estimating TE content when genome assemblies are not available, it exhibits several biases. One of these is that dnaPipeTE seems to consistently underestimate satellite content (compared to repeat masker on assembled genomes; see Goubert et al. 2015). Satellites comprise a significant portion of many animal genomes and are likely significant contributors to differences in genome size. This should have a stronger effect on results in species where satellites comprise a larger proportion of the genome relative to other repeats (e.g. Drosophila virilis, >40% of the genome (Flynn et al. 2020); Triatoma infestans, 25% of the genome (Pita et al. 2017) and many others). For example, the authors report that only 0.46% of the Triatoma infestans genome is "other repeats" (which include simple repeats and satellites). This contrasts with previous reports of {greater than or equal to}25% satellite content in Triatoma infestans (Pita et al. 2017). Similarly, this study's results for "other" repeat content appear to be consistently lower for Drosophila species relative to previous reports (e.g. de Lima & Ruiz-Ruano 2022). The most extreme case of this is for Drosophila albomicans where the authors report 0.06% "other" repeat content when previous reports have suggested that 18%->38% of the genome is composed of satellites (de Lima & Ruiz-Ruano 2022). It is conceivable that occasional drastic underestimates or overestimates for repeat content in some species could have a large effect on coevol results, but a minimal effect on more general trends (e.g. the overall relationship between repeat content and genome size).

Another bias of dnaPipeTE is that it does not detect ancient TEs as well as more recently active TEs (Goubert et al. 2015). Thus, the repeat content used for PIC and coevolve analyses here is inherently biased toward more recently inserted TEs. This bias could significantly impact the inference of long-term evolutionary trends.

Reviewer #3 (Public review):

Summary:<br /> How mechanical forces transmitted by blood flow contribute to normal cardiac development remains incompletely understood. Using the unique advantages of the zebrafish model system, Chen et al make the fundamental discovery that endocardial expression of id2b is induced by blood flow and required for normal atrioventricular canal (AVC) valve development and myocardial contractility by regulating calcium dynamics. Mechanistically, the authors suggest that Id2b binds to Tcf3b in endocardial cells, which relieves Tcf3b-mediated transcriptional repression of Neuregulin 1 (NRG1). Nrg1 then induces expression of the L-type calcium channel component LRRC1. This study significantly advances our understanding of flow-mediated valve formation and myocardial function.

Strengths:<br /> Strengths of the study are the significance of the question being addressed, use of the zebrafish model, and data quality (mostly very nice imaging). The text is also well-written and easy to understand.

Weaknesses:<br /> Weaknesses include a lack of rigor for key experimental approaches, which led to skepticism surrounding the main findings. Specific issues were the use of morpholinos instead of genetic mutants for the bmp ligands, cilia gene ift88, and tcf3b, lack of an explicit model surrounding BMP versus blood flow induced endocardial id2b expression, use of bar graphs without dots, the artificial nature of assessing the physical interaction of Tcf3b and Id2b in transfected HEK293 cells, and artificial nature of examining the function of the tcf3b binding sites upstream of nrg1.

Reviewer #3 (Public Review):

It is a strength of the paper that it analyses the function of dopamine neurons (DANs) at the level of single, identified neurons, and uses tools to address specific dopamine receptors (DopRs), exploiting the unique experimental possibilities available in larval Drosophila as a model system. Indeed, the result of their screening for transgenic drivers covering single or small groups of DANs and their histological characterization provides the community with a very valuable resource. In particular the transgenic driver to cover the DANc1 neuron might turn out useful. However, I wonder in which fraction of the preparations an expression pattern as in Figure 1f/ S1c is observed, and how many preparations the authors have analyzed. Also, given the function of DANs throughout the body, in addition to the expression pattern in the mushroom body region (Figure 1f) and in the central nervous system (Figure S1c) maybe attempts can be made to assess expression from this driver throughout the larval body (same for Dop2R distribution).

A first major weakness is that the main conclusion of the paper, which pertains to associative memory (last sentence of the abstract, and throughout the manuscript), is not justified by their evidence. Why so? Consider the paradigm in Figure 2g, and the data in Figure 2h (22 degrees, the control condition), where the assay and the experimental rationale used throughout the manuscript are introduced. Different groups of larvae are exposed, for 30min, to an odour paired with either i) quinine solution (red bar), ii) distilled water (yellow bar), or iii) sucrose solution (blue bar); in all cases this is followed by a choice test for the odour on one side and a distilled-water blank on the other side of a testing Petri dish. The authors observe that odour preference is low after odour-quinine pairing, intermediate after odour-water pairing and high after odour-sucrose pairing. The differences in odour preference relative to the odour-water case are interpreted as reflecting odour-quinine aversive associations and odour-sucrose appetitive associations, respectively. However, these differences could just as well reflect non-associative effects of the 30-min quinine or sucrose exposure per se (for a classical discussion of such types of issues see Rescorla 1988, Annu Rev Neurosci, or regarding Drosophila Tully 1988, Behav Genetics, or with some reference to the original paper by Honjo & Furukubo-Tokunaga 2005, J Neurosci that the authors reference, also Gerber & Stocker 2007, Chem Sens).<br /> As it stands, therefore, the current 3-group type of comparison does not allow conclusions about associative learning.

A second major weakness is apparent when considering the sketch in Figure 2g and the equation defining the response index (R.I.) (line 480). The point is that the larvae that are located in the middle zone are not included in the denominator. This can inflate scores and is not appropriate. That is, suppose from a group of 30 animals (line 471) only 1 chooses the odour side and 29, bedazzled after 30-min quinine or sucrose exposure or otherwise confused by a given opto- or thermogenetic treatment, stay in the middle zone... a P.I. of 1.0 would result.

Unless experimentally demonstrated, claims that the thermogenetic effector shibire/ts reduces dopamine release from DANs are questionable. This is because firstly, there might be shibire/ts-insensitive ways of dopamine release, and secondly because shibire/ts may affect co-transmitter release from DANs.<br /> To implicate a role of dopamine in DANs, previous work used e.g. RNAi against the dopamine-synthesizing TH enzyme (Rohwedder et al, cited).

It is not clear whether the genetic controls when using the Gal4/ UAS system are the homozygous, parental strains (XY-Gal4/ XY-Gal4 and UAS-effector/ UAS-effector), or as is standard in the field the heterozygous driver (XY-Gal4/ wildtype) and effector controls (UAS-effector/ wildtype) (in some cases effector controls appear to be missing, e.g. Figure 4d, Figure S4e, Figure S5c).

As recently suggested by Yamada et al 2024, bioRxiv, high cAMP can lead to synaptic depression (sic). That would call into question the interpretation of low-Dop2R leading to high-cAMP, leading to high-dopamine release, and thus the authors interpretation of the matching effects of low-Dop2R and driving DANs.

Reviewer #3 (Public Review):

Summary:

This manuscript utilized zebrafish bcas2 mutants to study the role of bcas2 in primitive hematopoiesis and further confirms that it has a similar function in mice. Moreover, they showed that bcas2 regulates the transition of hematopoietic differentiation from angioblasts via activating Wnt signaling. By performing a series of biochemical experiments, they also showed that bcas2 accomplishes this by sequestering b-catenin within the nucleus, rather than through its known function in pre-mRNA splicing.

Strengths:

The work is well-performed, and the manuscript is well-written.

Weaknesses:

Several issues need to be clarified.

(1) Is wnt signaling also required during hematopoietic differentiation from angioblasts? Can the authors test angioblast and endothelial markers in embryos with wnt inhibition? Also, can the authors add export inhibitor LMB to the mouse mutants to test if sequestering of b-catenin by bcas2 is conserved during primitive hematopoiesis in mice?

(2) Bcas2 is required for primitive myelopoiesis in ALM. Does bcas2 play a similar function in primitive myelopoiesis, or is bcas2/b-catenin interaction more important for hematopoietic differentiation in PLM?

(3) Is it possible that CC1-2 fragment sequester b-catenin? The different phenotypes between this manuscript and the previous article (Yu, 2019) may be due to different mutations in bcas2. Is it possible that the bcas2 mutation in Yu's article produces a complete CC1-2 fragment, which might sequester b-catenin?

(4) Can the author clarify what embryos the arrows point to in SI Figure 2D? In SI Figure 6B and B', can the author clarify how the nucleus and cytoplasm are bleached? In B, the nucleus also appears to be bleached.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

Reviewer #3 (Public review):

This valuable manuscript demonstrates the long-held prediction that the glycosyltransferase UGGT slows degradation of endoplasmic reticulum (ER)-associated degradation substrates through a mechanism involving re-glucosylation of asparagine-linked glycans following release from the calnexin/calreticulin lectins. The evidence supporting this conclusion is solid using genetically-deficient cell models and well established biochemical methods to monitor the degradation of trafficking-incompetent ER-associated degradation substrates, although this could be improved by better defining of the importance of UGGT in the secretion of trafficking competent substrates. This work will be of specific interest to those interested in mechanistic aspects of ER protein quality control and protein secretion.

The authors have attempted to address my comments from the previous round of review, although some issues still remain. For example, the authors indicate that it is difficult to assess how UGGT1 influences degradation of secretion competent proteins, but this is not the case. This can be easily followed using metabolic labeling experiments, where you would get both the population of protein secreted and degraded under different conditions. Thus, I still feel that addressing the impact of UGGT1 depletion on the ER quality control for secretion competent protein remains an important point that could be better addressed in this work.

Further, in the previous submission, the authors showed that UGGT2 depletion demonstrates a similar reduction of ATF6 activation to that observed for UGGT1 depletion, although UGGT2 depletion does not reduce ATF6 protein levels like what is observed upon UGGT1 depletion. In the revised manuscript, they largely remove the UGGT2 data and only highlight the UGGT1 depletion data. While they are somewhat careful in their discussion, the implication is that UGGT1 regulates ATF6 activity by controlling its stability. The fact that UGGT2 has a similar effect on activity, but not stability, indicates that these enzymes may have other roles not directly linked to ATF6 stability. It is important to include the UGGT2 data and explicitly highlight this point in the discussion. Its fine to state that figuring out this other function is outside the scope of this work but removing it does not seem appropriate.

As I mentioned in my previous review, I think that this work is interesting and addresses an important gap in experimental evidence supporting a previously asserted dogma in the field. I do think that the authors would be better suited for highlighting the limitations of the study, as discussed above. Ultimately, though, this is an important addition to the literature.

Reviewer #3 (Public review):

Summary:

In this paper, Wang et al. provides the most comprehensive description and comparison of the expression of the different genes required to synthesize, transport and recycle the most common neurotransmitters (Glutamate, Acetylcholine, GABA, Serotonin, Dopamine, Octopamine and Tyramine) used by hermaphrodite and male C. elegans. This paper will be a seminal reference in the field. Building and contrasting observations from previous studies using fosmid, multicopy reporters and single cell sequencing, they now describe CRISPR/Cas-9-engineered reporter strains that, in combination with the multicolor pan-neuronal labeling of all C. elegans neurons (NeuroPAL), allows rigorous elucidation of neurotransmitter expression patterns. These novel reporters also illuminate previously unappreciated aspects of neurotransmitter biology in C. elegans, including sexual dimorphism of expression patterns, co-transmission and the elucidation of cell-specific pathways that might represent new forms of neurotransmission.

Strengths:

The authors set to establish neurotransmitter identities in C. elegans males and hermaphrodites via varying techniques, including integration of previous studies, examination of expression patterns and generation of endogenous CRISPR-labeled alleles. Their study is comprehensive, detailed and rigorous, and achieve the aims. It is an excellent reference for the field, particularly those interested in biosynthetic pathways of neurotransmission and their distribution in vivo, in neuronal and non-neuronal cells.

Weaknesses:

No weaknesses noted. The authors do a great job linking their characterizations with other studies and techniques, leading credence to their findings. As the authors note, there are sexually dimorphic differences across animals, and varying expression patterns of enzymes. While it is unlikely there will be huge differences in the reported patterns across individual animals, it is possible that these expression patterns could vary developmentally, or based on physiological or environmental conditions.

Reviewer #3 (Public Review):

Summary:

The authors invent song and syllable discrimination tasks they use to train deep networks. These networks they then use as a basis for routine song analysis and song evaluation tasks. For the analysis, they consider both data from their own colony and from another colony the network has not seen during training. They validate the analysis scores of the network against expert human annotators, achieving a correlation of 80-90%.

Strengths:

(1) Robust Validation and Generalizability: The authors demonstrate a good performance of the AVN across various datasets, including individuals exhibiting deviant behavior. This extensive validation underscores the system's usefulness and broad applicability to zebra finch song analysis, establishing it as a potentially valuable tool for researchers in the field.

(2) Comprehensive and Standardized Feature Analysis: AVN integrates a comprehensive set of interpretable features commonly used in the study of bird songs. By standardizing the feature extraction method, the AVN facilitates comparative research, allowing for consistent interpretation and comparison of vocal behavior across studies.

(3) Automation and Ease of Use. By being fully automated, the method is straightforward to apply and should introduce barely an adoption threshold to other labs.

(4) Human experts were recruited to perform extensive annotations (of vocal segments and of song similarity scores). These annotations released as public datasets are potentially very valuable.

Weaknesses:

(1) Poorly motivated tasks. The approach is poorly motivated and many assumptions come across as arbitrary. For example, the authors implicitly assume that the task of birdsong comparison is best achieved by a system that optimally discriminates between typical, deaf, and isolated songs. Similarly, the authors assume that song development is best tracked using a system that optimally estimates the age of a bird given its song. My issue is that these are fake tasks since clearly, researchers will know whether a bird is an isolated or a deaf bird, and they will also know the age of a bird, so no machine learning is needed to solve these tasks. Yet, the authors imagine that solving these placeholder tasks will somehow help with measuring important aspects of vocal behavior. Along similar lines, authors assume that a good measure of similarity is one that optimally performs repeated syllable detection (i.e. to discriminate same syllable pairs from different pairs). The authors need to explain why they think these placeholder tasks are good and why no better task can be defined that more closely captures what researchers want to measure. Note: the standard tasks for self-supervised learning are next word or masked word prediction, why are these not used here?

(2) The machine learning methodology lacks rigor. The aims of the machine learning pipeline are extremely vague and keep changing like a moving target. Mainly, the deep networks are trained on some tasks but then authors evaluate their performance on different, disconnected tasks. For example, they train both the birdsong comparison method (L263+) and the song similarity method (L318+) on classification tasks. However, they evaluate the former method (LDA) on classification accuracy, but the latter (8-dim embeddings) using a contrast index. In machine learning, usually, a useful task is first defined, then the system is trained on it and then tested on a held-out dataset. If the sensitivity index is important, why does it not serve as a cost function for training? Also, usually, in solid machine learning work, diverse methods are compared against each other to identify their relative strengths. The paper contains almost none of this, e.g. authors examined only one clustering method (HDBSCAN).

(3) Performance issues. The authors want to 'simplify large-scale behavioral analysis' but it seems they want to do that at a high cost. (Gu et al 2023) achieved syllable scores above 0.99 for adults, which is much larger than the average score of 0.88 achieved here (L121). Similarly, the syllable scores in (Cohen et al 2022) are above 94% (their error rates are below 6%, albeit in Bengalese finches, not zebra finches), which is also better than here. Why is the performance of AVN so low? The low scores of AVN argue in favor of some human labeling and training on each bird.

(4) Texas bias. It is true that comparability across datasets is enhanced when everyone uses the same code. However, the authors' proposal essentially is to replace the bias between labs with a bias towards birds in Texas. The comparison with Rockefeller birds is nice, but it amounts to merely N=1. If birds in Japanese or European labs have evolved different song repertoires, the AVN might not capture the associated song features in these labs well.

(5) The paper lacks an analysis of the balance between labor requirement, generalizability, and optimal performance. For tasks such as segmentation and labeling, fine-tuning for each new dataset could potentially enhance the model's accuracy and performance without compromising comparability. E.g. How many hours does it take to annotate hundred song motifs? How much would the performance of AVN increase if the network were to be retrained on these? The paper should be written in more neutral terms, letting researchers reach their own conclusions about how much manual labor they want to put into their data.

(6) Full automation may not be everyone's wish. For example, given the highly stereotyped zebra finch songs, it is conceivable that some syllables are consistently mis-segmented or misclassified. Researchers may want to be able to correct such errors, which essentially amounts to fine-tuning AVN. Conceivably, researchers may want to retrain a network like the AVN on their own birds, to obtain a more fine-grained discriminative method.

(7) The analysis is restricted to song syllables and fails to include calls. No rationale is given for the omission of calls. Also, it is not clear how the analysis deals with repeated syllables in a motif, whether they are treated as two-syllable types or one.

(8) It seems not all human annotations have been released and the instruction sets given to experts (how to segment syllables and score songs) are not disclosed. It may well be that the differences in performance between (Gu et al 2023) and (Cohen et al 2022) are due to differences in segmentation tasks, which is why these tasks given to experts need to be clearly spelled out. Also, the downloadable files contain merely labels but no identifier of the expert. The data should be released in such a way that lets other labs adopt their labeling method and cross-check their own labeling accuracy.

(9) The failure modes are not described. What segmentation errors did they encounter, and what syllable classification errors? It is important to describe the errors to be expected when using the method.

(10) Usage of Different Dimensionality Reduction Methods: The pipeline uses two different dimensionality reduction techniques for labeling and similarity comparison - both based on the understanding of the distribution of data in lower-dimensional spaces. However, the reasons for choosing different methods for different tasks are not articulated, nor is there a comparison of their efficacy.

(11) Reproducibility: are the measurements reproducible? Systems like UMAP always find a new embedding given some fixed input, so the output tends to fluctuate.

Reviewer #3 (Public Review):

In this manuscript, Zhou et al. present a computational model of memory replay. Their model (CMR-replay) draws from temporal context models of human memory (e.g., TCM, CMR) and claims replay may be another instance of a context-guided memory process. During awake learning, CMR replay (like its predecessors) encodes items alongside a drifting mental context that maintains a recency-weighted history of recently encoded contexts/items. In this way, the presently encoded item becomes associated with other recently learned items via their shared context representation - giving rise to typical effects in recall such as primacy, recency, and contiguity. Unlike its predecessors, CMR-replay has built-in replay periods. These replay periods are designed to approximate sleep or wakeful quiescence, in which an item is spontaneously reactivated, causing a subsequent cascade of item-context reactivations that further update the model's item-context associations.

Using this model of replay, Zhou et al. were able to reproduce a variety of empirical findings in the replay literature: e.g., greater forward replay at the beginning of a track and more backward replay at the end; more replay for rewarded events; the occurrence of remote replay; reduced replay for repeated items, etc. Furthermore, the model diverges considerably (in implementation and predictions) from other prominent models of replay that, instead, emphasize replay as a way of predicting value from a reinforcement learning framing (i.e., EVB, expected value backup).

Overall, I found the manuscript clear and easy to follow, despite not being a computational modeller myself. (Which is pretty commendable, I'd say). The model also was effective at capturing several important empirical results from the replay literature while relying on a concise set of mechanisms - which will have implications for subsequent theory-building in the field.

With respect to weaknesses, additional details for some of the methods and results would help the readers better evaluate the data presented here (e.g., explicitly defining how the various 'proportion of replay' DVs were calculated).

For example, for many of the simulations, the y-axis scale differs from the empirical data despite using comparable units, like the proportion of replay events (e.g., Figures 1B and C). Presumably, this was done to emphasize the similarity between the empirical and model data. But, as a reader, I often found myself doing the mental manipulation myself anyway to better evaluate how the model compared to the empirical data. Please consider using comparable y-axis ranges across empirical and simulated data wherever possible.

In a similar vein to the above point, while the DVs in the simulations/empirical data made intuitive sense, I wasn't always sure precisely how they were calculated. Consider the "proportion of replay" in Figure 1A. In the Methods (perhaps under Task Simulations), it should specify exactly how this proportion was calculated (e.g., proportions of all replay events, both forwards and backwards, combining across all simulations from Pre- and Post-run rest periods). In many of the examples, the proportions seem to possibly sum to 1 (e.g., Figure 1A), but in other cases, this doesn't seem to be true (e.g., Figure 3A). More clarity here is critical to help readers evaluate these data. Furthermore, sometimes the labels themselves are not the most informative. For example, in Figure 1A, the y-axis is "Proportion of replay" and in 1C it is the "Proportion of events". I presumed those were the same thing - the proportion of replay events - but it would be best if the axis labels were consistent across figures in this manuscript when they reflect the same DV.

Reviewer #3 (Public review):

Summary:

The authors aim to establish that cipargamin can be used for the treatment of infection caused by Babesia organisms.

Strengths:

The study provides strong evidence that cipargamin is effective against various Babesia species. In vitro, growth assays were used to establish that cipargamin is effective against Babesia bovis and Babesia gibsoni. Infection of mice with Babesia microti demonstrated that cipargamin is as effective as the combination of atovaquone plus azithromycin. Cipargamin protected mice from lethal infection with Babesia rodhaini. Mutations that confer resistance to cipargamin were identified in the gene encoding ATP4, a P-type Na ATPase that was found in other apicomplexan parasites, thereby validating ATP4 as the target of cipargamin.

Weaknesses:

Cipargamin was tested in vivo at a single dose administered daily for 7 days. Despite the prospect of using cipargamin for the treatment of human babesiosis, there was no attempt to identify the lowest dose of cipagarmin that protects mice from Babesia microti infection. Exposure to cipargamin can induce resistance, indicating that cipargamin should not be used alone but in combination with other drugs. There was no attempt at testing cipargamin in combination with other drugs, particularly atovaquone, in the mouse model of Babesia microti infection. Given the difficulty in treating immunocompromised patients infected with Babesia microti, it would have been informative to test cipargamin in a mouse model of severe immunosuppression (SCID or rag-deficient mice).

Reviewer #3 (Public review):

This important study by Bohorquez et al examines the determinants necessary for concentrating the spatial modulator of cell division, MinD, at the future site of division and the cell poles. Proper localization of MinD is necessary to bring the division inhibitor, MinC, in proximity to the cell membrane and cell poles where it prevents aberrant assembly of the division machinery. In contrast to E. coli, in which MinD oscillates from pole to pole courtesy of a third protein MinE, how MinD localization is achieved in B. subtilis - which does not encode a MinE analog - has remained largely a mystery. The authors present compelling data indicating that MinD dimerization is dispensable for membrane localization but required for concentration at the cell poles. Dimerization is also important for interactions between MinD and MinC, leading to the formation of large protein complexes. Computational modeling, specifically a Monte Carlo simulation, supports a model in which differences in diffusion rates between MinD monomers and dimers lead to the concentration of MinD at cell poles. Once there, interaction with MinC increases the size of the complex, further reinforcing diffusion differences. Notably, interactions with MinJ-which has previously been implicated in MinCD localization, are dispensable for concentrating MinD at cell poles although MinJ may help stabilize the MinCD complex at those locations.

Reviewer #3 (Public Review):

Summary:

The research by Qianqian Ju et al. found that the knockdown of TAK1 promoted ESCC migration and invasion, whereas overexpression of TAK1 resulted in the opposite outcome. These in vitro findings could be recapitulated in a xenograft metastasis mouse model.<br /> Mechanistically, TAK1 phosphorylates PLCE1 S1060 in the cells, decreasing PLCE1 enzyme activity and repressing PIP2 hydrolysis. As a result, reducing DAG and inositol IP3, thereby suppressing signal transduction of PKC/GSK 3β/β Catenin. Consequently, cancer metastasis-related genes were impeded by TAK1.<br /> Overall, this study offers some intriguing observations. Providing a potential druggable target for developing agents for dealing with ESCC.

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

The weaknesses part of this research are:

Most of the experiments were done in protein overexpression conditions, with the protein level increasing hundreds of folds in the cell, producing an artificial environment that would sometimes generate false positive results.

Reviewer #3 (Public review):

Summary:

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

Strengths:

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

Reviewer #3 (Public review):

Summary:

In this study, the authors probe the connections between clustering of the Met4/32 transcription factors (TFs), clustering of their regulatory targets, and transcriptional regulation. While there is an increasing number of studies on TF clustering in vitro and in vivo, there is an important need to probe whether clustering plays a functional role in gene expression. Another important question is whether TF clustering leads to the clustering of relevant gene targets in vivo. Here the authors provide several lines of evidence to make a compelling case that Met4/32 and their target genes cluster and that this leads to an increase in transcription of these genes in the induced state. First, they found that, in the induced state, Met4/32 forms co-localized puncta in vivo. This is supported by in vitro studies showing that these TFs can form condensates in vitro with Med32 being the driver of these condensates. They found that two target genes, MET6 and MET13 have a higher probability of being co-localized with Met4 puncta compared with non-target loci. Using a targeted DNA methylation assay, they found that MET13 and MET6 show Met4-dependent long-range interactions with other Met4-regulated loci, consistent with the clustering of at least some target genes under induced conditions. Finally, by inserting a Met4-regulated reporter gene at variable distances from MET6, they provide evidence that insertion near this gene is a modest hotspot for activity.

Comments on revised version:

In this revised manuscript, the authors have achieved a good balance between revising the text/figures, and explaining why some lines of experiments proposed by reviewers are either not practical or beyond the scope of this work. I think that the revised study is an important contribution to understanding the function of transcription factors, TF condensates, and gene localization in a stress-responsive system.

Reviewer #3 (Public review):

Summary:

Yu et al harness the capabilities of mesoscopic 2P imaging to record simultaneously from populations of neurons in several visual cortical areas and measure their correlated variability. They first divide neurons in 65 classes depending on their tuning to moving gratings. They found the pairs of neurons of the same tuning class show higher noise correlations (NCs) both within and across cortical areas. Based on these observations and a model they conclude that visual information is broadcast across areas through multiple, discrete channels with little mixing across them.<br /> NCs can reflect indirect or direct connectivity, or shared afferents between pairs of neurons, potentially providing insight on network organization. While NCs have been comprehensively studied in neurons pairs of the same area, the structure of these correlations across areas is much less known. Thus, the manuscripts present novel insights on the correlation structure of visual responses across multiple areas.

Strengths:

The measurements of shared variability across multiple areas are novel. The results are mostly well presented and many thorough controls for some metrics are included.

Weaknesses:

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

Specifically:

Major concerns:

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

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

In the new version of the manuscript, behavioral modulations are explicitly considered in Figure S8. New analyses show that most of the variance of the neuronal responses is driven by the stimulus, rather than by behavioural variable. However, they new analyses still do not address if the shared noise correlation in cotuned neurons is also independent of behavioral modulations .

As behavioral modulations are not considered this confound affects the conclusions and the conclusion that activity in communicated unmixed across areas ( results in Figure 4), as it would result in larger NCs the more similar the tuning of the neurons is, independently of any connectivity feature. It seems that this alternative hypothesis can explain the results without the need of discrete broadcasting channels or any particular network architecture and should be addressed to support the main claims.

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

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

Reviewer #3 (Public Review):

Summary:

In this manuscript, Mehrhof and Nord study a large dataset of participants collected online (n=958 after exclusions) who performed a simple effort-based choice task. They report that the level of effort and reward influence choices in a way that is expected from prior work. They then relate choice preferences to neuropsychiatric syndromes and, in a smaller sample (n<200), to people's circadian preferences, i.e., whether they are a morning-preferring or evening-preferring chronotype. They find relationships between the choice bias (a model parameter capturing the likelihood to accept effort-reward challenges, like an intercept) and anhedonia and apathy, as well as chronotype. People with higher anhedonia and apathy and an evening chronotype are less likely to accept challenges (more negative choice bias). People with an evening chronotype are also more reward sensitive and more likely to accept challenges in the evening, compared to the morning.

Strengths:

This is an interesting and well-written manuscript which replicates some known results and introduces a new consideration related to chronotype relationships which have not been explored before. It uses a large sample size and includes analyses related to transdiagnostic as well as diagnostic criteria.

Weaknesses:

The authors do not explore how chronotype and depression are related (does one mediate the effect of the other etc). Both variables are included in the same model in the revised article now which is a great improvement, but it also means psychopathology and circadian rhythms are treated as distinct phenomena and their relationship in predicting effort-reward preferences is not examined.

Reviewer #3 (Public review):

Summary:

Here Li et al. examine pup-directed behavior in virgin Mandarin voles. Some males and females tend towards infanticide, others tend towards pup care. c-Fos staining showed more oxytocin cells activated in the paraventricular nucleus (PVN) of the hypothalamus in animals expressing pup care behaviors than in infanticidal animals. Optogenetic stimulation of PVN oxytocin neurons (with an oxytocin-specific virus to express the opsin transgene) increased pup-care, or in infanticidal voles increased latency towards approach and attack. Suppressing activity of PVN oxytocin neurons promoted infanticide. Use of a recent oxytocin GRAB sensor (OT1.0) showed changes in medial prefrontal cortex (mPFC) signals as measured with photometry in both sexes. Activating mPFC oxytocin projections increased latency to approach and attack in infanticidal females and males (similar to the effects of peripheral oxytocin injections), whereas in pup caring animals only males showed a decrease to approach. Inhibiting these projections increased infanticidal behaviors in both females and males, and no effect on pup caretaking.

Strengths:

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

The authors have done a good job responding to the comments on their preprint. I'd ask them to check their z-scored values, as the mean of a z-scored value should be 0 over time. Also I'm not sure I agree that the fiber photometry system "can automatically exclude effects of motion artifacts"; yes that is a function of imaging at a different wavelength but that process is also prone to error and imperfect.

Reviewer #3 (Public review):

Summary:

In this manuscript the authors explore the contribution of metabolism to the response of two subpopulations of macrophages to bacterial pathogens commonly encountered in the human lung, as well as the influence of priming signals typically produced at a site of inflammation. The two subpopulations are resident airway macrophages (AM) isolated via bronchoalveolar lavage and monocyte-derived macrophages (MDM) isolated from human blood and differentiated using human serum. The two cell types were primed using IFNγ and Il-4, which are produced at sites of inflammation as part of initiation and resolution of inflammation respectively, followed by stimulation with either heat-killed tuberculosis (Mtb) or LPS to simulate interaction with a bacterial pathogen that is either gram-negative in the case of Mtb or gram-positive in the case of LPS. The authors use human cells for this work, which makes use of widely reported and thoroughly described priming signals, as well as model antigens. This makes the observations on the functional response of these two subpopulations relevant to human health and disease to a greater extent that the mouse models typically used to interrogate theses interactions. To examine the relationship between metabolism and functional response, the authors measure rates of oxidative phosphorylation and glycolysis under baseline conditions, primed using IFNγ or IL-4, and primed and stimulated with Mtb or LPS.

The authors addressed most of the initial critiques. The dose of IFNγ used was justified, figure legends were harmonized, a contextual definition was provided for the term "functional plasticity," and the airway macrophage population was partially characterized by flow cytometry. However, some concerns remain relating to the clarity of methods and use of statistics. The authors have not adequately explained how % change was calculated in Figure 1, in either the figure legend or the methods section. Additionally, the use of multiple statistical analyses on the same data set in figures 4 and 5, with data exclusion resulting in lower p values, is not satisfactorily justified.

Strengths:

• The data indicate that both populations of macrophages increase metabolic rates when primed, but MDMs decrease their rates of oxidative phosphorylation after IL-4 priming and bacterial exposure while AMs do not.

• It is demonstrated that glycolysis rates are directly linked to the expression of surface molecules involved in T-cell stimulation and while secretion of TNFα in AM is dependent on glycolysis, in MDM this is not the case. IL-10 secretion does not appear regulated by glycolysis in either population. It is also demonstrated that Mtb and LPS stimulation produces responses that are not metabolically consistent across the two macrophage populations. The Mtb-induced response in MDMs differed from the LPS response, in that it relies on glycolysis, while this relationship is reversed in AMs. The difference in metabolic contributions to functional outcomes between these two macrophage populations is significant, despite acknowledgement of the reductive nature of the system by the authors.

• The observations that AM and MDM rely on glycolysis for production of cytokines during a response to bacterial pathogens in the lung, but that only AM shift to Warburg Metabolism following exposure to IL-4, are supported by the data and a significant contribution the study of the innate immune response.

Weaknesses:

Critiques:

• It is still difficult to interpret the metabolism data due to inconsistent normalization. It appears that in the case of rate measurement the data is normalized to unstimulated macrophages where values are set to one, but in the case of % change the values from unstimulated cells are not set to 100% and the methods say that values were calculated using primed controls, which is ambiguous. It is therefore unclear how exactly the % change values were determined. This makes it difficult to conclude whether the changes in glycolysis and oxidative phosphorylation in primed cells after stimulation are proportional to changes in unprimed cells. This would suggest that the majority of the observed effect on metabolism comes from priming itself and not from the subsequent stimulation as the authors claim.

• The use of repeated statistical analyses with different comparison groups in the same figure/data set (e.g., in Fig.4) is still not justified. The current approach, using two-way ANOVA, removing a third of the dataset, and then applying another two-way ANOVA, produces the desired p values, but is not appropriate.

Conclusion:

Overall, this study reveals how inflammatory and anti-inflammatory cytokine priming contributes to the metabolic reprogramming of AM and MDM populations. Their conclusions regarding the relationship between cytokine secretion and inflammatory molecule expression in response to bacterial stimuli are supported by the data. The involvement of metabolism in innate immune cell function is relevant when devising treatment strategies that target the innate immune response during infection. The data presented in this paper further our understanding of that relationship and advance the field of innate immune cell biology.

Reviewer #3 (Public review):

General Comments

This work probes the control of walking in cats at different speeds and different states (split-belt and regular treadmill walking). Since the time of Sherrington there has been ongoing debate on this issue. The authors provide modeling data showing that they could reproduce data from cats walking on a specialized treadmill allowing for regular and split-belt walking. The data suggest that a non-oscillating state-machine regime best explains slow walking - where phase transitions are handled by external inputs into the spinal network. They then show at higher speeds a flexor-driven and then a classical half-center regime dominates. In spinal animals, it appears that a non-oscillating state-machine regime best explains the experimental data. The model is adapted from their previous work and raises interesting questions regarding the operation of spinal networks, that, at low speeds, challenge assumptions regarding central pattern generator function. This is an outstanding study which will be of general interest to the neuroscience community.

Strengths

The study has several strengths. Firstly the detailed model has been well established by the authors and provides details that relate to experimental data such as commissural interneurons (V0c and V0d), along with V3 and V2a interneuron data. Sensory input along with descending drive is also modelled and moreover the model reproduces many experimental data findings. Moreover, the idea that sensory feedback is more crucial at lower speeds, also is confirmed by presynaptic inhibition increasing with descending drive. The inclusion of experimental data from split-belt treadmills, and the ability of the model to reproduce findings here is a definite plus.

Weaknesses

Conceptually, this is a compelling study which provides interesting modeling data regarding the idea that the network can operate in different regimes, especially at lower speeds. The modelling data speaks for itself, but on the other hand, sensory feedback also provides generalized excitation of neurons which in turn project to the CPG. That is they are not considered part of the CPG proper. The authors have discussed this possibility in their revised paper.

Reviewer #3 (Public Review):

Summary:

It has been proposed that the FOI is a method of using parasite genetics to determine changes in transmission in areas with high asymptomatic infection. The manuscript attempts to use queuing theory to convert multiplicity of infection estimates (MOI) into estimates of the force of infection (FOI), which they define as the number of genetically distinct blood-stage strains. They look to validate the method by applying it to simulated results from a previously published agent-based model. They then apply these queuing theory methods to previously published and analysed genetic data from Ghana. They then compare their results to previous estimates of FOI.

Strengths:

It would be great to be able to infer FOI from cross-sectional surveys which are easier and cheaper than current FOI estimates which require longitudinal studies. This work proposes a method to convert MOI to FOI for cross-sectional studies. They attempt to validate this process using a previously published agent-based model which helps us understand the complexity of parasite population genetics.

Weaknesses:

(1) I fear that the work could be easily over-interpreted as no true validation was done, as no field estimates of FOI (I think considered true validation) were measured. The authors have developed a method of estimating FOI from MOI which makes a number of biological and structural assumptions. I would not call being able to recreate model results that were generated using a model that makes its own (probably similar) defined set of biological and structural assumptions a validation of what is going on in the field. The authors claim this at times (for example, Line 153 ) and I feel it would be appropriate to differentiate this in the discussion.

(2) Another aspect of the paper is adding greater realism to the previous agent-based model, by including assumptions on missing data and under-sampling. This takes prominence in the figures and results section, but I would imagine is generally not as interesting to the less specialised reader. The apparent lack of impact of drug treatment on MOI is interesting and counterintuitive, though it is not really mentioned in the results or discussion sufficiently to allay my confusion. I would have been interested in understanding the relationship between MOI and FOI as generated by your queuing theory method and the model. It isn't clear to me why these more standard results are not presented, as I would imagine they are outputs of the model (though happy to stand corrected - it isn't entirely clear to me what the model is doing in this manuscript alone).

(3) I would suggest that outside of malaria geneticists, the force of infection is considered to be the entomological inoculation rate, not the number of genetically distinct blood-stage strains. I appreciate that FOI has been used to explain the latter before by others, though the authors could avoid confusion by stating this clearly throughout the manuscript. For example, the abstract says FOI is "the number of new infections acquired by an individual host over a given time interval" which suggests the former, please consider clarifying.

(4) Line 319 says "Nevertheless, overall, our paired EIR (directly measured by the entomological team in Ghana (Tiedje et al., 2022)) and FOI values are reasonably consistent with the data points from previous studies, suggesting the robustness of our proposed methods". I would agree that the results are consistent, given that there is huge variation in Figure 4 despite the transformed scales, but I would not say this suggests a robustness of the method.

(5) The text is a little difficult to follow at times and sometimes requires multiple reads to understand. Greater precision is needed with the language in a few situations and some of the assumptions made in the modelling process are not referenced, making it unclear whether it is a true representation of the biology.

Reviewer #3 (Public Review):

The manuscript by Talbi R et al. generated transgenic mice to assess the reproduction function of MC4R in Kiss1 neurons in vivo and used electrophysiology to test how MC4R activation regulated Kiss1 neuronal firing in ARH and AVPV/PeN. This timely study is highly significant in the field of neuroendocrinology research for the following reasons.

(1) The authors' findings are significant in the field of reproductive research. Despite the known presence of MC4R signaling in Kiss1 neurons, the exact mechanisms of how MC4R signaling regulates different Kiss1 neuronal populations in the context of sex hormone fluctuations are not completely understood. The authors reported that knocking out Mc4r from Kiss1 neurons replicates the reproductive impairment of MC4RKO mice, and Mc4r expression in Kiss1 neurons in the MC4R null background partially restored the reproductive impairment. MC4R activation excites Kiss1 ARH neurons and inhibits Kiss1 AVPV/PeN neurons (except for elevated estradiol).

(2) Reproduction dysfunction is one of obesity comorbidities. MC4R loss-of-function mutations cause obesity phenotype and impaired reproduction. However, it's hard to determine the causality. The authors carefully measured the body weight of the different mouse models (Figure 1C, Figure 2A, Figure 3B). For example, the Kiss1-MC4RKO females showed no body weight difference at the age of puberty onset. This clearly demonstrated the direct function of MC4R signaling in reproduction but was not a consequence of excessive adiposity.

(3) Gene expression findings in the "KNDy" system are in line with the reproduction phenotype.

(4) The electrophysiology results reported in this manuscript are innovative and provide more details of MC4R activation and Kiss1 neuronal activation.

Overall, the authors have presented sufficient background in a clear and logically organized structure, clearly stated the key question to be addressed, used the appropriate methodology, produced significant and innovative main findings, and made a justified conclusion.

Reviewer #3 (Public Review):

Dr Jin and colleagues revisit DLK and its established multifactorial roles in neuronal development, axonal injury, and neurodegeneration. The ambitious aim here is to understand the DLK-dependent gene network in the brain and, to pursue this, they explore the role of DLK in hippocampal glutamatergic neurons using conditional knockout and induced overexpression mice. They produce evidence that dorsal CA1 and dentate gyrus neurons are vulnerable to elevated expression of DLK, while CA3 neurons appear unaffected. Then they identify the DLK-dependent translatome featured by conserved molecular signatures and cell-type specificity. Their evidence suggests that increased DLK signaling is associated with possible STMN4 disruptions to microtubules, among else. They also produce evidence on cultured hippocampal neurons showing that expression levels of DLK are associated with changes in neurite outgrowth, axon specification, and synapse formation. They posit that downstream translational events related to DLK signaling in hippocampal glutamatergic neurons are a generalizable paradigm for understanding neurodegenerative diseases.

Strengths

This is an interesting paper based on a lot of work and a high number of diverse experiments that point to the pervasive roles of DLK in the development of select glutamatergic hippocampal neurons. One should applaud the authors for their work in constructing sophisticated molecular cre-lox tools and their expert Ribotag analysis, as well as technical skill and scholarly treatment of the literature. I am somewhat more skeptical of interpretations and conclusions on spatial anatomical selectivity without stereological approaches and also going directly from (extremely complex) Ribotag profiling patterns to relevance based on immunohistochemistry and no additional interventions to manipulate (e.g. by knocking down or blocking) their top Ribotag profile hits. Also, it seems to this reviewer that major developmental claims in the paper are based on gene translational profiling dependent on DLK expression, not DLK activation, despite some evidence in the paper that there is a correlation between the two. Therefore, observed patterns and correlations may or may not be physiologically or pathologically relevant. Generalizability to neurodegenerative diseases is an overreach not justified by the scope, approach, and findings of the paper.

Weaknesses and Suggestions:

The authors state that the rationale for the translatomic studies is to "to gain molecular understanding of gene expression associated with DLK in glutamatergic neurons" and to characterize the "DLK-dependent molecular and cellular network", However, a problem with the experimental design is the selection of an anatomical region at a time point featured by active neurodegeneration. Therefore, it is not straightforward that the differentially expressed genes or pathways caused by DLK overexpression changes could be due to processes related to neurodegeneration. Indeed, the authors find enrichment of signals related to pathways involved in extracellular matrix organization, apoptosis, unfolded protein responses, the complement cascade, DNA damage responses, and depletion of signals related to mitochondrial electron transport, etc., all of which could be the consequence of neurodegeneration regardless of cause. A more appropriate design to discover DLK-dependent pathways might be to look at a region and/or a time point that is not confounded by neurodegeneration.

In a related vein, the authors ask "if the differentially expressed genes associated with DLK(iOE) might show correlation to neuronal vulnerability" and, to answer this question, they select the set of differentially expressed genes after DLK overexpression and assess their expression patterns in various regions under normal conditions. It looks to me that this selection is already confounded by neurodegeneration which could be the cause for their downregulation. Therefore, such gene profiles may not be directly linked to neuronal vulnerability. A similar issue also relates to the conclusion that "...the enrichment of DLK-dependent translation of genes in CA1 suggests that the decreased expression of these genes may contribute to CA1 neuron vulnerability to elevated DLK".

To understand the role and relevance of the DLK overexpression model, there should be a discussion of to what extent it corresponds to endogenous levels of DLK expression or DLK-MAPK pathway activation under baseline or pathological conditions.

The authors posit that "dorsal CA1 neurons are vulnerable to elevated DLK expression, while neurons in CA3 appear largely resistant to DLK overexpression". This statement assumes that DLK expression levels start at a similar baseline among regions. Do the authors have any such data? Ideally, they should show whether DLK expression and p-c-Jun (as a marker of downstream DLK signaling) are the same or different across regions in both WT and overexpression mice. For example, what are the DLK/p-c-Jun expression levels in regions other than CA1 in Supplementary Figures 2-3 and how do they compare with each other? Normalization to baseline for each region does not allow such a comparison. Also, in Supplementary Figure 6, analyses and comparisons between regions are done at a time point when degeneration has already started. Ideally, these should be done at P10.

Illustration of proposed selective changes in hippocampal sector volume needs to be very carefully prepared in view of the substantial claims on selective vulnerability. In 2A under P15 and especially P60, it is difficult to see the difference - this needs lower magnification and a lot of care that anteroposterior levels are identical because hippocampal sector anatomy and volumes of sectors vary from level to level. One wonders if the cortex shrinks, too. This is important.

One cannot be sure that there is selective death of hippocampal sectors with DLK overexpression versus, say, rearrangement of hippocampal architecture. One may need stereological analysis, otherwise this substantial claim appears overinterpreted.

Is the GFAP excess reflective of neuroinflammation? What do microglial markers show? The presence of neuroinflammation does not bode well with apoptosis. Speaking of which, TUNEL in one cell in Supplementary Figure 4E is not strong evidence of a more widespread apoptotic event in CA1.

In several places in the paper (as illustrated in Figure 4B, Supplementary Figure 2B, etc.): the unit of biological observation in animal models is typically not a cell, but an organism, in which averaged measures are generated. This is a significant methodological problem because it is not easy to sample neurons without involving stereological methods. With the approach taken here, there is a risk that significance may be overblown.

Other Comments and Questions:

Supplementary Figure 9: The authors state that data points are shown for individual ROIs - ideally, they should also show averages for biological replicates. Can the authors confirm that statistical analyses are based on biological replicates (mice) and not ROIs?

For in vitro experiments, what is the effect of DLK overexpression on neuronal viability and density? Could these variables confound effects on synaptogenesis/synapse maturation?

Correlations between c-jun expression and phosphorylation are extremely important and need to be carefully and convincingly documented. I am a bit concerned about Supplementary Figure 6 images, especially 6B-CA1 (no difference between control and KO, too small images) and 6D (no p-c-Jun expression at all anywhere in the hippocampus at P15?).

Reviewer #3 (Public review):

Summary:

The manuscript by Chen et al examines the structure of the inactive LRRK2 bound to microtubules using cryo-EM tomography. Mutations in this protein have been shown to be linked to Parkinson's Disease. It is already shown that the active-like conformation of LRRK2 binds to the MT lattice, but this investigation shows that full-length LRRk2 can oligomerize on MTs in its autoinhibited state with different helical parameters than were observed with the active-like state. The structural studies suggest that the autoinhibited state is less stable on MTs.

Strengths:

The protein of interest is very important biomedically and a novel conformational binding to microtubules in the proposed.

Weaknesses:

(1) The structures are all low resolution.

(2) There are no measurements of the affinity of the various LRRK2 molecules (with and without inhibitors) to microtubules. This should be addressed through biochemical sedimentation assay.

Reviewer #3 (Public review):

Summary:

Non-opioid analgesics derived from human amniotic membrane (AM) product represents a novel and unique approach to analgesia that may avoid the traditional harms associated with opioids. Here, the study investigators demonstrate that HC-HAPTX3 is the primary bioactive component of the AM product FLO responsible for anti-nociception in mouse-model and in-vitro dorsal root ganglion (DRG) cell culture experiments. The mechanism is demonstrated to be via CD44 with an acute cytoskeleton rearrangement that is induced that inhibits Na+ and Ca++ current through ion channels. Taken together, the studies reported in the manuscript provide supportive evidence clarifying the mechanisms and efficacy of HC-HAPTX3 antinociception and analgesia.

Strengths:

Extensive experiments including murine behavioral paw withdrawal latency and Catwalk test data demonstrating analgesic properties. The breadth and depth of experimental data are clearly supporting mechanisms and antinociceptive properties.

Weaknesses:

A few changes to the text of the manuscript would be recommended but no major weaknesses were identified.

Reviewer #3 (Public Review):

Summary:

Most previous studies have focused on the contributions of Abeta and amyloid plaques in the neuronal degeneration associated with Alzheimer's disease, especially in the context of impaired synaptic transmission and plasticity which underlies the impaired cognitive functions, a hallmark in AD. But processes independent of Abeta and plaques are much less explored, and to some extent, the contributions of these processes are less well understood. Luo et all addressed this important question with an array of approaches, and their findings generally support the contribution of beta-CTF-dependent but non-Abeta-dependent process to the impaired synaptic properties in the neurons. Interestingly, the above process appears to operate in a cell-autonomous manner. This cell-autonomous effect of beta-CTF as reported here may facilitate our understanding of some potentially important cellular processes related to neurodegeneration. Although these findings are valuable, it is key to understand the probability of this process occurring in a more natural condition, such as when this process occurs in many neurons at the same time. This will put the authors' findings into a context for a better understanding of their contribution to either physiological or pathological processes, such as Alzheimer's. The experiments and results using the cell system are quite solid, but the in vivo results are incomplete and hence less convincing (see below). The mechanistic analysis is interesting but primitive and does not add much more weight to the significance. Hence, further efforts from the authors are required to clarify and solidify their results, in order to provide a complete picture and support for the authors' conclusions.

Strengths:

(1) The authors have addressed an interesting and potentially important question

(2) The analysis using the cell system is solid and provides strong support for the authors' major conclusions. This analysis has used various technical approaches to support the authors' conclusions from different aspects and most of these results are consistent with each other.

Weaknesses:

(1) The relevance of the authors' major findings to the pathology, especially the Abeta-dependent processes is less clear, and hence the importance of these findings may be limited.

(2) In vivo analysis is incomplete, with certain caveats in the experimental procedures and some of the results need to be further explored to confirm the findings.

(3) The mechanistic analysis is rather primitive and does not add further significance.

Reviewer #3 (Public review):

Summary:

Schmidt et al., aimed to provide an extremely comprehensive demonstration of the influence cardiac electromagnetic fields have on the relationship between age and the aperiodic slope measured from electroencephalographic (EEG) and magnetoencephalographic (MEG) data.

Strengths:

Schmidt et al., used a multiverse approach to show that the cardiac influence on this relationship is considerable, by testing a wide range of different analysis parameters (including extensive testing of different frequency ranges assessed to determine the aperiodic fit), algorithms (including different artifact reduction approaches and different aperiodic fitting algorithms), and multiple large datasets to provide conclusions that are robust to the vast majority of potential experimental variations.

The study showed that across these different analytical variations, the cardiac contribution to aperiodic activity measured using EEG and MEG is considerable, and likely influences the relationship between aperiodic activity and age to a greater extent than the influence of neural activity.

Their findings have significant implications for all future research that aims to assess aperiodic neural activity, suggesting control for the influence of cardiac fields is essential.

Weaknesses:

Figure 4I: The regressions explained here seem to contain a very large number of potential predictors. Based on the way it is currently written, I'm assuming it includes all sensors for both the ECG component and ECG rejected conditions?

I'm not sure about the logic of taking a complete signal, decomposing it with ICA to separate out the ECG and non-ECG signals, then including these latent contributions to the full signal back into the same regression model. It seems that there could be some circularity or redundancy in doing so. Can the authors provide a justification for why this is a valid approach?

I'm not sure whether there is good evidence or rationale to support the statement in the discussion that the presence of the ECG signal in reference electrodes makes it more difficult to isolate independent ECG components. The ICA algorithm will still function to detect common voltage shifts from the ECG as statistically independent from other voltage shifts, even if they're spread across all electrodes due to the referencing montage. I would suggest there are other reasons why the ICA might lead to imperfect separation of the ECG component (assumption of the same number of source components as sensors, non-Gaussian assumption, assumption of independence of source activities).

The inclusion of only 32 channels in the EEG data might also have reduced the performance of ICA, increasing the chances of imperfect component separation and the mixing of cardiac artifacts into the neural components, whereas the higher number of sensors in the MEG data would enable better component separation. This could explain the difference between EEG and MEG in the ability to clean the ECG artifact (and perhaps higher-density EEG recordings would not show the same issue).

In addition to the inability to effectively clean the ECG artifact from EEG data, ICA and other component subtraction methods have also all been shown to distort neural activity in periods that aren't affected by the artifact due to the ubiquitous issue of imperfect component separation (https://doi.org/10.1101/2024.06.06.597688). As such, component subtraction-based (as well as regression-based) removal of the cardiac artifact might also distort the neural contributions to the aperiodic signal, so even methods to adequately address the cardiac artifact might not solve the problem explained in the study. This poses an additional potential confound to the "M/EEG without ECG" conditions.

Literature Analysis, Page 23: was there a method applied to address studies that report reducing artifacts in general, but are not specific to a single type of artifact? For example, there are automated methods for cleaning EEG data that use ICLabel (a machine learning algorithm) to delete "artifact" components. Within these studies, the cardiac artifact will not be mentioned specifically, but is included under "artifacts".

Statistical inferences, page 23: as far as I can tell, no methods to control for multiple comparisons were implemented. Many of the statistical comparisons were not independent (or even overlapped with similar analyses in the full analysis space to a large extent), so I wouldn't expect strong multiple comparison controls. But addressing this point to some extent would be useful (or clarifying how it has already been addressed if I've missed something).

Methods:

Applying ICA components from 1Hz high pass filtered data back to the 0.1Hz filtered data leads to worse artifact cleaning performance, as the contribution of the artifact in the 0.1Hz to 1Hz frequency band is not addressed (see Bailey, N. W., Hill, A. T., Biabani, M., Murphy, O. W., Rogasch, N. C., McQueen, B., ... & Fitzgerald, P. B. (2023). RELAX part 2: A fully automated EEG data cleaning algorithm that is applicable to Event-Related-Potentials. Clinical Neurophysiology, result reported in the supplementary materials). This might explain some of the lower frequency slope results (which include a lower frequency limit <1Hz) in the EEG data - the EEG cleaning method is just not addressing the cardiac artifact in that frequency range (although it certainly wouldn't explain all of the results).

It looks like no methods were implemented to address muscle artifacts. These can affect the slope of EEG activity at higher frequencies. Perhaps the Riemannian Potato addressed these artifacts, but I suspect it wouldn't eliminate all muscle activity. As such, I would be concerned that remaining muscle artifacts affected some of the results, particularly those that included high frequency ranges in the aperiodic estimate. Perhaps if muscle activity were left in the EEG data, it could have disrupted the ability to detect a relationship between age and 1/f slope in a way that didn't disrupt the same relationship in the cardiac data (although I suspect it wouldn't reverse the overall conclusions given the number of converging results including in lower frequency bands). Is there a quick validity analysis the authors can implement to confirm muscle artifacts haven't negatively affected their results? I note that an analysis of head movement in the MEG is provided on page 32, but it would be more robust to show that removing ICA components reflecting muscle doesn't change the results. The results/conclusions of the following study might be useful for objectively detecting probable muscle artifact components: Fitzgibbon, S. P., DeLosAngeles, D., Lewis, T. W., Powers, D. M. W., Grummett, T. S., Whitham, E. M., ... & Pope, K. J. (2016). Automatic determination of EMG-contaminated components and validation of independent component analysis using EEG during pharmacologic paralysis. Clinical neurophysiology, 127(3), 1781-1793.

Reviewer #3 (Public Review):

Summary:

This work offers a novel perspective on the question of how hippocampal networks can adaptively generate different spatial maps and replays/preplays of the corresponding place cells, without any such maps pre-existing in the network architecture or its inputs. Unlike previous modeling attempts, the authors do not pre-tune their model neurons to any particular place fields. Instead, they build a random, moderately-clustered network of excitatory (and some inhibitory) cells, similar to CA3 architecture. By simulating spatial exploration through border-cell-like synaptic inputs, the model generates place cells for different "environments" without the need to reconfigure its synaptic connectivity or introduce plasticity. By simulating sleep-like random synaptic inputs, the model generates sequential activations of cells, mimicking preplays. These "preplays" require small-world connectivity, so that weakly connected cell clusters are activated in sequence. Using a set of electrophysiological recordings from CA1, the authors confirm that the modeled place cells and replays share many features with real ones. In summary, the model demonstrates that spontaneous activity within a small-world structured network can generate place cells and replays without the need for pre-configured maps.

Strengths:

This work addresses an important question in hippocampal dynamics. Namely, how can hippocampal networks quickly generate new place cells when a novel environment is introduced? And how can these place cells preplay their sequences even before the environment is experienced? Previous models required pre-existing spatial representations to be artificially introduced, limiting their adaptability to new environments. Other models depended on synaptic plasticity rules which made remapping slower than what is seen in recordings. This modeling work proposes that quickly-adaptive intrinsic spiking sequences (preplays) and spatially tuned spiking (place cells) can be generated in a network through randomly clustered recurrent connectivity and border-cell inputs, avoiding the need for pre-set spatial maps or plasticity rules. The proposal that small-world architecture is key for place cells and preplays to adapt to new spatial environments is novel and of potential interest to the computational and experimental community.

The authors do a good job of thoroughly examining some of the features of their model, with a strong focus on excitatory cell connectivity. Perhaps the most valuable conclusion is that replays require the successive activation of different cell clusters. Small-world architecture is the optimal regime for such a controlled succession of activated clusters.

The use of pre-existing electrophysiological data adds particular value to the model. The authors convincingly show that the simulated place cells and preplay events share many important features with those recorded in CA1 (though CA3 ones are similar).

Weaknesses:

To generate place cell-like activity during a simulated traversal of a linear environment, the authors drive the network with a combination of linearly increasing/decreasing synaptic inputs, mimicking border cell-like inputs. These inputs presumably stem from the entorhinal cortex (though this is not discussed). The authors do not explore how the model would behave when these inputs are replaced by or combined with grid cell inputs which would be more physiologically realistic.

Even though the authors claim that no spatially-tuned information is needed for the model to generate place cells, there is a small location-cue bias added to the cells, depending on the cluster(s) they belong to. Even though this input is relatively weak, it could potentially be driving the sequential activation of clusters and therefore the preplays and place cells. In that case, the claim for non-spatially tuned inputs seems weak. This detail is hidden in the Methods section and not discussed further. How does the model behave without this added bias input?

Unlike excitation, inhibition is modeled in a very uniform way (uniform connection probability with all E cells, no I-I connections, no border-cell inputs). This goes against a long literature on the precise coordination of multiple inhibitory subnetworks, with different interneuron subtypes playing different roles (e.g. output-suppressing perisomatic inhibition vs input-gating dendritic inhibition). Even though no model is meant to capture every detail of a real neuronal circuit, expanding on the role of inhibition in this clustered architecture would greatly strengthen this work.

For the modeling insights to be physiologically plausible, it is important to show that CA3 connectivity (which the model mimics) shares the proposed small-world architecture. The authors discuss the existence of this architecture in various brain regions but not in CA3, which is traditionally thought of and modeled as a random or fully connected recurrent excitatory network. A thorough discussion of CA3 connectivity would strengthen this work.

Reviewer #3 (Public Review):

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

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

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

Reviewer #3 (Public Review):

Summary:

The authors nicely show that the translation and ribosome stalling within the ToiL uORF upstream of the co-transcribed topAI-yjhQ toxin-antitoxin genes unmask the topAI translational initiation site, thereby allowing ribosome loading and preventing premature Rho-dependent transcription termination in the topAI region. Although similar translational/transcriptional attenuation has been reported in other systems, the base pairing between the leader sequence and the repressed region by the long RNA looping is somehow unique in toiL-topAI-yjhQP. The experiments are solidly executed, and the manuscript is clear in most parts with areas that could be improved or better explained. The real impact of such a study is not easy to appreciate due to a lack of investigation on the physiological consequences of topAI-yjhQP activation upon antibiotic exposure (see details below).

Strengths:

>Conclusion/model is supported by the integrated approaches consisting of genetics, in vivo SHAPE-seq and Ribo-Seq.

>Provide an elegant example of cis-acting regulatory peptides to a growing list of functional small proteins in bacterial proteomes.

Reviewer #3 (Public Review):

The question about whether L1 exhibits normal/homeostatic expression in the brain (and in general) is interesting and important. L1 is thought to be repressed in most somatic cells (with the exception of some stem/progenitor compartments). However, to our knowledge, this has not been authoritatively / systematically examined and the literature is still developing with respect to this topic. The full gamut of biological and pathobiological roles of L1 remains to be shown and elucidated and this area has garnered rapidly increasing interest, year-by-year. With respect to the brain, L1 (and repeat sequences in general) have been linked with neurodegeneration, and this is thought to be an aging-related consequence or contributor (or both) of inflammation. This study provides an impressive and apparently comprehensive imaging analysis of differential L1 ORF1p expression in mouse brain (with some supporting analysis of the human brain), compatible with a narrative of non-pathological expression of retrotransposition-competent L1 sequences. We believe this will encourage and support further research into the functional roles of L1 in normal brain function and how this may give way to pathological consequences in concert with aging. However, we have concerns with conclusions drawn, in some cases regardless of the lack of statistical support from the data. We note a lack of clarity about how the 3rd party pre-trained machine learning models perform on the authors' imaging data (validation/monitoring tests are not reported), as well as issues (among others) with the particular implementation of co-immunoprecipitation (ORF1p is not among the highly enriched proteins and apparently does not reach statistical significance for the comparison) - neither of which may be sufficiently rigorous.

Reviewer #3 (Public review):

The authors offer an interesting computational study on the dynamics of PROTAC-driven protein degradation. They employed a combination of protein-protein docking, structural alignment, atomistic MD simulations, and post-analysis to model a series of CRBN-dBET-BRD4 ternary complexes, as well as the entire degradation machinery complex. These degraders, with different linker properties, were all capable of forming stable ternary complexes but had been shown experimentally to exhibit different degradation capabilities. While in the initial models of the degradation machinery complex, no surface Lys residue(s) of BRD4 were exposed sufficiently for the crucial ubiquitination step, MD simulations illustrated protein functional dynamics of the entire complex and local side-chain arrangements to bring Lys residue(s) to the catalytic pocket of E2/Ub for reactions. Using these simulations, the authors were able to present a hypothesis as to how linker property affects degradation potency. They were able to roughly correlate the distance of Lys residues to the catalytic pocket of E2/Ub with observed DC50/5h values. This is an interesting and timely study that presents interesting tools that could be used to guide future PROTAC design or optimization.

Reviewer #3 (Public review):

Summary:

The authors aimed to investigate the multifaceted roles of the Inferior Colliculus (IC) in auditory and cognitive processes in monkeys. Through extracellular recordings during a sound duration-based novelty detection task, the authors observed a "climbing effect" in neuronal firing rates, suggesting an enhanced response during sensory prediction. Observations of reward prediction errors within the IC further highlight its complex integration in both auditory and reward processing. Additionally, the study indicated IC neuronal activities could be involved in decision-making processes.

Strengths:

This study has the potential to significantly impact the field by challenging the traditional view of the IC as merely an auditory relay station and proposing a more integrative role in cognitive processing. The results provide valuable insights into the complex roles of the IC, particularly in sensory and cognitive integration, and could inspire further research into the cognitive functions of the IC.

Weaknesses:

Major Comments:

(1) Structural Clarity and Logic Flow:<br /> The manuscript investigates three intriguing functions of IC neurons: sensory prediction, reward prediction, and cognitive decision-making, each of which is a compelling topic. However, the logical flow of the manuscript is not clearly presented and needs to be well recognized. For instance, Figure 3 should be merged into Figure 2 to present population responses to the order of sounds, thereby focusing on sensory prediction. Given the current arrangement of results and figures, the title could be more aptly phrased as "Beyond Auditory Relay: Dissecting the Inferior Colliculus's Role in Sensory Prediction, Reward Prediction, and Cognitive Decision-Making."

(2) Clarification of Data Analysis:<br /> Key information regarding data analysis is dispersed throughout the results section, which can lead to confusion. Providing a more detailed and cohesive explanation of the experimental design would significantly enhance the interpretation of the findings. For instance, including a detailed timeline and reward information for the behavioral paradigms shown in Figures 1C and D would offer crucial context for the study. More importantly, clearly presenting the analysis temporal windows and providing comprehensive statistical analysis details would greatly improve reader comprehension.

(3) Reward Prediction Analysis:<br /> The conclusion regarding the IC's role in reward prediction is underdeveloped. While the manuscript presents evidence that IC neurons can encode reward prediction, this is only demonstrated with two example neurons in Figure 6. A more comprehensive analysis of the relationship between IC neuronal activity and reward prediction is necessary. Providing population-level data would significantly strengthen the findings concerning the IC's complex functionalities. Additionally, the discussion of reward prediction in lines 437-445, which describes IC neuron responses in control experiments, does not sufficiently demonstrate that IC neurons can encode reward expectations. It would be valuable to include the responses of IC neurons during trials with incorrect key presses or no key presses to better illustrate this point.

Reviewer #3 (Public Review):

Summary:

In the context of the SCOURGE consortium's research, the authors conduct a GWAS meta-analysis on 4,702 hospitalized individuals of admixed American descent suffering from COVID-19. This study identified four significant genetic associations, including two loci initially discovered in Latin American cohorts. Furthermore, a trans-ethnic meta-analysis highlighted an additional novel risk locus in the CREBBP gene, underscoring the critical role of genetic diversity in understanding the pathogenesis of COVID-19.

Strengths:

(1) The study identified two novel severe COVID-19 loci (BAZ2B and DDIAS) by the largest GWAS meta-analysis for COVID-19 hospitalization in admixed Americans.<br /> (2) With a trans-ethnic meta-analysis, an additional risk locus near CREBBP was identified.

Weaknesses:

(1) The GWAS power is limited due to the relatively small number of cases.

(2) There is no replication study for the novel severe COVID-19 loci, which may lead to false positive findings.

(3) The variants selected for the PGS appear arbitrary and may not leverage the GWAS findings.

(4) The TWAS models were predominantly trained on European samples, and there is no replication study for the findings as well.

Reviewer #3 (Public review):

In this important work, the authors show compelling evidence that the Rapid Alkalinisation Factor1 (RALF1) peptide acts as an interlink between pectin methyl esterification status and FERONIA receptor-like kinase in mediating extracellular sensing. Moreover, the RALF1-mediated pectin perception is surprisingly independent of LRX-mediated extracellular sensing in roots. The authors also show that the peptide directly binds demethylated pectin and the positively charged amino acids are required for pectin binding as well as for its physiological activity.

Some present findings are surprising; previously, the FERONIA extracellular domain was shown to bind pectin directly, and the mode of operation in the pollen tube involves the LRX8-RALF4 complex, which seems not the case for RALF1 in the present study. Although some aspects remain controversial, this work is a very valuable addition to the ongoing debate about this elusive complex regulation and signaling.

The authors drafted the manuscript well, so I do not have a lot of criticism or suggestions. The experiments are well-designed, executed, and presented, and they solidly support the authors' claims.

Reviewer #3 (Public review):

Summary:

This report analyzes the structure of vimentin, GFAP, and keratin intermediate filament networks in cells that have been subjected to hypotonic stress and other treatments that either alter the ionic strength of the cytoplasm or change the charge density of the intermediate filament.

Strengths:

These experiments expand on the work of references 8 and 9, which showed that the vimentin network rapidly dissociates after hypotonic shock. The cellular imaging uses sophisticated super-resolution techniques and produces some striking images.

Weaknesses:

A fundamental weakness of this study lies in the interpretation, and lack of biochemical evidence for the provocative hypothesis raised that the assembly state of intermediate filaments in a cell is governed by coulomb interactions between charged filaments. Several essential experiments need to be done before this striking hypothesis can be plausibly supported.

(1) First the assembly and disassembly of vimentin filaments needs to be done in vitro systems. If the hypothesis is correct then these same effects will happen with purified vimentin intermediate filaments. These proteins are readily purified from bacterial expression systems and there's a wealth of biophysical data on them, so verifying the predictions of this cell-based model can be realistically tested with purified proteins.

(2) Interpretation of these results explicitly avoids a role for post-translational modifications of vimentin, which are well described and related to filament assembly state. For example, reference 9, which is one of the first discoveries that the vimentin network dissociates under osmotic stress explicitly shows that the vimentin network remains intact if either calcium influx or calpain proteolytic activity is prevented. Hypoosmotic stress will still lead to the same dilution, but the filaments remain intact, apparently contradicting the major interpretation of this paper.

(3) The third issue is that the role of polyelectrolyte effects and especially the importance of divalent cations is scarcely mentioned in the interpretation. There are many studies of how strongly vimentin intermediate filaments interact with divalent cations, in a manner that is relatively insensitive to ionic strength, and these effects need to be taken into account to interpret the cellular data or the hypothesis that coulomb repulsion is the major driver for vimentin disassembly.

Reviewer #3 (Public Review):

Summary:

The manuscript by Hallam et al describes the analysis of various biomarkers in patients undergoing complement factor I supplementation treatment (PPY988 gene therapy) as part of the FOCUS Phase I/II clinical trial. The authors used validated methods (multiplexed assays and OLINK proteomics) for measuring multiple soluble complement proteins in the aqueous humour (AH) and vitreous humour (VH) of 28 patients over a series of time points, up to and including 96 weeks. Based on biomarker comparisons, the levels of FI synthesised by PPY988 were believed to be insufficient to achieve the desired level of complement inhibition. Subsequent comparative experiments showed that PPY988-delivered FI was much less efficacious than Pegceptacoplan (FDA-approved complement inhibitor under the name SYFORVE) when tested in an artificial VH matrix.

Strengths:

The manuscript is well written with data clearly presented and appropriate statistics used for the analysis itself. It's great to see data from real clinical samples that can help support future studies and therapeutic design. The identification that complement biomarker levels present in the AH do not represent the levels found in the VH is an important finding for the field, given the number of complement-targeting therapies in development and the desperate need for good biomarkers for target engagement. This study also provides a wealth of baseline complement protein measurements in both human AH and VH (and companion measurements in plasma) that will prove useful for future studies.

Weaknesses:

Perhaps the conclusions drawn regarding the lack of observed efficacy are not fully justified. The authors focus on the hypothesis that not enough FI was synthesised in these patients receiving the PPY988 gene therapy, suggesting a delivery/transduction/expression issue. But beyond rare CFI genetic variants, most genetic associations with AMD imply that it is a FI-cofactor disease. A hypothesis supported by the authors' own experiments when they supplement their artificial VH matrix with FH and achieve a significantly greater breakdown of C3b than achieved with PPY988 treatment alone. Justification around doubling FI levels driving complement turnover refers to studies conducted in blood, which has an entirely different complement protein profile than VH. In Supplemental Table 5 we see there is approx. 10-fold more FH than FI (533ug/ml vs 50ug/ml respectively) so increasing FI levels will have a direct effect. Yet in Supplemental Table 3 we see there is more FI than FH in VH (608ng/ml vs 466ng/ml respectively). Therefore, adding more FI without more co-factors would have a very limited effect. Surely this demonstrates that the study was delivering the wrong payload, i.e. FI, which hit a natural ceiling of endogenous co-factors within the eye?

Reviewer #3 (Public Review):

Wu et al. present cryo-EM structures of the potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and presumably sodium-bound states at 3.2 Å, 2.5 Å, 2.8 Å, and 2.9 Å. The work builds on a large body of structural work on Kv1.2 and related voltage-gated potassium channels. The manuscript presents a plethora of structural work, and the authors are commended on the breadth of the studies. The structural studies are well-executed. Although the findings are mostly confirmatory, they do add to the body of work on this and related channels. Notably, the authors present structures of DTx-bound Kv1.2 and of Kv1.2 in a low concentration of potassium (which may contain sodium ions bound within the selectivity filter). These two structures add considerable new information. The DTx structure has been markedly improved in the revised version and the authors arrive at well-founded conclusions regarding its mechanism of block. Overall, the manuscript is well-written, a nice addition to the field, and a crowning achievement for the Sigworth lab.

Reviewer #3 (Public review):

Summary:

The article explores the brain's ability to generalize information, with a specific focus on the entorhinal cortex (EC) and its role in learning and representing structural regularities that define relationships between entities in networks. The research provides empirical support for the longstanding theoretical and computational neuroscience hypothesis that the EC is crucial for structure generalization. It demonstrates that EC codes can generalize across non-spatial tasks that share common structural regularities, regardless of the similarity of sensory stimuli and network size.

Strengths:

(1) Empirical Support: The study provides strong empirical evidence for the theoretical and computational neuroscience argument about the EC's role in structure generalization.

(2) Novel Approach: The research uses an innovative methodology and applies the same methods to three independent data sets, enhancing the robustness and reliability of the findings.

(3) Controlled Analysis: The results are robust against well-controlled data and/or permutations.

(4) Generalizability: By integrating data from different sources, the study offers a comprehensive understanding of the EC's role, strengthening the overall evidence supporting structural generalization across different task environments.

Weaknesses:

A potential criticism might arise from the fact that the authors applied innovative methods originally used in animal electrophysiology data (Samborska et al., 2022) to noisy fMRI signals. While this is a valid point, it is noteworthy that the authors provide robust simulations suggesting that the generalization properties in EC representations can be detected even in low-resolution, noisy data under biologically plausible assumptions. I believe this is actually an advantage of the study, as it demonstrates the extent to which we can explore how the brain generalizes structural knowledge across different task environments in humans using fMRI. This is crucial for addressing the brain's ability in non-spatial abstract tasks, which are difficult to test in animal models.

While focusing on the role of the EC, this study does not extensively address whether other brain areas known to contain grid cells, such as the mPFC and PCC, also exhibit generalizable properties. Additionally, it remains unclear whether the EC encodes unique properties that differ from those of other systems. As the authors noted in the discussion, I believe this is an important question for future research.

Reviewer #3 (Public review):

Summary:

Overall, this is a clearly written manuscript with nice hypothesis testing in a non-model organism that addresses the mechanism of Wolbachia-mediated male killing. The authors aim to determine how five previously identified male-killing genes (encoded in the prophage region of the wHm Wolbachia strain) impact the native host, Homona magnanima moths. This work builds on the authors' previous studies in which:<br /> (1) They tested the impact of these same wHm genes via heterologous expression in Drosophila melanogaster.<br /> (2) They examined the activity of other male-killing genes (e.g., from the wFur Wolbachia strain in its native host: Ostrinia furnacalis moths).

Advances here include identifying which wHm gene most strongly recapitulates the male-killing phenotype in the native host (rather than in Drosophila), and the finding that the Hm-Oscar protein has the potential for male-killing in a diverse set of lepidopterans, as inferred by the cell-culture assays.

Strengths:

Strengths of the manuscript include the reverse genetics approaches to dissect the impact of specific male-killing loci, and the use of a "masculinization" assay in Lepidopteran cell lines to determine the impact of interactions between specific masc and oscar homologs.

Weaknesses:

My major comments are related to the lack of statistics for several experiments (and the data normalization process), and opportunities to make the manuscript more broadly accessible.

The manuscript I think would be much improved by providing more details regarding some of the genes and cross-lineage comparisons. I know some of this is reported in previous publications, but some summary and/or additional analysis would make this current manuscript much more approachable for a broader audience, and help guide readers to specific important findings. For example, a graphic and/or more detail on how the wmk/oscar homologs (within and across Wolbachia strains) differ (e.g., domains, percent divergence, etc) would be helpful for contextualizing some of the results. I recognize the authors discuss this in parts (e.g., lines 223-227), but it does require some bouncing between sections to follow. Similarly, the experiments presented in Figure 4 indicate that Hm-oscar has broad spectrum activity: how similar are the masc proteins from these various lepidopterans? Are they highly conserved? Rapidly evolving? Do the patterns of masc protein evolution provide any hints at how Oscar might be interacting with masc?

It is clear from Figure 1 that the combinations of wmk homologs do not cause male killing on their own. Did the authors test if any of the wmk homologs impact the MK phenotype of oscar? It looks like a previous study tested this in wFur (noted in lines 250-252), but given that the authors also highlight the differences between the wFur-oscar and Hm-oscar proteins, this may be worth testing in this system. Related to this, what is the explanation for why there would be 4 copies of wmk in Hm?

Why are some of the broods male-biased (2/3) rather than ~50:50? (Lines 170-175, Figure 2a). For example, there is a strong male bias in un-hatched oscar-injected and naturally infected embryos, whereas the control uninfected embryos have normal 50:50 sex ratios. It is difficult to interpret the rate of male-killing given that the sex ratios of different sets of zygotes are quite variable.

Figure 2b - it appears there are both male and female bands in the HmOsc male lane. I think this makes sense (likely a partial phenotype due to the nature of the overexpression approach), but this is worth highlighting, especially in the context of trying to understand how much of the MK phenotype might be recapitulated through these methods. Related, there is no negative control for this PCR.

It appears the RNA-seq analysis (Figure 3) is based on a single biological replicate for each condition. And, there are no statistical comparisons that support the conclusions of a shift in dosage compensation. Finally, it is unclear what exactly is new data here: the authors note "The expression data of the wHm-t-infected and non-infected groups were also calculated based on the transcriptome data included in Arai et al. (2023a)" - So, are the data in Figure 3c and 3d a re-print of previous data? The level of dosage compensation inferred by visually comparing the control conditions in 3b and 3d does not appear consistent. With only one biological replicate library per condition, what looks like a re-print of previous data, and no statistical comparisons, this is a weakly supported conclusion.

In Figure 4: There are no statistics to support the conclusions presented here. Additionally, the data have gone through a normalization process, but it is difficult to follow exactly how this was done. The control conditions appear to always be normalized to 100 ("The expression levels of BmImpM in the Masc and Hm-Oscar/Oscar co-transfected cells were normalized by setting each Masc-transfected cell as 100"). I see two problems with this approach:<br /> (1) This has eliminated all of the natural variation in BmImpM expression, which is likely not always identical across cells/replicates.<br /> (2) How then was the percentage of BmImpM calculated for each of the experimental conditions? Was each replicate sample arbitrarily paired with a control sample? This can lead to very different outcomes depending on which samples are paired with each other. The most appropriate way to calculate the change between experimental and control would be to take the difference between every single sample (6 total, 3 control, 3 experimental) and the mean of the control group. The mean of the control can then be set at 100 as the authors like, but this also maintains the variability in the dataset and then eliminates the issue of arbitrary pairings. This approach would also then facilitate statistical comparisons which is currently missing.

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

A closer examination of the process that governs the behavioural effect in the present investigation would have been of even greater significance. The authors acknowledge the distinction between object familiarity vs object recognition, but a direct assessment would benefit the field's understanding of the current role of engrams on behaviour.

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

The impact of the paper is somewhat limited by the use of only one sex. Do the authors expect an identical process to be engaged in females in the present set of studies?

Reviewer #3 (Public Review):

Summary:

This study by Glica and colleagues utilized EEG (i.e., Beta power, Gamma power, and aperiodic activity) and 7T MRS (i.e., MRS IE ratio, IE balance) to reevaluate the neural noise hypothesis in Dyslexia. Supported by Bayesian statistics, their results show solid 'no evidence' of EI balance differences between groups, challenging the neural noise hypothesis. The work will be of broad interest to neuroscientists, and educational and clinical psychologists.

Strengths:

Combining EEG and 7T MRS, this study utilized both the indirect (i.e., Beta power, Gamma power, and aperiodic activity) and direct (i.e., MRS IE ratio, IE balance) measures to reevaluate the neural noise hypothesis in Dyslexia.

Weaknesses:

The authors may need to provide more data to assess the quality of the MRS data.

Reviewer #3 (Public Review):

Summary:

The authors find that Sst-EPN neurons, which project to the lateral habenula, encode information about response directionality (left vs right) and outcome (rewarded vs unrewarded). Surprisingly, impairment of vesicular signaling in these neurons onto their LHb targets did not impair probabilistic choice behavior.

Strengths:

Strengths of the current work include extremely detailed and thorough analysis of data at all levels, not only of the physiological data but also an uncommonly thorough analysis of behavioral response patterns.

Weaknesses:

Overall, I saw very few weaknesses, with only two issues, both of which should be possible to address without new experiments:

(1) The authors note that the neural response difference between rewarded and unrewarded trials is not an RPE, as it is not affected by reward probability. However, the authors also show the neural difference is partly driven by the rapid motoric withdrawal from the port. Since there is also a response component that remains different apart from this motoric difference (Figure 2, Supplementary Figure 1E), it seems this is what needs to be analyzed with respect to reward probability, to truly determine whether there is no RPE component. Was this done?

(2) The current study reaches very different conclusions than a 2016 study by Stephenson-Jones and colleagues despite using a similar behavioral task to study the same Sst-EPN-LHb circuit. This is potentially very interesting, and the new findings likely shed important light on how this circuit really works. Hence, I would have liked to hear more of the authors' thoughts about possible explanations of the differences. I acknowledge that a full answer might not be possible, but in-depth elaboration would help the reader put the current findings in the context of the earlier work, and give a better sense of what work still needs to be done in the future to fully understand this circuit.

For example, the authors suggest that the Sst-EPN-LHb circuit might be involved in initial learning, but play less of a role in well-trained animals, thereby explaining the lack of observed behavioral effect. However, it is my understanding that the probabilistic switching task forces animals to continually update learned contingencies, rendering this explanation somewhat less persuasive, at least not without further elaboration (e.g. maybe the authors think it plays a role before the animals learn to switch?).

Also, as I understand it, the 2016 study used manipulations that likely impaired phasic activity patterns, e.g. precisely timed optogenetic activation/inhibition, and/or deletion of GABA/glutamate receptors. In contrast, the current study's manipulations - blockade of vesicle release using tetanus toxin or deletion of VGlut2, would likely have blocked both phasic and tonic activity patterns. Do the authors think this factor, or any others they are aware of, could be relevant?

Reviewer #3 (Public Review):

Summary:

The work submitted by Dr. Jeong-Oh Shin and co-workers aims to investigate the therapeutic efficacy of rhPTH(1-34) and R25CPTH(1-34) on bone regeneration and osseointegration of titanium implants using a postmenopausal osteoporosis animal model.

In my opinion the findings presented are not strongly supported by the provided data since the methods utilized do not allow to significantly support the primary claims.

Strengths:

Strengths include certain good technologies utilized to perform histological sections (i.e. the EXAKT system).

Reviewer #3 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

Reviewer #3 (Public Review):

Summary:

In the manuscript by Greter, et al., entitled "Acute targeted induction of gut-microbial metabolism affects host clock genes and nocturnal feeding" the authors are attempting to demonstrate that an acute exposure to a non-nutritive disaccharide (lactulose) promotes microbial metabolism that feeds back onto the host to impact circadian networks. The premise of the study is interesting and the authors have performed several thoughtful experiments to dissect these relationships, providing valuable insights for the field. However, the work presented does not necessarily support some of the conclusions that are drawn. For instance, lactulose is administered during the fasting period to mimic the impact of a feeding bout on the gut microbiota, but it would be important to perform this treatment during the fed state as well to show that the effects on food intake, etc. do not occur. To truly draw the conclusion that the current outcomes are directly connected to and mediated via an impact on the host circadian clock, it would be ideal to perform these studies in a circadian gene knock-out animal (i.e., Cry1 or Cry2 KO mice, or perhaps Bmal-VilCre tissue-specific KO mice). If the effects are lost in these animals, this would more concretely connect the current findings to the circadian clock gene network. Despite these reservations, the work is promising.

Strengths:

Attempting to disentangle nutrient acquisition from microbial fermentation and its impact on diurnal dynamics of gut microbes on host circadian rhythms is an important step for providing insights into these host-microbe interactions.

The authors utilize a novel approach in leveraging lactulose coupled with germ-free animals and metabolic cages fitted with detectors that can measure microbial byproducts of fermentation, particularly hydrogen, in real-time.

The authors consider several interesting aspects of lactulose delivery, including how it shifts osmotic balance as well as provides calculations that attempt to explain the caloric contribution of fermentation to the animal in the context of reduced food intake. This provides interesting fundamental insights into the role of microbial outputs on host metabolism.

Weaknesses:

While the authors have done a large amount of work to examine the osmotic vs. metabolic influence of lactulose delivery, the authors have not accounted for the enlarged cecum and increased cecal surface area in germ-free mice. The authors could consider an additional control of cecectomy in germ-free mice.

The authors have examined GI hormones as one possible mechanism for how food intake is altered by microbial fermentation of lactulose. However, the authors measure PYY and GLP-1 only at a single time point, stating that there are no differences between groups. Given the goal of the studies is to tie these findings back into circadian rhythms, it would be important to show if the diurnal patterns of these GI hormones are altered.

Considerations of other factors, such as conjugated vs. deconjugated bile acids, microbial bile salt hydrolase activity, and bile acid resorption, might be an important consideration for how lactulose elicits more influence on ileal circadian clock genes relative to cecum and colon.

Measurements of GI transit time (both whole gut and regional) would be an important for consideration for how lactulose might be impacting the ileum vs. cecum vs. colon.

Reviewer #3 (Public Review):

Septins in several systems are thought to guide the location of exocytosis, and they have been found to interact with the exocyst vesicle-tethering complex in some cells. However, it is not known whether such interactions are direct or indirect. Moreover, septin-exocyst physical associations were not detected in several other systems, including yeasts, making it unclear whether such interactions reflect a conserved septin-exocytosis link or whether they may missed if they depend on septin polymerization or association into higher-order structures. Singh et. al., set out to define whether and how septins influence the exocyst during S. pombe cytokinesis. Based on three lines of evidence, the authors conclude that septins directly bind to exocyst subunits to regulate localization of the exocyst and vesicle secretion during cytokinesis.<br /> The conclusions are consistent with the data presented, but some interpretations need to be clarified and extended:

(1) The first line of evidence examines septin and exocyst localization during cytokinesis in wild-type and septin-mutant or exocyst-mutant yeast. Quantitative imaging convincingly shows that the detailed localization of the exocyst at the division site is perturbed in septin mutants, and that this is accompanied by modest accumulation of vesicles and vesicle cargos. Whether that is sufficient to explain the increased thickness of the division septum in septin mutants remains unclear.

(2) The second line of evidence involves a comprehensive Alphafold2 analysis of potential pair-wise interactions between septin and exocyst subunits. This identifies several putative interactions in silico, but it is unclear whether the identified interaction surfaces would be available in the full septin or exocyst complexes.

(3) The third line of evidence uses co-immunoprecipitation and yeast two hybrid assays to show that several physical interactions predicted by Alphafold2 can be detected, leading the authors to conclude that they have identified direct interactions. However, both methods leave open the possibility that the interactions are indirect and mediated by other proteins in the fission yeast extract (co-IP) or budding yeast cell (two-hybrid).

(4) Based on prior studies it would be expected that the large majority of both septins and exocyst subunits are present in cells and extracts as stoichiometric complexes. Thus, one would expect any septin-exocyst interaction to yield associations detectable with multiple subunits, yet co-IPs were not detectded in some combinations. It is therefore unclear whether the interactions reflect associations between fully-formed functional complexes or perhaps between transient folding intermediates.

Reviewer #3 (Public Review):

Summary:

The authors describe how E. coli in the late stationary phase have an active TCA cycle and respiration. Mutation of crp results in the down-regulation of TCA cycle genes and an upregulation of anabolic pathways and reduced persisters. Mutation of a variety of metabolic genes also resulted in fewer persisters in the late-stationary phase.

Strengths:

The work is vast, including metabolomic analysis and characterization of a large number of mutant strains. The identification of active respiration being required for persister cell survival in the late stationary phase is interesting. The induction of anabolic pathways resulting in the sensitization of bacteria to antibiotics is possibly the most interesting part of the paper.

Weaknesses:

The authors try to draw too many conclusions and it's difficult to identify what their actual findings are. For instance, they do not have any interesting findings with aminoglycosides but include the data and spend a lot of time discussing it, but it is really a distraction. The correlation between the induction of anabolic pathways in the crp mutant in the late stationary phase and the reduction in persisters is potentially very interesting but is buried in the paper with the vast quantities of data, and observations and conclusions that are often not well substantiated.

The discussion section is particularly difficult to read and I recommend a large overhaul to increase clarity. For instance, what are the authors trying to conclude in section (iii) of the discussion? That persisters in the stationary phase have higher energy than other cells? Is there data to support that? All sections are similarly lacking in clarity.

The large number of mutants characterized is a strength, but the quality of the data provided for those experiments is poor. Did some of these mutants lose fitness in the deep stationary phase in the absence of antibiotics? Did some reach a far lower cfu/ml in the stationary phase? These details are important and without them, it is difficult to interpret the data.

There is ample analysis of persister formation in mutants in the pts/CRP pathway that is not discussed (Zeng et al PNAS 2022, Parsons et al PNAS, 2024).

The authors do not discuss ROS production and antibiotic killing in these experiments. Presumably, the WT would have a greater propensity to produce ROS in response to antibiotics than the crp mutant, but it survives better. Is ROS not involved in antibiotic killing in these conditions?

Reviewer #3 (Public Review):

In this work, Bryant, et al. investigate genetic interactions between non-essential members of the outer membrane protein biogenesis pathway and other genes in the genome using a transposon-directed insertion sequencing (TraDIS) approach in E. coli K-12. The authors identify interactions with other components of the envelope including LPS, peptidoglycan, and enterobacterial common antigen biogenesis, and they tie these interactions to specific members of the outer membrane biogenesis pathway. Although many of these interactions are known and have been previously investigated in the field, the study provides several synthetic phenotypes that could be useful for further investigations.

The strengths of the paper include their unbiased, TraDIS approach, and follow up on the interactions they observe. The interactions with genes of unknown function also are of interest as they may suggest experiments to find the functions of these genes. The largest weakness of this paper is the use of a gene deletion allele for bamB that is known to be polar leading to decreased expression of an essential gene. This largely invalidates all results related to DNA replication. In addition, it is a weakness that the paper does not adequately address its place in the field through discussion of existing results on the interactions they investigate.

Reviewer #3 (Public Review):

Summary:

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

Previous comments:

Strengths:

When considered separately the new mutants are an improvement over the MOs and the paper contains a lot of new data.

Weaknesses:

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

Comment on the revised version:

The authors addressed some of our comments, but not our main criticisms, which we reiterate here:

(1) The authors argue that morpholino studies are unreliable and here they made new mutants to solve this uncertainty for dap 1/2. However, creating stable mutant lines to largely confirm previous results obtained by using morpholino knock-down phenotypes does not justify publication in eLife.

(2) The authors argue that since it has not been shown conclusively that craniofacial defects in wnt11 and dap1/2 mutants are secondary to gastrulation defects there is no solid evidence preventing them from investigating these craniofacial defects. However, since it is extremely likely that the rod-like ethmoid plates of wnt11f2- and dact1/2 mutants focused on here are secondary to gastrulation defects previously described by others (Heisenberg and NussleinVolhard 1997; Waxman et al., 2004), the burden of proof is on the authors to provide much stronger evidence against this interpretation.

(3) The data for calpain overexpression remains too preliminary.

Reviewer #3 (Public Review):

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

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

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

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

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

Reviewer #3 (Public Review):

Summary:<br /> The authors introduce a tool that employs thermal cameras to automatically detect urine and feces deposits in rodents. The detection process involves a heuristic to identify potential thermal regions of interest, followed by a transformer network-based classifier to differentiate between urine, feces, and background noise. The tool's effectiveness is demonstrated through experiments analyzing social preference, stress response, and temporal dynamics of deposits, revealing differences between male and female mice.

Strengths:<br /> The method effectively automates the identification of deposits<br /> The application of the tool in various behavioral tests demonstrates its robustness and versatility.<br /> The results highlight notable differences in behavior between male and female mice

Weaknesses:<br /> The definition of 'start' and 'end' periods for statistical analysis is arbitrary. A robustness check with varying time windows would strengthen the conclusions.<br /> The paper could better address the generalizability of the tool to different experimental setups, environments, and potentially other species.<br /> The results are based on tests of individual animals, and there is no discussion of how this method could be generalized to experiments tracking multiple animals simultaneously in the same arena (e.g., pair or collective behavior tests, where multiple animals may deposit urine or feces).