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    1. Reviewer #1 (Public review):

      Summary:

      Here, the authors attempt to show that CCL5 is increased after stroke, possibly due to decreased miR-324, and that this is a modifiable system to decrease stroke damage. By bidirectionally manipulating CCL5 levels through direct injection of CCL5; a CCL5 blocking antibody; miR324; miR324 antagomir; or CCR5-blocking Maraviroc, they broadly show improvement with lower CCL5 levels. This includes infarct size, behavioral analysis, and immunohistochemical analysis of astrocytes, microglia, and neurons. They further try to mechanistically tie miR324 and CCL5 in astrocytes specifically to stroke-induced changes using a neuronal/astrocytic coculture system. They argue that decreasing CCL5 leads to increased ERK and CREB phosphorylation as a potential neuroprotective mechanism. CCL5 is one potential ligand for CCR5, and recent work identified CCR5 as a targetable mechanism by clinically-approved drug Maraviroc to enhance stroke recovery. Particularly given the high level of interest in CCR5 in stroke recovery, the focus on CCL5 - one of CCR5's potential ligands - and its miR regulation is an exciting expansion of this area of stroke biology.

      Strengths:

      The authors' findings that decreasing CCL5 acutely after stroke shows behavioral improvement appear robust. This broadly replicates work from other groups, although the finding that miR324 manipulation can phenocopy direct CCL5 manipulation is novel and intriguing. However, many of their other claims are difficult to evaluate based on a combination of missing methodological information, inappropriate statistical testing, and a flawed culture system.

      Weaknesses:

      Broadly speaking, the manuscript takes a zoomed-out view of what is fundamentally highly localized biology.

      (1) miRNA-based regulation, by definition, has to include miR and mRNA in the same cell type; as the authors note, CCL5 is expressed in many cells. It is therefore impossible to propose any interaction on the basis of the tissue-level changes described; any evidence of in vivo cell-type specificity would dramatically improve the claims.

      (2) The authors treat an extensive area of ipsilesional cortex uniformly as "IP". Astrocytic and microglial responses to localized injuries such as stroke are highly location-dependent and undoubtedly change dramatically within this area. The presented data cannot be interpreted without confirmation that these were taken at identical distances from the injury, and what that distance was. These do not appear to be adjacent to the injury, where the responses would presumably be the most informative. Similarly, it is difficult to interpret the neuronal Sholl and spine data without more information on where within the large IP region these neurons were found.

      The authors attempt to narrow in on cell-type specificity via culture. However, astrocytes are notoriously prone to a dramatic change in culture and require careful methods (immunopanning; see eg doi: 10.1016/j.neuron.2011.07.022) to maintain much resemblance to their in vivo counterpart. It is difficult to conclude much about the role of astrocytes in the CCL5 pathway based on the use of this shaking-based culture system, particularly in the absence of cell-type specific validation in vivo.

      There is missing methodological information, including infarct size measurements, TUNEL staining, and statistical testing. The TTC figures look very odd, like a collection of overlapping stars have been placed on the images rather than the natural relatively smooth infarct edges one would expect. It is unclear if the infarct volume measurements accounted for edema, as is standard; there is no description of the protocol used for quantification. It is also unclear if the infarct volume measurement comparisons were also done with t-tests vs ANOVA, as the statistical test used is not listed in the figure legends. In numerous cases where statistical testing is listed, repeated t-tests between subgroups are used vs the more appropriate ANOVA (assuming normality; nonparametric testing as appropriate), making it difficult to have confidence in the results.

    1. Reviewer #1 (Public review):

      Summary:

      The authors of this study use an optimization algorithm approach, based on the established Nelder-Mead method, to infer polymer models that best match input bulk Hi-C contact data. The procedure infers the best parameters of a generic polymer model that combines loop-extrusion (LE) dynamics and compartmentalisation of chromatin types driven by weak biochemical affinities. Using this and DNA FISH, the authors investigate the chromatin structure of the MYC locus in leukaemia cells, showing that loop extrusion alone cannot explain local pathogenic chromatin rearrangements. Finally, they study the locus single-cell heterogeneity and time dynamics.

      In the revised manuscript the authors have adequately addressed my questions and comments. The exception concerns point #5 of my original review:

      (5) Besides cumulative probability distributions, I asked the authors to show the TAD2-TAD4 (model vs. exp) distances in Fig. 3c as relative frequency histograms. This allows readers to more accurately evaluate whether model and experimental distributions have same shape and variance.

    1. Reviewer #1 (Public Review):

      The individual roles of both cosolvents and intrinsically disordered proteins (IDPs) in desiccation have been well established, but few studies have tried to elucidate how these two factors may contribute synergistically. The authors quantify the synergy for the model and true IDPs involved with desiccation and find that only the true IDPs have strong desiccation tolerance and synergy with cosolvents. Using these as model systems, they quantify the local (secondary structure vis-a-vi CD spectroscopy) and global dimensions (vis-a-vi the Rg of SAXS experiments) and find no obvious changes with the co-solvents. Instead, they focus on the gelation of one of the IDPs and, using theory and experiments, suggest that the co-solvents may enable desiccation tolerance, an interesting hypothesis to guide future in vivo desiccation studies. A few minor points that remained unclear to this reviewer and that were noted previously have been reasonably addressed in this revision.

      Strengths:

      This paper is quite extensive and has significant strengths worth highlighting. Notably, the number and type of methods employed to study IDPs are quite unusual, employing CD spectroscopy, SAXS measurements, and DSC. The use of the TFE is an exciting integration of the physical chemistry of cosolvents into the desiccation field is a nice approach and a clever way of addressing the gap of the lack of conformational changes depending on the cosolvents. Furthermore, I think this is a major point and strength of the paper; the underlying synergy of cosolvents and IDPs may lie in the thermodynamics of the dehydration process.

      Figure S6A is very useful. I encourage readers who are confused about the DSC analysis, interpretation, and calculation to refer to it.

      Weaknesses:

      All minor weaknesses were addressed in this revision.

    1. Reviewer #1 (Public review):

      Summary:

      The manuscript by Sztangierska et al explores how the Hsp70 chaperone together with its JDP-NEF cofactors and Hsp104 disentangle aggregated proteins. Specifically, the study provides mechanistic findings that explain what role the NEF class Hsp110 has in protein disaggregation. The results explain several previous observations related to Hsp110 in protein disaggregation. Importantly, the study provides compelling evidence that Hsp110 acts early in the disaggregation process.

      Strengths:

      (1) This is a very well performed study with multiple in vitro experiments that provide convincing support for the claims.

      (2) An important finding is that the study places Hsp110 function early in the disaggregation process.

      (3) The study has an important value in that it picks up on a number of observations in the field that have not been explored or directly tested by experiment. The presented results settle questions and controversy regarding Hsp110 function in disaggregation.

      Weaknesses:

      (1) While the key finding of this manuscript is that it places Hsp110 early in the disaggregation process, the other findings are advancing the field less.

    1. Reviewer #1 (Public review):

      Summary:

      Matsui et al. present an experimental pipeline for visualizing molecular machinery of synapses in the brain, which includes numerous techniques, starting with generating labeled antibodies and recombinant mice, continuing with HPF and FIB milling and finishing with tilt series collection and 3D image processing. This pipeline represents a breakthrough in preparation of brain tissue for high resolution imaging and can be used in future tomographic research to reconstruct molecular details of synaptic complexes as well as pre- and post-synaptic assemblies. This methodology can also be adapted for a broader range of tissue preparations and signifies the next step towards better structural understanding of how molecular machineries operate in natural conditions.

      Strengths:

      The manuscript is very well written, contains a detailed description of methodology, provides nice illustrations and will be an outstanding guide for future research.

      Weaknesses:

      None noted.

    1. Reviewer #1 (Public review):

      Summary:

      This paper by Yang et al. established an in vitro triple co-culture BBB model and demonstrated its advantages compared with the mono or double co-culture BBB model. Further, the authors used their established in vitro BBB model and combined it with other methodologies to investigate the specific signaling mechanisms that co-culture with astrocytes but also neurons enhancing the integrity of endothelial cells.

      Strengths:

      The results persuasively demonstrated that the established triple co-culture BBB model well mimicked several important characteristics of BBB compared with the mono-culture BBB model, including better barrier function and in vivo/in vitro correlation. The use of human-derived immortalized cells made the model construction process faster and more efficient and had a better in vivo correlation without the complications of species differences. This model is expected to be a useful high-throughput evaluation tool for CNS drug development.

      Moreover, the authors used a variety of experiments to prove that the triple co-culture model also reflected the interactions between NVU cells, including promoting endothelial cell proliferation and the formation of intercellular junctions. Interestingly, the authors found that neurons also released GDNF to promote barrier properties of brain endothelial cells, as most current research has focused on the promoting effect of astrocytes-derived GDNF on BBB. Meanwhile, the author also validated the functions of GDNF for BBB integrity in vivo by silencing GDNF in mouse brains. Overall, the experiments and data presented support the claim that neurons, alongside astrocytes, contribute to the promoting effects of the barrier function of endothelial cells through GDNF secretion.

      Weaknesses:

      While the authors explained that the use of human-derived immortalized cells has been justified as more reproducible and efficient in constructing the model, the TEER value of the triple co-culture model remains lower than that of the physiological statement. Future research may need to explore additional methods to further enhance the barrier function of the model.

    1. Reviewer #1 (Public review):

      Herzog and colleagues investigated the interactions between working memory (WM) task condition (updating, maintenance) and BMI (body-mass-index), while considering selected dopaminergic genes (COMT, Taq1A, C957T, DARPP-32). Emerging evidence suggest that there might be a specific negative association with BMI in the updating but not maintenance condition, with potential bearings to reversal reward learning in obesity. The inclusion of multiple dopaminergic genes is a strength in the present study, considering the complexity of the interactions between tonic and phasic dopamine across the brain that may distinctly associate with the component processes of WM. Here, the finding was that BMI was negatively associated with WM performance regardless of the condition (updating, maintenance), but in models including moderation by either Taq1A or DARPP-32 (but not by COMT and C957T) an interaction by task condition was observed. Furthermore, a two-way interaction effect between BMI and genotype was observed exclusively in the updating condition. These findings are in line with the accounts by which striatal dopamine as reflected by Taq1A and DARPP-32 play an important role in working memory updating, while cortical dopamine as reflected by COMT is mainly associated with maintenance. The authors conclude that the genetic moderation reflects a compound effect of having high BMI and an advantageous allele in Taq1A or DARPP-32 to working memory updating specifically.

      These data increment the accumulating evidence that the dopamine system plays an important role in obesity. The result that Taq1A and DARPP-32 moderated the interaction between WM condition and BMI required intricate post hoc analysis to understand the bearings to updating. The authors found that Taq1A or DARPP-32 genotype moderated the negative association between BMI and WM exclusively in update condition (significant two-way interaction effect), suggesting that the BMI-WM associations in other conditions were similar across genotypes. Importantly, visual inspection of the relationship between WM and BMI (Fig 4 & 5) suggests more prevalent positive effects of the putatively advantageous Taq1A-A1 and DARPP-32-AA genotypes to the overall negative relationship between WM and BMI in updating, but not in the other conditions. Given that an overall negative relationship was statistically supported across all conditions (model 1), a plausible interpretation would be that updating condition stands out in terms of a positive moderation by putative advantageous genotypes, rather than compound negative consequences of BMI and genotype in updating. Statistical testing stratified by Taq1A genotype confirmed that the interaction with task condition was driven by the carriers of the advantageous genotype, whereas stratification by DARPP-32 genotype revealed a significant task-condition interaction in both A/A- and G-carriers. Taken together, the present results highlight inter-subject variability in the associations between obesity, dopamine, and working memory, which can sometimes be captured using blood-based dopamine markers. This finding indicates that not all individuals with obesity show the same patterns of dopamine-related alterations and underscores the necessity to address inter-individual variability in future research and treatment efforts.

    1. Reviewer #1 (Public Review):

      In the current study, Papandreou et al. developed an iPSC-based midbrain dopaminergic neuronal cell model of Beta-Propeller Protein-Associated Neurodegeneration (BPAN), which is caused by mutations in the WDR45 gene and is known to impair autophagy. They also noted defective autophagy and abnormal BPAN-related gene expression signatures. Further, they performed a drug screening and identified five cardiac glycosides. Treatment with these drugs effectively in improved autophagy defects and restored gene expression. Seeing the autophagy defects and impaired expression of BPAN-related genes adds strength to this study. Importantly, this work shows the value of iPSC-based modeling in studying disease and finding therapeutic strategies for genetic disorders, including BPAN.

    1. Reviewer #2 (Public review):

      Prior work by the Sehgal group has shown that a small group of neurons in the fly brain (anterior posterior (ap) α'β' mushroom body neurons (MBNs)) promote sleep and sleep-dependent appetitive memory specifically under fed conditions (Chouhan et al., (2021) Nature). Here, Li, Chouhan et al. combine cell-specific transcriptomics with measurements of sleep and memory to identify molecular processes underlying this phenomenon. They define transcriptional changes in ap α'β' MBNs and suggest a role for two genes downregulated following memory induction (Polr1F and Regnase-1) in regulating sleep and memory.

      The transcriptional analyses in this manuscript are impressive. The authors have now included additional experiments that define acute and developmental roles for Polr1F and Regnase-1 respectively in regulating sleep. They have also provided additional data to strengthen their conclusion that Polr1F knockdown in α'β' mushroom body neurons enhances sleep.

      The resubmitted work represents a convincing investigation of two novel sleep-regulatory proteins that may also play important roles in memory formation.

      The authors have comprehensively addressed my comments, which I very much appreciate. I congratulate them on this excellent work.

    1. Reviewer #1 (Public review):

      This is a very nice study of Belidae weevils using anchored phylogenomics that presents a new backbone for the family and explores, despite a limited taxon sampling, several evolutionary aspects of the group. I find that the methodology is appropriate, and all analytical steps are well presented. The paper is well written and presents interesting aspects of Belidae systematics and evolution. The major weakness of the study being the very limited taxon sampling that has deep implications for the discussion of ancestral estimations.

    1. Reviewer #1 (Public Review):

      Summary:

      The study investigated how root cap cell corpse removal affects the ability of microbes to colonize Arabidopsis thaliana plants. The findings demonstrate how programmed cell death and its control in root cap cells affect the establishment of symbiotic relationships between plants and fungi. Key details on molecular mechanisms and transcription factors involved are also given. The study suggests reevaluating microbiome assembly from the root tip, thus challenging traditional ideas about this process. While the work presents a key foundation, more research along the root axis is recommended to gain a better understanding of the spatial and temporal aspects of microbiome recruitment.

      Comments on revised version:

      The authors have positively addressed all the critical points I raised in the previous review.

    1. Reviewer #1 (Public review):

      In this manuscript, the role of orexin receptors in dopamine transmission is studied. It extends previous findings suggesting an interplay of these two systems in regulating behaviour by first characterising the expression of orexin receptors in the midbrain and then disrupting orexin transmission in dopaminergic neurons by deleting its predominant receptor, OX1R (Ox1R fl/fl, Dat-Cre tg/wt mice). Electrophysiological and calcium imaging data suggest that orexin A acutely and directly stimulates SN and VTA dopaminergic neurons, but does not seem to induce c-Fos expression. Behavioural effects of depleting OX1R from dopaminergic neurons includes enhanced novelty-induced locomotion and exploration, relative to littermate controls (Ox1R fl/fl, Dat-Cre wt/wt). However, no difference between groups is observed in tests that measure reward processing, anxiety, and energy homeostasis. To test whether depletion of OX1R alters overall orexin-triggered activation across the brain, PET imaging is used in OX1R∆DAT knockout and control mice. This analysis reveals that several regions show a higher neuronal activation after orexin injection in OX1R∆DAT mice, but the authors focus their follow up study on the dorsal bed nucleus of the stria terminalis (BNST) and lateral paragigantocellular nucleus (LPGi). Dopaminergic inputs and expression of dopamine receptors type-1 and -2 (DRD1 & DRD2) is assessed and compared to control demonstrating moderate decrease of DRD1 and DRD2 expression in BNST of OX1R∆DAT mice and unaltered expression of DRD2, with absence of DRD1 expression in LPGi of both groups. Overall, this study is valuable for the information it provides on orexin receptor expression and function on behaviour and for the new tools it generated for the specific study of this receptor in dopaminergic circuits.

      Strengths:

      The use of a transgenic line that lacks OX1R in dopamine-transporter expressing neurons is a strong approach to dissect the direct role of orexin in modulating dopamine signalling in the brain. The battery of behavioural assays to study this line provides a valuable source of information for researchers interested in the role of orexin in animal physiology.

      Weaknesses:

      This study falls short in providing evidence for an anatomical substrate of the altered behaviour observed in mice lacking orexin receptor subtype 1 in dopaminergic neurons. How orexin transmission in dopaminergic neurons regulates the expression of postsynaptic dopamine receptors (as observed in BNST of OX1R∆DAT mice) is an intriguing question poorly discussed. Whether disruption of orexin activity alters dopamine release in target areas is an important point not addressed.

    1. Reviewer #1 (Public review):

      Summary:

      The authors investigated the anatomical features of the synaptic boutons in layer 1 of the human temporal neocortex. They examined the size of each synapse, the macular or perforated appearance, the size of the synaptic active zone, the number and volume of the mitochondria, and the number of synaptic and dense core vesicles, also differentiating between the readily releasable, the recycling, and the resting pool of synaptic vesicles. The coverage of the synapse by astrocytic processes was also assessed, and all the above parameters were compared to other layers of the human temporal neocortex. The authors conclude that the subcellular morphology of the layer 1 synapses are suitable for the functions of the neocortical layer, i.e. the synaptic integration within the cortical column. The low glial coverage of the synapses might allow increased glutamate spillover from the synapses, enhancing synpatic crosstalk within this cortical layer.

      Strengths:

      The strengths of this paper are the abundant and very precious data about the fine structure of the human neocortical layer 1. Quantitative electron microscopy data (especially that derived from the human brain) are very valuable since this is a highly time- and energy-consuming work. The techniques used to obtain the data, as well as the analyses and the statistics performed by the authors are all solid, strengthen this manuscript, and mainly support the conclusions drawn in the discussion.

      Weaknesses:

      There are several weaknesses in this work. First, the authors should check and review extensively for improvements to the use of English. Second, several additional analyses performed on the existing data could substantially elevate the value of the data presented. Much more information could be gained from the existing data about the functions of the investigated layer, of the cortical column, and about the information processing of the human neocortex. Third, several methodological concerns weaken the conclusions drawn from the results.

    1. Reviewer #1 (Public review):

      Assessment:

      This important work advances our understanding of navigation and path integration in mammals by using a clever behavioral paradigm. The paper provides compelling evidence that mice are able to create and use a cognitive map to find "short cuts" in an environment, using only the location of rewards relative to the point of entry to the environment and path integration, and need not rely on visual landmarks.

      Summary:

      The authors have designed a novel experimental apparatus called the 'Hidden Food Maze (HFM)' and a beautiful suite of behavioral experiments using this apparatus to investigate the interplay between allothetic and idiothetic cues in navigation. The results presented provide a clear demonstration of the central claim of the paper, namely that mice only need a fixed start location and path integration to develop a cognitive map. The experiments and analyses conducted to test the main claim of the paper -- that the animals have formed a cognitive map -- are conclusive. While I think the results are quite interesting and sound, one issue that needs to be addressed is the framing how landmarks are used (or not), as discussed below, although I believe this will be a straight forward issue for the authors to address.

      Strengths:

      The 90 degree rotationally symmetric design and use of 4 distal landmarks and 4 quadrants with their corresponding rotationally equivalent locations (REL) lends itself to teasing apart the influence of path integration and landmark-based navigation in a clever way. The authors use a really complete set of experiments and associated controls to show that mice can use a start location and path integration to develop a cognitive map and generate shortcut routes to new locations.

      Weaknesses:

      There were no major weaknesses identified that were not addressed during revisions.

    1. Reviewer #1 (Public review):

      Summary:

      This study focuses on characterizing a previously identified gene, encoding the secreted protein Ppe1, that may play a role in rice infection by the blast fungus Magnaporthe oryzae. Magnaporthe oryzae is a hemibiotrophic fungus that infects living host cells before causing disease. Infection begins with the development of a specialized infection cell, the appressorium, on the host leaf surface. The appressorium generates enormous internal turgor that acts on a thin penetration peg at the appressorial base, forcing it through the leaf cuticle. Once through this barrier, the peg elaborates into bulbous invasive hyphae that colonizes the first infected cell before moving to neighboring cells via plasmodesmata. During this initial biotrophic growth stage, invasive hyphae invaginate the host plasma membrane, which surrounds growing hyphae as the extra-invasive hyphae membrane (EIHM). To avoid detection, the fungus secretes apoplastic effectors into the EIHM matrix via the conventional ER-Golgi secretion pathway. The fungus also forms a plant-derived structure called the biotrophic interfacial complex (BIC) that receives cytoplasmic effectors through an unconventional secretion route before they are delivered into the host cell. Together, these secreted effector proteins act to evade or suppress host innate immune responses. Here the authors contribute to our understanding of M. oryzae infection biology by showing how Ppe1, which localizes to both the appressorial penetration peg and to the appressorial-like transpressoria associated with invasive hyphal movements into adjacent cells, maximizes host cell penetration and disease development and is thus a novel contributor to rice blast disease.

      Strengths:

      A major goal of M. oryzae research is to understand how the fungus causes disease, either by determining the physiological underpinnings of the fungal infection cycle or by identifying effectors and their host targets. Such new knowledge may point the way to novel mitigation strategies. Here, the authors make an interesting discovery that bridges both fungal physiology and effector biology research by showing how a secreted protein Ppe1, initially considered an effector with potential host targets, associates with its own penetration peg (and transpressoria) to facilitate host invasion. In a previous study, the authors had identified a small family of small secreted proteins that may function as effectors. Here they suggest Ppe1 (and, later in the manuscript, Ppe2/3/5) localizes outside the penetration peg when appressoria develops on surfaces that permit penetration, but not on artificial hard surfaces that prevent peg penetration. Deleting the PPE1 gene reduced (although did not abolish) penetration, and a fraction of those that penetrated developed invasive hyphae that were reduced in growth compared to WT. Using fluorescent markers, the authors show that Ppe1 forms a ring underneath appressoria, likely where the peg emerges, which remained after invasive hyphae had developed. The ring structure is smaller than the width of the appressorium and also lies within the septin ring known to form during peg development. This so-called penetration ring also formed at the transpressorial penetration point as invasive hyphae moved to adjacent cells. This structure is novel, and required for optimum penetration during infection. Furthermore, Ppe1, which carries a functional signal peptide, may form on the periphery of the peg, together suggesting it is secreted and associated with the peg to facilitate penetration. Staining with aniline blue also suggests Ppe1 is outside the peg. Together, the strength of the work lies in identifying a novel appressorial penetration ring structure required for full virulence.

      Weaknesses:

      The main weakness of the paper is that, although Ppe1 is associated with the peg and optimizes penetration, the function of Ppe1 is not known. The work starts off considering Ppe1 a secreted effector, then a facilitator of penetration by associating with the peg, but what role it plays here is only often speculated about. For example, the authors consider at various times that it may have a structural role, a signaling role orchestrating invasive hyphae development, or a tethering role between the peg and the invaginated host plasma membrane (called throughout the host cytoplasmic membrane, a novel term that is not explained). However, more effort should be expended to determine which of these alternative roles is the most likely. Otherwise, as it stands, the paper describes an interesting phenomenon (the appressorial ring) but provides no understanding of its function.

      The inability to nail down the function of Ppe1 likely stems from two underlying assumptions with weak support. Firstly, the authors assume that Ppe1 is secreted and associated with the peg to form a penetration ring between the plant cell wall and cytoplasm membrane. However, the authors do not demonstrate it is secreted (for instance by blocking Ppe1 secretion and its association with the peg using brefeldin A). Also, they do not sufficiently show that Ppe1 localizes on the periphery of the peg. This is because confocal microscopy is not powerful enough to see the peg. The association they are seeing (for example in Figure 4) shows localization to the bottom of the appressorium and around the primary hyphae, but the peg cannot be seen. Here, the authors will need to use SEM, perhaps in conjunction with gold labeling of Ppe1, to show it is associating with the peg and, indeed, is external to the peg (rather than internal, as a structural role in peg rigidity might predict). It would also be interesting to repeat the microscopy in Figure 4C but at much earlier time points, just as the peg is penetrating but before invasive hyphae have developed - Where is Ppe1 then? Finally, the authors speculate, but do not show, that Ppe1 anchors penetration pegs on the plant cytoplasm membrane. Doing so may require FM4-64 staining, as used in Figure 2 of Kankanala et al, 2007 (DOI: 10.1105/tpc.106.046300), to show connections between Ppe1 and host membranes. Note that the authors also do not show that the penetration ring is a platform for effector delivery, as speculated in the Discussion.

      Secondly, the authors assume Ppe1 is required for host infection due to its association with the peg. However, its role in infection is minor. The majority of appressoria produced by the mutant strain penetrate host cells and elaborate invasive hyphae, and lesion sizes are only marginally reduced compared to WT (in fact, the lesion density of the 70-15 WT strain itself seems reduced compared to what would be expected from this strain). The authors did not analyze the lesions for spores to confirm that the mutant strains were non-pathogenic (non-pathogenic mutants sometimes form small pinprick-like lesions that do not sporulate). Thus, the pathogenicity phenotype of the knockout mutant is weak, which could contribute to the inability to accurately define the molecular and cellular function of Ppe1.

      In summary, it is important that the role of Ppe1 in infection be determined.

    1. Reviewer #1 (Public Review):

      The paper itself has a reasonable aim, to compare the inputs to the hippocampus from cortical regions across mammals. But for some reason, the conclusions that are reached are very limited. We know for example that the main laboratory rodents investigated, rats and mice, are nocturnal, live in underground tunnels, and have a very wide field of view with no fovea. In contrast, primates have a highly developed cortical system for vision and a fovea, and so have very different capabilities to rodents, as they have an ability to identify people or objects at a distance, and to remember where they have been seen. Despite this major difference in the visual cortical processing in these different mammals, somehow important points are missed in this paper about how the cortical processing is organised in these different mammals, and how this is reflected in the anatomy.

    1. Reviewer #1 (Public review):

      Summary:

      The "number sense" refers to an imprecise and noisy representation of number. Many researchers propose that the number sense confers a fixed (exogenous) subjective representation of number that adheres to scalar variability, whereby the variance of the representation of number is linear in the number.

      This manuscript investigates whether the representation of number is fixed, as usually assumed in the literature, or whether it is endogenous. The two dimensions on which the authors investigate this endogeneity are the subject's prior beliefs about stimuli values and the task objective. Using two experimental tasks, the authors collect data that are shown to violate scalar variability and are instead consistent with a model of optimal encoding and decoding, where the encoding phase depends endogenously on prior and task objectives. I believe the paper asks a critically important question. The literature in cognitive science, psychology, and increasingly in economics, has provided growing empirical evidence of decision-making consistent with efficient coding. However, the precise model mechanics can differ substantially across studies. This point was made forcefully in a paper by Ma and Woodford (2020, Behavioral & Brain Sciences), who argue that different researchers make different assumptions about the objective function and resource constraints across efficient coding models, leading to a proliferation of different models with ad-hoc assumptions. Thus, the possibility that optimal coding depends endogenously on the prior and the objective of the task, opens the door to a more parsimonious framework in which assumptions of the model can be constrained by environmental features. Along these lines, one of the authors' conclusions is that the degree of variability in subjective responses increases sublinearly in the width of the prior. And importantly, the degree of this sublinearity differs across the two tasks, in a manner that is consistent with a unified efficient coding model.

      Comments:

      (1) Modeling and implementation of estimation task

      The biggest concern I have with the paper is about the experimental implementation and theoretical account of the estimation task. The salient features of the experimental data (Figure 1C) are that the standard deviations of subjects' estimated quantities are hump-shaped in the true stimulus x and that the standard deviation, conditional on the true stimulus x, is increasing in prior width. The authors attribute these features to a Bayesian encoding and decoding model in which the internal representation of the quantity is noisy, and the degree of noise depends on the prior - as in models of efficient coding (Wei and Stocker 2015 Nature Neuro; Bhui and Gershman 2018 Psych Review; Hahn and Wei 2024 Nature Neuro).

      The concern I have is about the final "step" in the model, where the authors assume there is an additional layer of motor noise in selecting the response. The authors posit that the subject's selection of the response is drawn from a Gaussian with a mean set to the optimally decoded estimate x*(r), and variance set to a free parameter sigma_0^2. However, the authors also assume that the Gaussian distribution is "truncated to the prior range." This truncation is a nontrivial assumption, and I believe that on its own, it can explain many features of the data.

      To see this, assume that there is no noise in the internal representation of x, there is only motor noise. This corresponds to a special case of the authors' model in which υ is set to 0. The model then reduces to a simple account in which responses are drawn from a Gaussian distribution centered at the true value of x, but with asymmetric noise due to the truncation. I simulated such a model with sigma_0=7. The resulting standard deviations of responses for each value of x (based on 1000 draws for each value of x), across the three different priors, reproduce the salient patterns of the standard deviation in Figure 1C: i) within each condition, the standard deviation is hump-shaped and peaks at x=60 and ii) conditional on x, standard deviation increases in prior width. The takeaway is that this simple model with only truncated motor noise - and without any noisy or efficient coding of internal representations - provides an alternative channel through which the prior affects behavior.

      Of course, this does not imply that subjects' coding is not described by the efficient encoding and decoding model posited by the authors. However, it does suggest an important alternative mechanism for the authors' theoretical results in the estimation task. Moreover, some of the quantitative conclusions about the differences in behavior with the discrimination task would be greatly affected by the assumption of truncated motor noise.

      Turning to the experiment, a basic question is whether such a truncation was actually implemented in the design. That is, was the range of the slider bar set to the range of the prior? (The methods section states that the size on the screen of the slider was proportional to the prior width, but it was unclear whether the bounds of the slider bar changed with the prior). If the slider bar range did depend on the prior, then it becomes difficult to interpret the data. If not, then perhaps one can perform analyses to understand how much the motor noise is responsible for the dependence of the standard deviation on both x and the prior width. Indeed, the authors emphasize that their model is best fit at α=0.48, which would seem to imply that the best fitting value of υ is strictly positive. However, it would be important to clarify whether the estimation procedure allowed for υ=0, or whether this noise parameter was constrained to be positive (i.e., clarify whether the estimation assumed noisy and efficient coding of internal representations).

      (2) Differences across tasks

      A main takeaway from the paper is that optimal coding depends on the expected reward function in each task. This is the explanation for why the degree of sublinearity between standard deviation and prior width changes across the estimation and discrimination task. But besides the two different reward functions, there are also other differences across the two tasks. For example, the estimation task involves a single array of dots, whereas the discrimination task involves a pair of sequences of Arabic numerals. Related to the discussion above, in the estimation task the response scale is continuous whereas in the discrimination task, responses are binary. Is it possible that these other differences in the task could contribute to the observed different degrees of sublinearity? It is likely beyond the scope of the paper to incorporate these differences into the model, but such differences across the two tasks should be discussed as potential drivers of differences in observed behavior.

      If it becomes too difficult to interpret the data from the estimation task due to the slider bar varying with the prior range, then which of the paper's conclusions would still follow when restricting the analysis to the discrimination task?

      (3) Placement literature

      One closely related experiment to the discrimination task in the current paper can be found in Frydman and Jin (2022 Quarterly Journal of Economics). Those authors also experimentally vary the width of a uniform prior in a discrimination task using Arabic numerals, in order to test principles of efficient coding. Consistent with the current findings, Frydman and Jin find that subjects exhibit greater precision when making judgments about numbers drawn from a narrower distribution. However, what the current manuscript does is it goes beyond Frydman and Jin by modeling and experimentally varying task objectives to understand and test the effects on optimal coding. This contribution should be highlighted and contrasted against the earlier experimental work of Frydman and Jin to better articulate the novelty of the current manuscript.

    1. Reviewer #1 (Public review):

      Summary:

      In this work, the authors recorded the dynamics of the 5-HT with fiber photometry from CA1 in one hemisphere and LFP from CA1 in the other hemisphere. They observed an ultra-slow oscillation in the 5-HT signal during both wakefulness and NREM sleep. The authors have studied different phases of the ultra-slow oscillation to examine the potential difference in the occurrence of some behavioral state-related physiological phenomena (hippocampal ripples, EMG, and inter-area coherence).

      Strengths:

      The relation between the falling/rising phase of the ultra-slow oscillation and the ripples is sufficiently shown. There are some minor concerns about the observed relations that should be addressed with some further analysis.

      Systematic observations have started to establish a strong relation between the dynamics of neural activity across the brain and measures of behavioral arousal. Such relations span a wide range of temporal scales that are heavily inter-related. Ultra-slow time scales are specifically understudied due to technical limitations and neuromodulatory systems are the strongest mechanistic candidates for controlling/modulating the neural dynamics at these time scales. The hypothesis of the relation between a specific time scale and one certain neuromodulator (5-HT in this manuscript) could have a significant impact on the understanding of the hierarchy in the temporal scales of neural activity.

      Weaknesses:

      One major caveat of the study is that different neuromodulators are strongly correlated across all time scales and related to this, the authors need to discuss this point further and provide more evidence from the literature (if any) that suggests similar ultra-slow oscillations are weaker or lack from similar signals recorded for other neuromodulators such as Ach and NA.

      A major question that has been left out from the study and discussion is how the same level of serotonin before and after the peak could be differentially related to the opposite observed phenomenon. What are the possible parallel mechanisms for distinguishing between the rising and falling phases? Any neurophysiological evidence for sensing the direction of change in serotonin concentration (or any other neuromodulator), and is there any physiological functionality for such mechanisms?

    1. Reviewer #1 (Public review):

      Summary:

      In the manuscript by Hoisington et al., the authors utilized a novel conditional neuronal prosap2-interacting protein 1 (Prosapip1) knockout mouse to delineate the effects of both neuronal and dorsal hippocampal (dHP)-specific knockout of Prosapip1 impacts biochemical and electrophysiological neuroadaptations within the dHP that may mediate behaviors associated with this brain region.

      Strengths:

      (1) Methodological Strengths

      a. The generation and use of a conditional neuronal knockout of Prosapip1 is a strength. These mice will be useful for anyone interested in studying or comparing and contrasting the effects of loss of Prosapip1 in different brain regions or in non-neuronal tissues.

      b. The use of biochemical, electrophysiological, and behavioral approaches are a strength. By providing data across multiple domains, a picture begins to emerge about the mechanistic role for Prosapip1. While questions still remain, the use of the 3 domains is a strength.

      c. The use of both global, constitutive neuronal loss of Prosapip1 and postnatal dHP-specific knockout of Prosapip1 help support and validate the behavioral conclusions.

      (2) Strengths of the results

      a. It is interesting that loss of Prosapip1 leads to specific alterations in the expression of GluN2B and PSD95 but not GluA1 or GluN2A in a post-homogenization fraction that the author's term a "synaptic" fraction. Therefore, these results suggest protein-specific modulation of glutamatergic receptors within a "synaptic" fraction.

      b. The electrophysiological data demonstrate an NMDAR-dependent alteration in measures of hippocampal synaptic plasticity, including long-term potentiation (LTP) and NMDAR input/output. These data correspond with the biochemical data demonstrating a biochemical effect on GluN2B localization. Therefore, the conclusion that loss of Prosapip1 influences NMDAR function is well supported.

      c. The behavioral data suggest deficits in memory in particular novel object recognition and spatial memory, in the Prosapip1 knockout mice. These data are strongly bolstered by both the pan-neuronal knockout and the dHP Cre transduction.

      Weaknesses:

      (1) Methodological Weaknesses

      a. The synapsin-Cre mice may more broadly express Cre-recombinase than just in neuronal tissues. Specifically, according to Jackson Laboratories, there is a concern with these mice expressing Cre-recombinase germline. As the human protein atlas suggests that Prosapip1 protein is expressed extraneuronally, validation of neuron or at least brain-specific knockout would be helpful in interpreting the data. Having said that, the data demonstrating that the brain region-specific knockout has similar behavioral impacts helps alleviate this concern somewhat; however, there are no biochemical or electrophysiological readouts from these animals, and therefore an alternative mechanism in this adult knockout cannot be excluded.

      b. The use of the word synaptic and the crude fractionation make some of the data difficult to interpret/contextualize. It is unclear how a single centrifugation that eliminates the staining of a nuclear protein can be considered a "synaptic" fraction. This is highlighted by the presence of GAPDH in this fraction which is a cytosolically-enriched protein. While GAPDH may be associated with some membranes it is not a synaptic protein. There is no quantification of GAPDH against total protein to validate that it is not enriched in this fraction over control. Moreover, it should not be used as a loading control in the synaptic fraction. There are multiple different ways to enrich membranes, extrasynaptic fractions, and PSDs and a better discussion on the caveats of the biochemical fractionation is a minimum to help contextualize the changes in PSD95 and GluN2B.

      c. Also, the word synaptosomal on page 7 is not correct. One issue is this is more than synaptosomes and another issue is synaptosomes are exclusively presynaptic terminals. The correct term to use is synaptoneurosome, which includes both pre and postsynaptic components. Moreover, as stated above, this may contain these components but is most likely not a pure or even enriched fraction.

      d. The age at which the mice underwent injection of the Cre virus was not mentioned.

      (2) Weaknesses of results

      a. There were no measures of GluN1 or GluA2 in the biochemical assays. As GluN1 is the obligate subunit, how it is impacted by the loss of Prosapip1 may help contextualize the fact that GluN2B, but not GluN2A, is altered. Moreover, as GluA2 has different calcium permeance, alterations in it may be informative.

      b. While there was no difference in GluA1 expression in the "synaptic" fraction, it does not mean that AMPAR function is not impacted by the loss of Prosapip1. This is particularly important as Prosapip1 may interact with kinases or phosphatases or their targeting proteins. Therefore, measuring AMPAR function electrophysiologically or synaptic protein phosphorylation would be informative.

      c. There is a lack of mechanistic data on what specifically and how GluN2B and PSD95 expression is altered. This is due to some of the challenges with interpreting the biochemical fractionation and a lack of results regarding changes in protein posttranslational modifications.

      d. The loss of social novelty measures in both the global and dHP-specific Prosapip1 knockout mice were not very robust. As they were consistently lost in both approaches and as there were other consistent memory deficits, this does not impact the conclusions, but may be important to temper discussion to match these smaller deficits within this domain.

      e. Alterations in presynaptic paired-pulse ratio measures are intriguing and may point to a role for Prosapip1 in synapse development, as discussed in the manuscript. It would be interesting to delineate if these PPR changes also occur in the adult knockout to help detail the specific Prosapip1-induced neuroadaptations that link to the alterations in novelty-induced behaviors.

    1. Reviewer #1 (Public review):

      The authors investigate the function and neural circuitry of reentrant signals in the visual cortex. Recurrent signaling is thought to be necessary to common types of perceptual experience that are defined by long-range relationships or prior expectations. Contour illusions - where perceptual objects are implied by stimuli characteristics - are a good example of this. The perception of these illusions is thought to emerge as recurrent signals from higher cortical areas feedback onto the early visual cortex, to tell the early visual cortex that it should be seeing object contours where none are actually present.

      The authors test the involvement of reentrant cortical activity in this kind of perception using a drug challenge. Reentrance in the visual cortex is thought to rely on NMDAR-mediated glutamate signalling. The authors accordingly employ an NMDA antagonist to stop this mechanism, looking for the effect of this manipulation on visually evoked activity recorded in EEG.

      The motivating hypothesis for the paper is that NMDA antagonism should stop recurrent activity and that this should degrade perceptual activity supporting the perception of a contour illusion, but not other types of visual experience. Results in fact show the opposite. Rather than degrading cortical activity evoked by the illusion, memantine makes it more likely that machine learning classification of EEG will correctly infer the presence of the illusion.

      On the face of it, this is confusing, and the paper currently does not entirely resolve this confusion. But there are relatively easy ways to improve this. The authors would be well served by entertaining more possible outcomes in the introduction - there's good reason to expect a positive effect of memantine on perceptual brain activity, and I provide details on this below. The authors also need to further emphasize that the directional expectations that motivated E1 were, of course, adapted after the results from this experiment emerged. The authors presumably at least entertained the notion that E2 would reproduce E1 - meaning that E2 was motivated by a priori expectations that were ultimately met by the data.

      I broadly find the paper interesting, graceful, and creative. The hypotheses are clear and compelling, the techniques for both manipulation of brain state and observation of that impact are cutting edge and well suited, and the paper draws clear and convincing conclusions that are made necessary by the results. The work sits at the very interesting crux of systems neuroscience, neuroimaging, and pharmacology. I believe the paper can be improved in revision, but my suggestions are largely concerning presentation and nuance of interpretation.

      (1) I miss some treatment of the lack of behavioural correlate. What does it mean that metamine benefits EEG classification accuracy without improving performance? One possibility here is that there is an improvement in response latency, rather than perceptual sensitivity. Is there any hint of that in the RT results? In some sort of combined measure of RT and accuracy?

      (2) An explanation is missing, about why memantine impacts the decoding of illusion but not collinearity. At a systems level, how would this work? How would NMDAR antagonism selectively impact long-range connectivity, but not lateral connectivity? Is this supported by our understanding of laminar connectivity and neurochemistry in the visual cortex?

      (3) The motivating idea for the paper is that the NMDAR antagonist might disrupt the modulation of the AMPA-mediated glu signal. This is in line with the motivating logic for Self et al., 2012, where NMDAR and AMPAR efficacy in macacque V1 was manipulated via microinfusion. But this logic seems to conflict with a broader understanding of NMDA antagonism. NMDA antagonism appears to generally have the net effect of increasing glu (and ACh) in the cortex through a selective effect on inhibitory GABA-ergic cells (eg. Olney, Newcomer, & Farber, 1999). Memantine, in particular, has a specific impact on extrasynaptic NMDARs (that is in contrast to ketamine; Milnerwood et al, 2010, Neuron), and this type of receptor is prominent in GABA cells (eg. Yao et al., 2022, JoN). The effect of NMDA antagonists on GABAergic cells generally appears to be much stronger than the effect on glutamergic cells (at least in the hippocampus; eg. Grunze et al., 1996).

      This all means that it's reasonable to expect that memantine might have a benefit to visually evoked activity. This idea is raised in the GD of the paper, based on a separate literature from that I mentioned above. But all of this could be better spelled out earlier in the paper, so that the result observed in the paper can be interpreted by the reader in this broader context.

      To my mind, the challenging task is for the authors to explain why memantine causes an increase in EEG decoding, where microinfusion of an NMDA antagonist into V1 reduced the neural signal Self et al., 2012. This might be as simple as the change in drug... memantine's specific efficacy on extrasynaptic NMDA receptors might not be shared with whatever NMDA antagonist was used in Self et al. 2012. Ketamine and memantine are already known to differ in this way.

      (4) The paper's proposal is that the effect of memantine is mediated by an impact on the efficacy of reentrant signaling in visual cortex. But perhaps the best-known impact of NMDAR manipulation is on LTP, in the hippocampus particularly but also broadly. Perception and identification of the kanisza illusion may be sensitive to learning (eg. Maertens & Pollmann, 2005; Gellatly, 1982; Rubin, Nakayama, Shapley, 1997); what argues against an account of the results from an effect on perceptual learning? Generally, the paper proposes a very specific mechanism through which the drug influences perception. This is motivated by results from Self et al 2012 where an NMDA antagonist was infused into V1. But oral memantine will, of course, have a whole-brain effect, and some of these effects are well characterized and - on the surface - appear as potential sources of change in illusion perception. The paper needs some treatment of the known ancillary effects of diffuse NMDAR antagonism to convince the reader that the account provided is better than the other possibilities.

      (5) The cross-decoding approach to data analysis concerns me a little. The approach adopted here is to train models on a localizer task, in this case, a task where participants matched a kanisza figure to a target template (E1) or discriminated one of the three relevant stimuli features (E2). The resulting model was subsequently employed to classify the stimuli seen during separate tasks - an AB task in E1, and a feature discrimination task in E2. This scheme makes the localizer task very important. If models built from this task have any bias, this will taint classifier accuracy in the analysis of experimental data. My concern is that the emergence of the kanisza illusion in the localizer task was probably quite salient, respective to changes in stimuli rotation or collinearity. If the model was better at detecting the illusion to begin with, the data pattern - where drug manipulation impacts classification in this condition but not other conditions - may simply reflect model insensitivity to non-illusion features.

      I am also vaguely worried by manipulations implemented in the main task that do not emerge in the localizer - the use of RSVP in E1 and manipulation of the base rate and staircasing in E2. This all starts to introduce the possibility that localizer and experimental data just don't correspond, that this generates low classification accuracy in the experimental results and ineffective classification in some conditions (ie. when stimuli are masked; would collinearity decoding in the unmasked condition potentially differ if classification accuracy were not at a floor? See Figure 3c upper, Figure 5c lower).

      What is the motivation for the use of localizer validation at all? The same hypotheses can be tested using within-experiment cross-validation, rather than validation from a model built on localizer data. The argument may be that this kind of modelling will necessarily employ a smaller dataset, but, while true, this effect can be minimized at the expense of computational cost - many-fold cross-validation will mean that the vast majority of data contributes to model building in each instance.

      It would be compelling if results were to reproduce when classification was validated in this kind of way. This kind of analysis would fit very well into the supplementary material.

    1. Reviewer #1 (Public review):

      Summary:

      This study investigated the role of transcriptional and translational controls of gene expression in dorsal root ganglia and lumbar spinal cord in neuropathic pain in mice. Using ribosome profiling (Ribo-seq) and translating ribosome affinity purification (TRAP), they show changes in transcriptomic and translational gene expression at the peripheral and central levels rapidly after nerve injury. While translational changes in gene expression remained elevated for more than two months in both DRGs and the spinal cord, transcriptomic regulation was absent in the spinal cord long after the onset of neuropathy. Disrupting mRNA translation in dorsal horn neurons using antisense oligonucleotides reduced mechanical withdrawal threshold and facial expression of pain. Using fluorescent noncanonical amino acid tagging (FUNCAT), the authors further show that de novo protein expression primarily occurs in inhibitory neurons in the superficial dorsal horn after nerve injury. Accordingly, a selective increase in translational control of gene expression in spinal inhibitory neurons, or a subset of mainly inhibitory neurons expressing parvalbumin (PV), using transgenic mice, led to a decrease in the excitability of PV neurons and mechanical allodynia. In contrast, decreasing the translational control of spinal PV neurons prevented the alteration of the electrophysiological properties of the PV cells induced by nerve injury.

      Strengths:

      This is a well-written article that uncovers a previously unappreciated role of gene expression control in PV neurons, which seems to play an important part in the loss of inhibitory control of spinal circuits typically seen after peripheral nerve injury. The conclusions are generally well supported by the data.

      Weaknesses:

      The study would benefit from further clarifications in the methods section and a deeper analysis of gene expression changes in mRNA expression and ribosomal footprint observed after nerve injury.

      Antisense oligonucleotides used to reduce translation by disrupting eIF4E expression were administered i.c.v. It is unknown if the authors controlled for locomotor deficits, which might add confounds in the interpretation of behavioral results. A more local route should have been preferable to avoid targeting brain regions, which could potentially affect behavior.

      Only female mice were used for Ribo-Seq, TRAP, FUNCAT, and electrophysiology, but both sexes were used for behavior experiments.

      The conditional KO of 4E-BP1 using transgenic animals should be total in the targeted cells. However, only a partial reduction is reported in Figure S2 in GAD2, PV, Vglut2, or Tac1 cells. Again, proper methods for quantification of fluorescence in these experiments are lacking.

      The elegant knockdown of eIF4E using AAV-mediated shRNAmir shows a recovery of the electrophysiological intrinsic properties of PV neurons after injury. It is unclear if such manipulation would be sufficient to reverse mechanical allodynia in vivo.

    1. Mucho más completo mapa mental y mejor descripción verbal del mismo, que está asociado no solo a lo reciente del mapa, sino al nivel de detalle del mapa.

      Valdría la pena incorporar el contraste con otros paradigmas de computación/programación mostrados en el video mismo.

    1. Puede que la IA adquiera un poder inmenso en un futuro y se cumplan esos riesgos que a decir verdad, dan miedo, pero lo mismo sucedió con los computadores en su época, surgieron temores donde se decía que en unos años iban a superar nuestras capacidades humanas, cosa que no sucedió. Ahora bien, si hay un tema importante y es saber utilizar estas herramientas para complementar nuestras habilidades como seres humanos, no para que nos hagan las cosas y nos volvamos dependientes de estas. Por lo tanto, no me preocuparía tanto porque la IA nos reemplace o destruya en un futuro.

      Creo que es importante mantener una postura esperanzada pero crítica, no sólo frente a las tecnologías venideras, sino a las que tenemos "naturalizadas" ya. De este modo podemos saber cuándo es mejor la bici o el metro (subterraneo!) que el carro particular o no revisar notificaciones de redes sociales al despertar o saber cuándo es mejor escribir uno mismo a que lo haga una IA.

    2. Es importante que el mapa refleje los elementos estructurales del video (su tabla de contenido) de manera más detallada.

    1. Considero que nunca vamos a estar completamente preparados para la inteligencia artificial, es algo que cada día está tomando más control de decisión en las empresas no solo en el sector tecnología sino en todos los sectores.

      Quizás lo que tenemos que cuestionar es la noción misma de IA, al menos como las grandes empresas del Valle de Silicio (Sillicon Valley) de modo que podamos desenmascarár muchos de los mitos al respecto.

    2. No estamos preparados para la inteligencia artificial

      El mapa refleja varias de las tensiones del video y los impactos negativos de la IA para la sociedad.

      Es importante que el mapa mental refleje de manera más detallada la "tabla de contenido" del video, de forma que conceptos como "bosque oscuro" queden claramente articulados.

    3. No logré publicar el mapa en Internet Archive

      ¿Qué errores puntuales se presentaron durante esta publicación? Por favor documéntalo agregando capturas de pantalla.

    1. Debido a la magnitud de información y las redes neuronales que forma esta herramienta, puede que en algún momento esta logre satisfacer las necesidades que los seres humanos no han conseguido completar.

      ¿Cómo cuales necesidades?

      Respecto a lo que dijiste de la cura de una enfermedad, interesante lo que se de despligue de proteinas. Pienso mirar la complementariedad entre humanos y máquinas, así como las instituciones como las articulan.

    2. Creo que hay varios elementos del lenguaje en los cuales el mapa no profundiza, en particular cómo las redes neuronales funciona y cómo se construyen los modelos vectoriales del lenguaje.

    1. Reviewer #1 (Public Review):

      In the article by Dearlove et al., the authors present evidence in strong support of nucleotide ubiquitylation by DTX3L, suggesting it is a promiscuous E3 ligase with capacity to ubiquitylate ADP ribose and nucleotides. The authors include data to identify the likely site of attachment and the requirements for nucleotide modification.

      While this discovery potentially reveals a whole new mechanism by which nucleotide function can be regulated in cells, there are some weaknesses that should be considered. Is there any evidence of nucleotide ubiquitylation occurring cells? It seems possible, but evidence in support of this would strengthen the manuscript. The NMR data could also be strengthened as the binding interface is not reported or mapped onto the structure/model, this seems of considerable interest given that highly related proteins do have the same activity.

      The paper is for the most part well well-written and is potentially highly significant

      Comments on revised version:

      The revised manuscript has addressed many of the concerns raised and clarified a number of points. As a result the manuscript is improved.

      The primary concern that remains is the absence of biological function for Ub-ssDNA/RNA and the inability to detect it in cells. Despite this the manuscript will be of interest to those in the ubiquitin field and will likely provoke further studies and the development of tools to better assess the cellular relevance. As a result this manuscript is important.

      Minor issue:<br /> Figure 1A - the authors have now included the constructs used but it would be more informative if the authors lined up the various constructs under the relevant domains in the full-length protein.

    1. Reviewer #1 (Public review):

      Summary:

      Tian et al. describes how TIPE regulates melanoma progression, stemness, and glycolysis. The authors link high TIPE expression to increased melanoma cell proliferation and tumor growth. TIPE causes dimerization of PKM2, as well as translocation of PKM2 to the nucleus, thereby activating HIF-1alpha. TIPE promotes the phosphorylation of S37 on PKM2 in an ERK-dependent manner. TIPE is shown to increase stem-like phenotype markers. The expression of TIPE is positively correlated with the levels of PKM2 Ser37 phosphorylation in murine and clinical tissue samples. Taken together, the authors demonstrate how TIPE impacts melanoma progression, stemness, and glycolysis through dimeric PKM2 and HIF-1alpha crosstalk.

      The authors manipulated TIPE expression using both shRNA and overexpression approaches throughout the manuscript. Using these models, they provide strong evidence of the involvement of TIPE in mediating PKM2 Ser37 phosphorylation and dimerization. The authors also used mutants of PKM2 at S37A to block its interaction with TIPE and HIF-1alpha. In addition, an ERK inhibitor (U0126) was used to block the phosphorylation of Ser37 on PKM2. The authors show how dimerization of PKM2 by TIPE causes nuclear import of PKM2 and activation of HIF-1alpha and target genes. Pyridoxine was used to induce PKM2 dimer formation, while TEPP-46 was used to suppress PKM2 dimer formation. TIPE maintains stem cell phenotypes by increasing expression of stem-like markers. Furthermore, the relationship between TIPE and Ser37 PKM2 was demonstrated in murine and clinical tissue samples.

      The evaluation of how TIPE causes metabolic reprogramming can be better assessed using isotope tracing experiments and improved bioenergetic analysis.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript uses PS-coated and IgG-opsonized targets to model the engulfment of apoptotic cells and pathogens. It demonstrates that differential activation of the respiratory burst accounts for variations in cell morphology, adhesion, and migration following phagocytosis of different particles. Specifically, reactive oxygen species produced by phagosomes containing IgG-opsonized targets activate Rho GTPases. This activation triggers Formin- and ERM-dependent compaction of the cortical actin network, leading to rounded cell morphology, reduced membrane ruffling, disassembly of podosomes, and decreased migration. Some of these findings are validated in cells exposed to pathogens or soluble MAMPs.

      Strengths:

      The manuscript presents well-executed and controlled experiments. It proposes an intriguing model to explain the distinct behaviors of myeloid cells when confronted with different phagocytic cargoes and offers fresh insights into immune surveillance.

      Weaknesses:

      Certain aspects of the proposed model require further experimental evidence. The significance of the cellular behavioral differences in response to various phagocytic cargoes warrants further exploration within physiological contexts.

      Specific comments:

      How do reactive oxygen species lead to an increase in Rho activation while simultaneously reducing Rac activity? The underlying molecular mechanisms remain unresolved, although potential regulatory pathways are discussed.

      Given that the number of phagocytosed particles affects cell behavior (SF1), it is important to ensure that an equivalent number of particles are phagocytosed when comparing cells treated with PS-beads and IgG-beads (Figure 1a). How was this experimentally controlled, and how many particles are phagocytosed under each condition?

      Why were experiments conducted in BMDM, Raw264.7, and PMN cells under different conditions? For Raw264.7 and PMN cells, cell behavior was only compared between those treated with IgG-RBC and untreated cells. What occurs to these cells when they are exposed to PS-beads as opposed to IgG-beads?

      How long does it take for cells treated with IgG-beads to recover and regain their mobility and surveillance activity? Does this recovery occur following a reduction in reactive oxygen species production?

      A contractile actin cortex usually requires the activity of both Formin and myosin II. It is a bit surprising that inhibitors of ROCK and myosin II, when added to Raw cells engulfing IgG-RBC, did not affect podosome disassembly. Is the cytoskeletal rearrangement observed in Figure 2 also independent of myosin II activity?

    1. Reviewer #1 (Public review):

      Summary:

      The paper examined livestock abortion, as it is an important disease syndrome that affects productivity and livestock economies. If livestock abortion remains unexamined it poses risks to public health.

      Several pathogens are associated with livestock abortions but across Africa however the livestock disease surveillance data rarely include information from abortion events, little is known about the aetiology and impacts of livestock abortions, and data are not available to inform prioritisation of disease interventions. Therefore the current study seeks to examine the issue in detail and proposes some solutions.

      The study took place in 15 wards in northern Tanzania spanning pastoral, agropastoral and smallholder agro-ecological systems. The key objective is to investigate the causes and impacts of livestock abortion.

      The data collection system was set up such that farmers reported abortion cases to the field officers of the Ministry of Livestock and Fisheries livestock<br /> The reports were made to the investigation teams. The team only included abortion of those that the livestock field officers could attend to within 72 hours of the event occurring.

      Also a field investigation was carried out to collect diagnostic samples from aborted materials. In addition aborting dams and questionnaires were administer to collect data on herd/flock management. Laboratory diagnostic tests were carried out for a range of abortigenic pathogens

      Over the period of the study 215 abortion events in cattle (n=71), sheep (n=44) and goats (n=100) were investigated. In all 49 investigated cases varied widely across wards, with three .The Aetiological attribution, achieved for 19.5% of cases through PCR-based diagnostics, was significantly affected by delays in field investigation.

      The result also revealed that vaginal swabs from aborting dams provided a practical and sensitive source of diagnostic material for pathogen detection.

      Livestock abortion surveillance can generate valuable information on causes of zoonotic disease outbreaks, and livestock reproductive losses and can identify important pathogens that are not easily captured through other forms of livestock disease surveillance. The study demonstrated the feasibility of establishing an effective reporting and investigation system that could be implemented across a range of settings, including remote rural areas,

      Strengths:

      The paper combines both science and socio economic methodology to achieve the aim of the study.

      The methodology was well presented and the sequence was great. The authors explain where and how the data was collected. Figure 2 was used to describe the study area which was excellently done. The section on Investigation of cases was well written. The sample analysis was also well written. The authors devoted a section to summarizing the investigated cases and description of the livestock 221-study population. The logic model has been well presented

      Weaknesses:

      All the weaknesses identified have been resolved by the the authors

    1. Reviewer #1 (Public review):

      Summary:

      Deletion of the hrp2 and hrp3 loci in P. falciparum poses an immediate public health threat. This manuscript provides a more complete understanding of the dynamic nature with which these deletions are generated. By delving into the likely mechanisms behind their generation, the authors also provide interesting insight into general Plasmodium biology that can inform our broader understanding of the parasite's genomic evolution.

      Strengths:

      The sub-telomeric regions of P. falciparum (where hrp2 and hrp3 are located) are notoriously difficult to study with short-read sequence data. The authors take an appropriate, targeted approach toward studying the loci of interest, which includes read-depth analysis and local haplotype reconstruction. They additionally use both long-read and short-read data to validate their major findings. There is an extensive set of supplementary plots, which helps clarify several aspects of the data.

      Weaknesses:

      The revised version of this manuscript has helpfully expanded the details regarding methodology, however, publication of the tool PathWeaver (which is used for local haplotype reconstruction) remains in preparation.

    1. Reviewer #3 (Public review):

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

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

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

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

      Comments on latest version:

      The authors have effectively addressed the minor issues raised in the most recent round of non-public reviews. This reviewer has no additional recommendations.

    1. Reviewer #1 (Public review):

      In the revision of their paper, N'Guessan et al have improved the report of their study of expression QTL (eQTL) mapping in yeast using single cells. The authors make use of advances in single cell RNAseq (scRNAseq) in yeast to increase the efficiency with which this type of analysis can be undertaken. Building on prior research led by the senior author that entailed genotyping and fitness profiling of almost 100,000 cells derived from a cross between two yeast strains (BY and RM) they performed scRNAseq on a subset of ~5% (n = 4,489) individual cells. To address the sparsity of genotype data in the expression profiling they used a Hidden Markov Model (HMM) to infer genotypes and then identify the most likely known lineage genotype from the original dataset. To address the relationship between variance in fitness and gene expression the authors partition the variance to investigate the sources of variation. They then perform eQTL mapping and study the relationship between eQTL and fitness QTL identified in the earlier study.

      This paper seeks to address the question of how quantitative trait variation and expression variation are related. scRNAseq represents an appealing approach to eQTL mapping as it is possible to simultaneously genotype individual cells and measure expression in the same cell. As eQTL mapping requires large sample sizes to identify statistical relationships, the use of scRNAseq is likely to dramatically increase the statistical power of such studies. However, there are several technical challenges associated with scRNAseq and the authors' study is focused on addressing those challenges. Most of the points raised by my review of the initial version have been addressed. However, one point remains and one additional point should be considered.

      (1) Given that the authors overcame many technical and analytical challenges in the course of this research, the study would be greatly strengthened through analysis of at least one, and ideally several, more conditions which would expand the conclusions that could be drawn from the study and demonstrate the power of using scRNAseq to efficiently quantify expression in different environments.

      (2) In this version the authors have introduced the use of data imputation using a published algorithm, DISCERN. This has greatly increased the variation explained by their model as presented in figure 3. However, it is possible that the explained variance is now an overestimation as a result of using the imputed expression data. I think that it would be appropriate to present figure 3 using the sparse data presented in the initial version of the paper and the newly presented imputed data so that the reader can draw their own conclusions about the interpretation.

    1. Reviewer #1 (Public review):

      The goal of this work is to understand the clinical observation of a subgroup of diabetics who experience extremely high levels of blood glucose levels after a period of high carbohydrate intake. These symptoms are similar to the onset of Type 1 diabetes but, crucially, have been observed to be fully reversible in some cases.

      The authors interpret these observations by analyzing a simple yet insightful mathematical model in which β-cells temporarily stop producing insulin when exposed to high levels of glucose. For a specific model realization of such dynamics (and for specific parameter values) they show that such dynamics lead to two distinct stable states. One is the relatively normal/healthy state in which β-cells respond appropriately to glucose by releasing insulin. In contrast, when enough β-cells "refuse" to produce insulin in a high-glucose environment, there is not enough insulin to reduce glucose levels, and the high-glucose state remains locked in because the high-glucose levels keep β-cells in their inactive state. The presented mathematical analysis shows that in their model the high-glucose state can be entered through an episode of high glucose levels and that subsequently the low-glucose state can be re-entered through prolonged insulin intake.

      The strength of this work is twofold. First, the intellectual sharpness of translating clinical observations of ketosis-prone type 2 diabetes (KPD) into the need for β-cell responses on intermediate timescales. Second, the analysis of a specific model clearly establishes that the clinical observations can be reproduced with a model in which β-cells dynamics reversibly enter a non-insulin-producing state in a glucose-dependent fashion.

      The likely impact of this work is a shift in attention in the field from a focus on the short and long-term dynamics in glucose regulation and diabetes progression to the intermediate timescales of β-cell dynamics. I expect this to lead to much interest in probing the assumptions behind the model to establish what exactly the process is by which patients enter a 'KPD state'. Furthermore, I expect this work to trigger much research on how KPD relates to "regular" type 2 diabetes and to lead to experimental efforts to find/characterize previously overlooked β-cell phenotypes.

      In summary, the authors claim that observed clinical dynamics and possible remission of KPD can be explained through introducing a temporarily inactive β-cell state into a "standard model" of diabetes. The evidence for this claim comes from analyzing a mathematical model and clearly presented. Importantly, the authors point out that this does not mean their model is correct. Other hypotheses are that:

      - Instead of switching to an inactive state, individual β-cells could adjust how they respond to high glucose levels. If this response function changes reversibly on intermediate timescales the clinical observations could be explained without a reversible inactive state.

      - Kidney function is indirectly impaired through chronic high glucose levels. The apparent rapid glucose increase might then not highlight a new type of β-cell phenotype but would reflect rapid changes in kidney function.

      - In principle, the remission could be due to a direct response of β-cells to insulin and not mediated through the lowering of glucose levels.

      Crucially, the hypothesized reversibly inactive state of β-cells remains to be directly observed. One of the key contributions of this theoretical work is directing experimental focus towards looking for reversible β-cell phenotypes.

    1. Reviewer #1 (Public review):

      Summary:

      Barlow and coauthors utilized the high-parameter imaging platform of CODEX to characterize the cellular composition of immune cells in situ from tissues obtained from organ donors with type 1 diabetes, subjects presented with autoantibodies who are at elevated risk, or non-diabetic organ donor controls. The panels used in this important study were based on prior publications using this technology, as well as a priori and domain-specific knowledge of the field by the investigators. Thus, there was some bias in the markers selected for analysis. The authors acknowledge that these types of experiments may be complemented moving forward with the inclusion of unbiased tissue analysis platforms that are emerging that can conduct a more comprehensive analysis of pathological signatures employing emerging technologies for both high-parameter protein imaging and spatial transcriptomics.

      Strengths:

      In terms of major findings, the authors provide important confirmatory observations regarding a number of autoimmune-associated signatures reported previously. The high parameter staining now increases the resolution for linking these features with specific cellular subsets using machine learning algorithms. These signatures include a robust signature indicative of IFN-driven responses that would be expected to induce a cytotoxic T-cell-mediated immune response within the pancreas. Notable findings include the upregulation of indolamine 2,3-dioxygenase-1 in the islet microvasculature. Furthermore, the authors provide key insights as to the cell:cell interactions within organ donors, again supporting a previously reported interaction between presumably autoreactive T and B cells.

      Weaknesses:

      These studies also highlight a number of molecular pathways that will require additional validation studies to more completely understand whether they are potentially causal for pathology, or rather, epiphenomenon associated with increased innate inflammation within the pancreas of T1D subjects. Given the limitations noted above, the study does present a rich and integrated dataset for analysis of enriched immune markers that can be segmented and annotated within distinct cellular networks. This enabled the authors to analyze distinct cellular subsets and phenotypes in situ, including within islets that peri-islet infiltration and/or intra-islet insulitis.

      Despite the many technical challenges and unique organ donor cohort utilized, the data are still limited in terms of subject numbers - a challenge in a disease characterized by extensive heterogeneity in terms of age of onset and clinical and histopathological presentation. Therefore, these studies cannot adequately account for all of the potential covariates that may drive variability and alterations in the histopathologies observed (such as age of onset, background genetics, and organ donor conditions). In this study, the manuscript and figures could be improved in terms of clarifying how variable the observed signatures were across each individual donor, with the clear notion that non-diabetic donors will present with some similar challenges and variability.

    1. Reviewer #1 (Public review):

      Many labs world-wide now use the blind source deconvolution technique to identify the firing patterns of multiple motor units simultaneously in human subjects. This technique has had a truly transformative effective on our understanding of the structure of motor output in both normal subjects and, increasingly, in persons with neurological disorders. The key advance presented here is that the software provides real time identification of these firing patterns.

      The main strengths are the clarity of the presentation and the great potential that real-time decoding will provide. Figures are especially effective and statistical analyses are excellent.

    1. Reviewer #1 (Public review):

      This study describes a useful antibody-free method to map G-quadruplexes in vertebrate cells. The analysis of the data is solid but it remains primarily descriptive and does not substantially add to existing publications (such as PMID:34792172 for example). Nevertheless, the datasets generated here might constitute a good starting point for more functional studies.

      Comments on revised version:

      It is disappointing to see that the authors decided to brush aside most of the comments made by the three referees, even though these comments were largely consistent with each other. As a result, the revised manuscript is not substantially changed or improved. Legitimate concerns regarding the specificity of the Cut&Tag signals were not addressed and therefore remain. The sensitivity of the HBD-seq signals to a combination of RNase A and RNase H does not demonstrate that HBD-seq specifically reports the presence of RNA:DNA hybrids. The new Figure 9 comparing HepG4-seq to existing datasets does not unequivocally demonstrate the superiority of the Hemin-based strategy to map G4s.

    1. Reviewer #2 (Public review):

      Qin, Sanbo and Zhou, Huan-Xiang created a model, SeqDYN, to predict nuclear magnetic resonance (NMR) spin relaxation spectra of intrinsically disordered proteins (IDPs), based primarily on amino acid sequence. To fit NMR data, SeqDYN uses 21 parameters, 20 that correspond to each amino acid, and a sequence correlation length for interactions. The model demonstrates that local sequence features impact the dynamics of the IDP, as SeqDYN performs better than a one residue predictor, despite having similar numbers of parameters. SeqDYN is trained using 45 IDP sequences and is retrained using both leave-one-out cross validation and five-fold cross validation, ensuring the model's robustness. While SeqDYN can provide reasonably accurate predictions in many cases, the authors note that improvements can be made by incorporating secondary structure predictions, especially for alpha-helices that exceed the correlation length of the model. The authors apply SeqDYN to study nine IDPs and a denatured ordered protein, demonstrating its predictive power. The model can be easily accessed via the website mentioned in the text.

      The authors have adequately addressed the majority of my previous concerns. However, I still wonder if an attempt to fit the individual protein fitting parameter based on temperature and magnetic field strength would be possible. The authors would have 45 data points on which to fit such a parameter, which would only depend on two variables.

    1. Joint Public Review:

      The paper sought to determine the number of myosin 10 molecules per cell and localized to filopodia, where they are known to be involved in formation, transport within, and dynamics of these important actin-based protrusions. The authors used a novel method to determine the number of molecules per cell. First, they expressed HALO tagged Myo10 in U20S cells and generated cell lysates of a certain number of cells and detected Myo10 after SDS-PAGE, with fluorescence and a stained free method. They used a purified HALO tagged standard protein to generate a standard curve which allowed for determining Myo10 concentration in cell lysates and thus an estimate of the number of Myo10 molecules per cell. They also examined the fluorescence intensity in fixed cell images to determine the average fluorescence intensity per Myo10 molecule, which allowed the number of Myo10 molecules per region of the cell to be determined. They found a relatively small fraction of Myo10 (6%) localizes to filopodia. There are hundreds of Myo10 in each filopodia, which suggests some filopodia have more Myo10 than actin binding sites. Thus, there may be crowding of Myo10 at the tips, which could impact transport, the morphology at the tips, and dynamics of the protrusions themselves. Overall, the study forms the basis for a novel technique to estimate the number of molecules per cell and their localization to actin-based structures. The implications are broad also for being able to understand the role of myosins in actin protrusions, which is important for cancer metastasis and wound healing.

      Comments on latest version (from the Reviewing Editor):

      One of the main critiques that still remains is that the results were derived from experiments with overexpressed Myo10 and therefore are hard to extrapolate to physiological conditions. Measurement were also only performed in a single cell line. The authors counter this critique with the argument that their results provide insight into a system in which Myo10 is a limiting factor for controlling filopodia formation. They demonstrate that U20S cells do not express detectable levels of Myo10 and thus introducing Myo10 expression demonstrates how triggering Myo10 expression impacts filopodia. An example is given of how melanoma cells often heavily upregulate Myo10.

    1. Reviewer #1 (Public review):

      Summary:

      The authors provide an genome annotation resource of 33 insects using a motif-blind prediction methods for tissue-specific cis-regulatory modules. This is a welcome addition that may facilitate further research in new laboratory systems, and the approach seem to be relatively accurate, although it should be combined with other sources of evidence to be practical.

      Strengths:

      The paper clearly presents the resource, including the testing of candidate enhancers identified from various insects in Drosophila. This cross-species analysis, and the inherent suggestion that training datasets generated in flies can predict a cis-regulatory activity in distant insects, is interesting. While I can not be sure this approach will prevail in the future, for example with approaches that leverage the prediction of TF binding motifs, the SCRMShaw tool is certainly useful and worth of consideration for the large community of genome scientists working on insects.

      Weaknesses from the previous version were appropriately corrected in this revision, as the authors improved data availability including with genome annotation resources.

    1. Reviewer #1 (Public review):

      This study comes to an interesting conclusion: a polyunsaturated fatty acid, Lin-Glycine, increases the conductance of KCNQ1/KCNE1 channels by stabilizing a state of the selectivity filter that allows K+ conduction. The stabilization of a conducting state is well supported by single channel analysis, which shows that normally infrequent opening bursts occur more often in the presence of the PUFA. The linkage to PUFA action through the selectivity filter is supported by disruption of PUFA effects by mutation of residues which change conformation in two KCNQ1 structures from the literature. A definitive functional experiment is conducted by single channel recordings with selectivity filter domain mutation Y315F which ablates the Lin-Glycine effect on Gmax. The computational exploration of two selectivity filter structures proposed to interact distinctly with Lin-Glycine is informative. Both mutation results and simulations converge on the proposed selectivity filter mechanism, although other possibilities for Lin-Glycine binding and action might be possible. Overall, the major claim of the abstract is well-supported: "... that the selectivity filter in KCNQ1 is normally unstable ... and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state."

    1. Reviewer #1 (Public review):

      Summary:

      The authors demonstrate impairments induced by a high cholesterol diet on GLP-1R dependent glucoregulation in vivo as well as an improvement after reduction in cholesterol synthesis with simvastatin in pancreatic islets. They also map sites of cholesterol high occupancy and residence time on active versus inactive GLP-1Rs using coarse-grained molecular dynamics (cgMD) simulations and screened for key residues selected from these sites and performed detailed analyses of the effects of mutating one of these residues, Val229, to alanine on GLP-1R interactions with cholesterol, plasma membrane behaviour, clustering, trafficking and signalling in pancreatic beta cells and primary islets, and describe an improved insulin secretion profile for the V229A mutant receptor.

      These are extensive and very impressive studies indeed. I am impressed with the tireless effort exerted to understand the details of molecular mechanisms involved in the effects of cholesterol for GLP-1 activation of its receptor. In general the study is convincing, the manuscript well written and the data well presented. Some of the changes are small and insignificant which makes one wonder how important the observations are. For instance in figure 2 E (which is difficult to interpret anyway because the data are presented in percent, conveniently hiding the absolute results) does not show a significant result of the cyclodextrin except for insignificant increases in basal secretion. That is not identical to impairment of GLP-1 receptor signaling!

      To me the most important experiment of them all is the simvastatin experiment, but the results rest on very few numbers and there is a large variation. Apparently, in a previous study using more extensive reduction in cholesterol the opposite response was detected casting doubt on the significance of the current observation. I agree with the authors that the use of cyclodextrin may have been associated with other changes in plasma membrane structure than cholesterol depletion at the GLP-1 receptor. The entire discussion regarding he importance of cholesterol would benefit tremendously from studies of GLP-1 induced insulin secretion in people with different cholesterol levels before and after treatment with cholesterol-lowering agents. I suspect that such a study would not reveal major differences.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, the authors investigate a very interesting but often overlooked aspect of abstract vs. concrete processing in language. Specifically, they study if the differences in processing of abstract vs. concrete concepts in the brain is static or dependent on the (visual) context in which the words occur. This study takes a two-step approach to investigate how context might affect the perception of concepts. First, the authors analyze if concrete concepts, expectedly, activate more sensory systems while abstract concepts activate higher-order processing regions. Second, they measure the contextual situatedness vs. displacement of each word with respect to the visual scenes in which it is spoken and then evaluate if this contextual measure correlates with more activation in the sensory vs. higher-order regions respectively.

      Strengths:

      This study raises a pertinent and understudied question in language neuroscience. It also combines both computational and meta-analytic approaches.

    1. Reviewer #1 (Public review):

      Summary:

      The study made fundamental findings in investigations of the dynamic functional states during sleep. Twenty-one HMM states were revealed from the fMRI data, surpassing the number of EEG-defined sleep stages, which can define sub-states of N2 and REM. Importantly, these findings were reproducible over two nights, shedding new light on the dynamics of brain function during sleep.

      Strengths:

      The study provides the most compelling evidence on the sub-states of both REM and N2 sleep. Moreover, they showed these findings on dynamics states and their transitions were reproducible over two nights of sleep. These novel findings offered unique information in the field of sleep neuroimaging.

      Comments on revised version:

      Nice work! All my concerns have been addressed, and I have no further suggestions.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors aimed to develop and validate an automated, deep learning-based system for scoring the Rey-Osterrieth Complex Figure Test (ROCF), a widely used tool in neuropsychology for assessing memory deficits. Their goal was to overcome the limitations of manual scoring, such as subjectivity and time consumption, by creating a model that provides automatic, accurate, objective, and efficient assessments of memory deterioration in individuals with various neurological and psychiatric conditions.

      Strengths:

      Comprehensive Data Collection: The authors collected over 20,000 hand-drawn ROCF images from a wide demographic and geographic range, ensuring a robust and diverse dataset. This extensive data collection is critical for training a generalizable and effective deep learning model.

      Advanced Deep Learning Approach: Utilizing a multi-head convolutional neural network to automate ROCF scoring represents a sophisticated application of current AI technologies. This approach allows for detailed analysis of individual figure elements, potentially increasing the accuracy and reliability of assessments.

      Validation and Performance Assessment: The model's performance was rigorously evaluated against crowdsourced human intelligence and professional clinician scores, demonstrating its ability to outperform both groups. The inclusion of an independent prospective validation study further strengthens the credibility of the results.

      Robustness Analysis Efficacy: The model underwent a thorough robustness analysis, testing its adaptability to variations in rotation, perspective, brightness, and contrast. Such meticulous examination ensures the model's consistent performance across different clinical imaging scenarios, significantly bolstering its utility for real-world applications.

      Appraisal and discussion:

      By leveraging a comprehensive dataset and employing advanced deep learning techniques, they demonstrated the model's ability to outperform both crowdsourced raters and professional clinicians in scoring the ROCF. This achievement represents a significant step forward in automating neuropsychological assessments, potentially revolutionizing how memory deficits are evaluated in clinical settings. Furthermore, the application of deep learning to clinical neuropsychology opens avenues for future research, including the potential automation of other neuropsychological tests and the integration of AI tools into clinical practice. The success of this project may encourage further exploration into how AI can be leveraged to improve diagnostic accuracy and efficiency in healthcare.

      However, the critique regarding the lack of detailed analysis across different patient demographics, the inadequacy of network explainability, and concerns about the selection of median crowdsourced scores as ground truth raises questions about the completeness of their objectives. These aspects suggest that while the aims were achieved to a considerable extent, there are areas of improvement that could make the results more robust and the conclusions stronger.

      Comments on revised version:

      I appreciate the opportunity to review this revised submission. Having considered the other reviews, I believe this study presents an important advance in using AI methods for clinical applications, which is both innovative and has implications beyond a single subfield.

      The authors have developed a system using fundamental AI that appears sufficient for clinical use in scoring the Rey-Osterrieth Complex Figure (ROCF) test. In human neuropsychology, tests that generate scores like this are a key part of assessing patients. The evidence supporting the validity of the AI scoring system is compelling. This represents a valuable step towards evaluating more complex neurobehavioral functions.

      However, one area where the study could be strengthened is in the explainability of the AI methods used. To ensure the scores are fully transparent and consistent for clinical use, it will be important for future work to test the robustness of the approach, potentially by comparing multiple methods. Examining other latent variables that can explain patients' cognitive functioning would also be informative.

      In summary, I believe this study provides an important proof-of-concept with compelling evidence, while also highlighting key areas for further development as this technology moves towards real-world clinical applications.

    1. Reviewer #1 (Public review):

      Summary:

      This study investigated how traumatic brain injury affects oscillatory and single-unit hippocampal activity in awake-behaving rats.

      Strengths:

      The use of high-density laminar electrodes enabled precise localization of recording sites. To ensure an unbiased, rigorous approach, single-unit analysis was performed by a reviewer who was blind to experimental conditions. A proof of concept study was undertaken to characterize the pathology that resulted from the specific TBI model used in the main study. There was an effort to link abnormalities in hippocampal activity to memory disruption by running a cohort of rats on the Morris Water Maze task.

      Weaknesses:

      The paper is written as if the experiment was exploratory and not hypothesis-driven despite the fact that there is a wealth of experimental evidence about this TBI model that could have informed very specific predictions to test a hypothesis that is only hinted at in the discussion. The number of rats used for the spatial working memory experiment is not reported. Some of the statistics are not completely reported. It is also unclear what the rationale was for recording single units in a novel and familiar environment. Furthermore, this analysis comparing single-unit activity between familiar and novel environments is quite rudimentary. There are much more rigorous analyses to answer the question of how hippocampal single-unit firing patterns differ across changes in environments. There are details lacking about the number of units recorded per session and per rat, all of which are usually reported in studies that record single units. Spatial working memory assessment is delegated to a single panel of a supplementary figure. More importantly, there is no effort to dissociate between spatial working memory deficits and other motor, motivational, or sensory deficits that could have been driving the lower "memory score" in the experimental group.

    1. Joint Public Review

      Summary:

      The authors sought to elucidate the mechanism by which infections increase sleep in Drosophila. Their work is important because it further supports the idea that the blood-brain barrier is involved in brain-body communication, and because it advances the field of sleep research. Using knock-down and knock-out of cytokines and cytokine receptors specifically in the endocrine cells of the gut (cytokines) as well as in the glia forming the blood-brain barrier (BBB) (cytokines receptors), the authors show that cytokines, upd2 and upd3, secreted by entero-endocrine cells in response to infections increase sleep through the Dome receptor in the BBB. They also show that gut-derived Allatostatin (Alst) A promotes wakefulness by inhibiting Alst A signaling that is mediated by Alst receptors expressed in BBB glia. Their results suggest there may be additional mechanisms that promote elevated sleep during gut inflammation.<br /> The authors suggest that upd3 is more critical than upd2, which is not sufficiently addressed or explained. In addition, the study uses the gut's response to reactive oxygen molecules as a proxy for infection, which is not sufficiently justified. Finally, further verification of some fundamental tools used in this paper would further solidify these findings making them more convincing.

      Strengths:

      (1) The work addresses an important topic and proposes an intriguing mechanism that involves several interconnected tissues. The authors place their research in the appropriate context and reference related work, such as literature about sickness-induced sleep, ROS, the effect of nutritional deprivation on sleep, sleep deprivation and sleep rebound, upregulated receptor expression as a compensatory mechanism in response to low levels of a ligand, and information about Alst A.

      (2) The work is, in general, supported by well-performed experiments that use a variety of different tools, including multiple RNAi lines, CRISPR, and mutants, to dissect both signal-sending and receiving sides of the signaling pathway.

      (3) The authors provide compelling evidence that shows that endocrine cells from the gut are the source of the upd cytokines that increase daytime sleep, that the glial cells of the BBB are the targets of these upds, and that upd action causes the downregulation of Alst receptors in the BBB via the Jak/Stat pathways.

      Weaknesses:

      (1) There is a limited characterization of cell types in the midgut which are classically associated with upd cytokine production.

      (2) Some of the main tools used in this manuscript to manipulate the gut while not influencing the brain (e.g., Voilà and Voilà + R57C10-GAL80), are not directly shown to not affect gene expression in the brain. This is critical for a manuscript delving into intra-organ communication, as even limited expression in the brain may lead to wrong conclusions.

      (3) The model of gut inflammation used by the authors is based on the increase in reactive oxygen species (ROS) obtained by feeding flies food containing 1% H2O2. The use of this model is supported by the authors rather weakly in two papers (refs. 26 and 27 ): The paper by Jiang et al. (ref. 26) shows that the infection by Pseudomonas entomophila induces cytokine responses upd2 and 3, which are also induced by the Jnk pathway. In addition, no mention of ROS could be found in Buchon et al. (ref 27); this is a review that refers to results showing that ROS are produced by the NADPH oxidase DUOX as part of the immune response to pathogens in the gut. Thus, there is no strong support for the use of this model.

      (4) Likewise, there is no support for the use of ROS in the food instead a direct infection by pathogenic bacteria. Furthermore, it is known that ROS damages the gut epithelium, which in turn induces the expression of the cytokines studied. Thus the effects observed may not reflect the response to infection. In addition, Majcin Dorcikova et al. (2023). Circadian clock disruption promotes the degeneration of dopaminergic neurons in male Drosophila. Nat Commun. 2023 14(1):5908. doi: 10.1038/s41467-023-41540-y report that the feeding of adult flies with H2O2 results in neurodegeneration if associated with circadian clock defects. Thus, it would be important to discuss or present controls that show that the feeding of H2O2 does not cause neuronal damage.

      (5) The novelty of the work is difficult to evaluate because of the numerous publications on sleep in Drosophila. Thus, it would be very helpful to read from the authors how this work is different and novel from other closely related works such as: Li et al. (2023) Gut AstA mediates sleep deprivation-induced energy wasting in Drosophila. Cell Discov. 23;9(1):49. doi: 10.1038/s41421-023-00541-3.

    1. Reviewer #1 (Public review):

      Summary:

      This study represents valuable insight into the potential contribution of ciliation deficits and cholinergic neuron survival in an etiologically appropriate Parkinson's disease mouse model. The evidence presented is convincing, employing a validated methodology to assess measures across multiple brain regions and time points, with adequate observation numbers. Similarities between some of the data here and human patients further validate the model, and the study provides numerous avenues to aid future advances.

      Strengths:

      Overall, this study presents a thorough analysis of ciliary defects and cell loss in cholinergic neurons throughout the brain in the LRRK2 G2019S knockin mouse model of Parkinson's disease. The authors aimed to characterize ciliary defects in areas not only implicated in PD but also in cholinergic neuron function. Additionally, they repeated measures across age and sex, presenting a body of work that is more readily translatable to human disease states. The strengths of the paper included the breadth of brain regions tested and additional mechanistic contributions of LRRK2 that may correlate to their observed phenotypes. The study conveys to the reader the ciliary phenotype observed in all the cholinergic neurons assessed throughout the brains of knock-in LRRK2 mutant mice. Importantly, the pattern of changes is, in some instances, strikingly similar to PD, which strengthens the case for construct and face validation of the G2019S knock-in mouse model. Future investigations of the physiological and behavioural correlates/consequences of these changes will inform ongoing and, as yet untried, therapeutic intervention attempts.

      Weaknesses:

      At times, the claims are only partially substantiated by how the data are presented (e.g., inappropriate statistics within an age (t-tests, not ANOVA) and a lack of comparison between ages (despite referring to the progress of a phenotype). More appropriate statistical analyses and revisions to the data presentation are required to substantiate basic and more 'progressive' conclusions. Further, distributing the central claim over 10 figures dilutes the impact, many of which could be compressed into a couple of single figures (e.g., cell counts in all regions and ciliation). Also, a summary graphic showing the brain regions affected by ciliation alterations and cell loss at young, middle, and old age in the GS mice would be hugely beneficial. This peer would like to see more discussion of how the observed changes would impact circuit-level function and more speculation of the underlying mechanisms leading to the deficits. Minor changes to the abstract and introduction (to include more detail in the rationale and supporting evidence) are recommended, as summaries of existing literature are vague and could flow better between one statement and the next.

    1. Reviewer #1 (Public review):

      The manuscript by Griesius et al. addresses the dendritic integration of synaptic input in cortical GABAergic interneurons (INs). Dendritic properties, passive and active, of principal cells have been extensively characterized, but much less is known about the dendrites of INs. The limited information is particularly relevant in view of the high morphological and physiological diversity of IN types. The few studies that investigated IN dendrites focused on parvalbumin-expressing INs. In fact, in a previous study, the authors examined dendritic properties of PV INs, and found supralinear dendritic integration in basal, but not in apical dendrites (Cornford et al., 2019 eLife).

      In the present study, complementary to the prior work, the authors investigate whether dendrite-targeting IN types, NDNF-expressing neurogliaform cells, and somatostatin(SOM)-expressing O-LM neurons, display similar active integrative properties by combining clustered glutamate-uncaging and pharmacological manipulations with electrophysiological recording and calcium imaging from genetically identified IN types in mouse acute hippocampal slices.

      The main findings are that NDNF IN dendrites show strong supralinear summation of spatially- and temporally-clustered EPSPs, which is changed into sublinear behavior by bath application of NMDA receptor antagonists, but not by Na+-channel blockers. L-type calcium channel blockers abolished the supralinear behavior associated calcium transients but had no or only weak effect on EPSP summation. SOM IN dendrites showed similar, albeit weaker NMDA-dependent supralinear summation, but no supralinear calcium transients were detected in these INs. In summary, the study demonstrates that different IN types are endowed with active dendritic integrative mechanisms, but show qualitative and quantitative divergence in these mechanisms.

      While the research is conceptionally not novel, it constitutes an important incremental gain in our understanding of the functional diversity of GABAergic INs. In view of the central roles of IN types in network dynamics and information processing in the cortex, results and conclusions are of interest to the broader neuroscience community.

      The experiments are well designed, and closely follow the approach from the previous publication in parts, enabling direct comparison of the results obtained from the different IN types. The data is convincing and the conclusions are well-supported, and the manuscript is very well-written.

      I see only a few open questions and some inconsistencies in the presentation of the data in the figures (see details below).

    1. Reviewer #1 (Public review):

      Summary:

      This study provides convincing evidence on the infraslow oscillation of DG cells during NREM sleep, and how serotonergic innervation modulates hippocampal activity pattern during sleep and memory.

      Strengths and Weaknesses:

      The authors used state-of-the-art techniques to carry out these experiments. Given that the functional role of infraslow rhythm still remains to be studied, this study provides convincing evidence of the role of DG cells in regulating infraslow rhythm, sleep microarchitecture, and memory.

      I have a few minor comments.

      (1) Decreased infraslow rhythm during NREMs in the 5ht1a KO mice is striking. It would be helpful to know whether sleep-wake states, MAs, and transitions to REMs are changed.

      (2) It would be interesting to discuss whether the magnitude in changes of infraslow rhythm strength is correlated with memory performance (Figure 6).

      (3) The authors should cite the Oikonomou Neuron paper that describes slow oscillatory activity of DRN SERT neurons during NREM sleep.

      (4) The authors should clarify how they define the phasic pattern of the photometry signal.

    1. Reviewer #1 (Public review):

      Summary:

      This study is an important follow-up to their prior work - Wong et al. (2019), starting with clear questions and hypotheses, followed by a series of thoughtful and organized experiments. The method and results are convincing. Experiment 1 demonstrated the sensory preconditioned fear with few (8) or many (32) sound-light pairings. Experiments 2A and 2B showed the role of PRh NMDA receptors during conditioning for online integration, revealing that this contribution is present only after a few sound-light pairings, not after many sound-light pairings. Experiments 3A and 3B showed the contribution of PRh-BLA communication to online integration, again only after a few but not after many. Contrary to Experiments 3A and 3B, Experiments 4A and 4B showed the contribution of PRh-BLA communication to integration at test only after many but not few sound-light pairings.

      Strengths:

      Throughout the manuscript, the methods and results are clearly organized and described, and the use of statistics is solid, all contributing to the overall clarity of the research. The discussion section was also well-written, effectively comparing the current research with the prior work and offering insightful interpretations and potential future directions for this line of research. I have only a limited amount of concerns about some results and some details of experiments/statistics.

      Weaknesses:

      Could you provide further interpretation regarding line 171: the observation that sensory preconditioned fear increased with the number of sound-light pairings? Was this increase due to better sound-light association learning during Stage 1? Additionally, were there any experimental differences between Experiment 1 and the other experiments that might explain why freezing was higher in the P32 group compared to the P8 group? This pattern seemed to be absent in the other experiments. If we consider the hypothesis that the online integration mechanism is more active with fewer pairings and the chaining mechanism at the test is more prominent with many pairings, we wouldn't expect a difference between the P8 and P32 groups. Given the relatively small sample size in Experiment 1, the authors might consider conducting a cross-experiment analysis or something similar to investigate this further.

    1. Reviewer #1 (Public review):

      Summary:

      The behavioral strategies underlying decisions based on perceptual evidence are often studied in the lab with stimuli whose elements provide independent pieces of decision-related evidence that can thus be equally weighted to form a decision. In more natural scenarios, in contrast, the information provided by these pieces is often correlated, which impacts how they should be weighted. Tardiff, Kang & Gold set out to study decisions based on correlated evidence and compare the observed behavior of human decision-makers to normative decision strategies. To do so, they presented participants with visual sequences of pairs of localized cues whose location was either uncorrelated, or positively or negatively correlated, and whose mean location across a sequence determined the correct choice. Importantly, they adjusted this mean location such that, when correctly weighted, each pair of cues was equally informative, irrespective of how correlated it was. Thus, if participants follow the normative decision strategy, their choices and reaction times should not be impacted by these correlations. While Tardiff and colleagues found no impact of correlations on choices, they did find them to impact reaction times, suggesting that participants deviated from the normative decision strategy. To assess the degree of this deviation, Tardiff et al. adjusted drift-diffusion models (DDMs) for decision-making to process correlated decision evidence. Fitting these models to the behavior of individual participants revealed that participants considered correlations when weighing evidence, but did so with a slight underestimation of the magnitude of this correlation. This finding made Tardiff et al. conclude that participants followed a close-to-normative decision strategy that adequately took into account correlated evidence.

      Strengths:

      The authors adjust a previously used experimental design to include correlated evidence in a simple, yet powerful way. The way it does so is easy to understand and intuitive, such that participants don't need extensive training to perform the task. Limited training makes it more likely that the observed behavior is natural and reflective of everyday decision-making. Furthermore, the design allowed the authors to make the amount of decision-related evidence equal across different correlation magnitudes, which makes it easy to assess whether participants correctly take account of these correlations when weighing evidence: if they do, their behavior should not be impacted by the correlation magnitude.

      The relative simplicity with which correlated evidence is introduced also allowed the authors to fall back to the well-established DDM for perceptual decisions, which has few parameters, is known to implement the normative decision strategy in certain circumstances, and enjoys a great deal of empirical support. The authors show how correlations ought to impact these parameters, and which changes in parameters one would expect to see if participants mis-estimate these correlations or ignore them altogether (i.e., estimate correlations to be zero). This allowed them to assess the degree to which participants took into account correlations on the full continuum from perfect evidence weighting to complete ignorance. With this, they could show that participants in fact performed rational evidence weighting if one assumed that they slightly underestimated the correlation magnitude.

      Weaknesses:

      The experiment varies the correlation magnitude across trials such that participants need to estimate this magnitude within individual trials. This has several consequences:

      (1) Given that correlation magnitudes are estimated from limited data, the (subjective) estimates might be biased towards their average. This implies that, while the amount of evidence provided by each 'sample' is objectively independent of the correlation magnitude, it might subjectively depend on the correlation magnitude. As a result, the normative strategy might differ across correlation magnitudes, unlike what is suggested in the paper. In fact, it might be the case that the observed correlation magnitude underestimates corresponds to the normative strategy.

      (2) The authors link the normative decision strategy to putting a bound on the log-likelihood ratio (logLR), as implemented by the two decision boundaries in DDMs. However, as the authors also highlight in their discussion, the 'particle location' in DDMs ceases to correspond to the logLR as soon as the strength of evidence varies across trials and isn't known by the decision maker before the start of each trial. In fact, in the used experiment, the strength of evidence is modulated in two ways:<br /> (i) by the (uncorrected) distance of the cue location mean from the decision boundary (what the authors call the evidence strength) and<br /> (ii) by the correlation magnitude. Both vary pseudo-randomly across trials, and are unknown to the decision-maker at the start of each trial. As previous work has shown (e.g. Kiani & Shadlen (2009), Drugowitsch et al. (2012)), the normative strategy then requires averaging over different evidence strength magnitudes while forming one's belief. This averaging causes the 'particle location' to deviate from the logLR. This deviation makes it unclear if the DDM used in the paper indeed implements the normative strategy, or is even a good approximation to it.

      Given that participants observe 5 evidence samples per second and on average require multiple seconds to form their decisions, it might be that they are able to form a fairly precise estimate of the correlation magnitude within individual trials. However, whether this is indeed the case is not clear from the paper.

      Furthermore, the authors capture any underestimation of the correlation magnitude by an adjustment to the DDM bound parameter. They justify this adjustment by asking how this bound parameter needs to be set to achieve correlation-independent psychometric curves (as observed in their experiments) even if participants use a 'wrong' correlation magnitude to process the provided evidence. Curiously, however, the drift rate, which is the second critical DDM parameter, is not adjusted in the same way. If participants use the 'wrong' correlation magnitude, then wouldn't this lead to a mis-weighting of the evidence that would also impact the drift rate? The current model does not account for this, such that the provided estimates of the mis-estimated correlation magnitudes might be biased.

      Lastly, the paper makes it hard to assess how much better the participants' choices would be if they used the correct correlation magnitudes rather than underestimates thereof. This is important to know, as it only makes sense to strictly follow the normative strategy if it comes with a significant performance gain.

    1. rogorc raodenobrivi, aseve Tvisebrivikvlevis Sedegebi miuTiTebs, rom poli-tikaSi CarTul qalTa mimarT ZaladobasaqarTveloSi farTod gavrcelebulifenomenia, romelic misi gavrcelebu-lobis da simwvavis miuxedavad, iSviaTadxdeba sajaro ganxilvis sagani. kvlevaaCvenebs, rom problemis aRiareba da sa-Tanado ganxilva mniSvnelovnad aZlie-rebs im qalebs, romlebic am problemazesaubars ver bedaven. amitom rekomende-bulia, rom politikaSi CarTul qalTamimarT Zaladobis sakiTxs saTanado yu-radReba miaqcion, rogorc xelisufle-bis da politikuri partiebis warmomad-genlebma, aseve centralurma saarCevnokomisiam da mediam.2 da regularuladganixilon igi , rogorc centraluri daadgilobrivi xelisuflebis da poli-tikuri partiebis doneze, aseve sajarodebatebSi, gansakuTrebiT, winasaarCevnoperiodSi, rodesac Zaladobis riskebikidev ufro maRalia

      ვფიქრობ,რომ პოლიტიკში ჩართულ ქალთა მიმართ ძალადობის პრევენციისათვის მართლც აუცილებელია მსგავსი გამოკითხვები,კვლევები თუ სხვა სახის ღონისძიებები,რადგნ ეს მამოტივირებელია პლიტიკაში ჩართული ქალებისთვის,რომლებიც საკუთარ პრობლემებზე ღიად ვერ საუბრობენ

    1. Reviewer #1 (Public review):

      The manuscript by Chen et al. investigated the interaction between CHI3L1, a chitinase-like protein in the 18 glycosyl hydrolase family, and gut bacteria in the mucosal layers. The authors provided evidence to document the direct interaction between CHI3L1 and peptidoglycan, a major component of bacterial cell wall. Doing so, Chi3l1 produced by gut epithelial cells regulates the balance of gut microbiome and diminishes DSS-induced colitis, potentially through the colonization of protective gram-positive bacteria such as lactobacillus.

      The study is the first to systemically document the interactions between Chi3L1 and microbiome. Convincing data were shown to characterize the imbalance of gram-positive bacteria in the newly generated gut epithelial-specific Chi3L1 deficient mice. Comprehensive FMT experiments were performed to demonstrate the contributions of gut microbiome using the mouse colitis model. The manuscript is strengthened by additional mechanistic studies concerning the binding between Chi3l1 and peptidoglycan, and discussions on existing body of literature demonstrating that detrimental roles of Chi3l1 in mouse IBD model, which conflict with the current study.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript the authors investigate the contributions of the long noncoding RNA snhg3 in liver metabolism and MAFLD. The authors conclude that liver-specific loss or overexpression of Snhg3 impacts hepatic lipid content and obesity through epigenetic mechanisms. More specifically, the authors invoke that nuclear activity of Snhg3 aggravates hepatic steatosis by altering the balance of activating and repressive chromatin marks at the Pparg gene locus. This regulatory circuit is dependent on a transcriptional regulator SNG1.

      Strengths:

      The authors developed a tissue specific lncRNA knockout and KI models. This effort is certainly appreciated as few lncRNA knockouts have been generated in the context of metabolism. Furthermore, lncRNA effects can be compensated in a whole organism or show subtle effects in acute versus chronic perturbation, rendering the focus on in vivo function important and highly relevant. In addition, Snhg3 was identified through a screening strategy and as a general rule the authors the authors attempt to follow unbiased approaches to decipher the mechanisms of Snhg3.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript nicely outlines a conceptual problem with the bFAC model in A-motility, namely, how the energy derived from the inner membrane AglRQS motor transduced through the cell wall into mechanical force on the cell surface to drive motility? To address this, the authors make a significant contribution by identifying and characterizing a lytic transglycosylase (LTG) called AgmT. This work thus provides clues and a future framework work to address mechanical force transmission from the cytoplasm through the cell envelope to the cell surface.

      Strengths:

      (i) Convincing evidence shows AgmT functions as a LTG and, surprisingly, that mltG from E. coli complements the swarming defect of an agmT mutant.

      (ii) Show 13 other LTGs found in M. xanthus are not required for A-motility.

      (iii) Authors show agmT mutants develop morphological changes in response to treatment with a beta-lactam antibiotic, mecillinam.

      (iv) The use of single molecule tracking to monitor the assembly and dynamics of bFACs in WT and mutant backgrounds.

      (v) The authors understand the limitations of their work and do not overinterpret their data.

      Weaknesses:

      The authors provided more experiments and clearly addressed my prior concerns in their revised manuscript.

    1. Reviewer #1 (Public review):

      The authors present data on outer membrane vesicle (OMV) production in different mutants, but they state that this is beyond the scope of the current manuscript, which I disagree with. This data could provide valuable physiological context that is otherwise lacking. The preliminary blots suggest that YafK does not alter OMV biogenesis. I recommend repeating these blots with appropriate controls, such as blotting for proteins in the culture media, an IM protein, periplasmic protein and an OM protein to strengthen the reliability of these findings. Including this data in the manuscript, even if it does not directly support the initial hypothesis, would enhance the physiological relevance of the study. Currently, the manuscript relies completely on the experimental setup (labeling-mass spec) previously developed by the authors, which limits the broader scope and interpretability of this study.

      Additionally susceptibility of strains to detergents like SDS can be tested to provide a much needed physisological context to the study.

      In summary, the authors should consider revising the manuscript to improve clarity, substantiate their claims with more detailed evidence, and include additional experimental results that provide necessary physiological context to their study.

      Comments on the revised version:

      Regarding my comments from last review on a new figure on OMV analysis, The authors have redirected me to their previous response and have not performed the suggested control blots. I do not get their argument that this is for specialized audience. I do not have any more comments.

    1. Reviewer #1 (Public review):

      Summary:

      An investigation of the dynamics of a neural network model characterized by sparsely connected clusters of neuronal ensembles. The authors found that such a network could intrinsically generate sequence preplay and place maps, with properties like those observed in the real-world data.

      Strengths:

      Computational model and data analysis supporting the hippocampal network mechanisms underlying sequence preplay of future experiences and place maps.<br /> The revised version of the manuscript addressed all my comments and as a result is significantly improved.

      Weaknesses:

      None noted

    1. Reviewer #1 (Public review):

      Summary:

      Here, the authors propose that changes in m6A levels may be predictable via a simple model that is based exclusively on mRNA metabolic events. Under this model, m6A mRNAs are "passive" victims of RNA metabolic events with no "active" regulatory events needed to modulate their levels by m6A writers, readers, or erasers; looking at changes in RNA transcription, RNA export, and RNA degradation dynamics is enough to explain how m6A levels change over time.

      The relevance of this study is extremely high at this stage of the epi transcriptome field. This compelling paper is in line with more and more recent studies showing how m6A is a constitutive mark reflecting overall RNA redistribution events. At the same time, it reminds every reader to carefully evaluate changes in m6A levels if observed in their experimental setup. It highlights the importance of performing extensive evaluations on how much RNA metabolic events could explain an observed m6A change.

      Weaknesses:

      It is essential to notice that m6ADyn does not exactly recapitulate the observed m6A changes. First, this can be due to m6ADyn's limitations. The authors do a great job in the Discussion highlighting these limitations. Indeed, they mention how m6ADyn cannot interpret m6A's implications on nuclear degradation or splicing and cannot model more complex scenario predictions (i.e., a scenario in which m6A both impacts export and degradation) or the contribution of single sites within a gene.

      Secondly, since predictions do not exactly recapitulate the observed m6A changes, "active" regulatory events may still play a partial role in regulating m6A changes. The authors themselves highlight situations in which data do not support m6ADyn predictions. Active mechanisms to control m6A degradation levels or mRNA export levels could exist and may still play an essential role.

      (1) "We next sought to assess whether alternative models could readily predict the positive correlation between m6A and nuclear localization and the negative correlations between<br /> m6A and mRNA stability. We assessed how nuclear decay might impact these associations by introducing nuclear decay as an additional rate, δ. We found that both associations were robust to this additional rate (Supplementary Figure 2a-c)."<br /> Based on the data, I would say that model 2 (m6A-dep + nuclear degradation) is better than model 1. The discussion of these findings in the Discussion could help clarify how to interpret this prediction. Is nuclear degradation playing a significant role, more than expected by previous studies?

      (2) The authors classify m6A levels as "low" or "high," and it is unclear how "low" differs from unmethylated mRNAs.

      (3) The authors explore whether m6A changes could be linked with differences in mRNA subcellular localization. They tested this hypothesis by looking at mRNA changes during heat stress, a complex scenario to predict with m6ADyn. According to the collected data, heat shock is not associated with dramatic changes in m6A levels. However, the authors observe a redistribution of m6A mRNAs during the treatment and recovery time, with highly methylated mRNAs getting retained in the nucleus being associated with a shorter half-life, and being transcriptional induced by HSF1. Based on this observation, the authors use m6Adyn to predict the contribution of RNA export, RNA degradation, and RNA transcription to the observed m6A changes. However:

      (a) Do the authors have a comparison of m6ADyn predictions based on the assumption that RNA export and RNA transcription may change at the same time?

      (b) They arbitrarily set the global reduction of export to 10%, but I'm not sure we can completely rule out whether m6A mRNAs have an export rate during heat shock similar to the non-methylated mRNAs. What happens if the authors simulate that the block in export could be preferential for m6A mRNAs only?

      (c) The dramatic increase in the nucleus: cytoplasmic ratio of mRNA upon heat stress may not reflect the overall m6A mRNA distribution upon heat stress. It would be interesting to repeat the same experiment in METTL3 KO cells. Of note, m6A mRNA granules have been observed within 30 minutes of heat shock. Thus, some m6A mRNAs may still be preferentially enriched in these granules for storage rather than being directly degraded. Overall, it would be interesting to understand the authors' position relative to previous studies of m6A during heat stress.

      (d) Gene Ontology analysis based on the top 1000 PC1 genes shows an enrichment of GOs involved in post-translational protein modification more than GOs involved in cellular response to stress, which is highlighted by the authors and used as justification to study RNA transcriptional events upon heat shock. How do the authors think that GOs involved in post-translational protein modification may contribute to the observed data?

      (e) Additionally, the authors first mention that there is no dramatic change in m6A levels upon heat shock, "subtle quantitative differences were apparent," but then mention a "systematic increase in m6A levels observed in heat stress". It is unclear to which systematic increase they are referring to. Are the authors referring to previous studies? It is confusing in the field what exactly is going on after heat stress. For instance, in some papers, a preferential increase of 5'UTR m6A has been proposed rather than a systematic and general increase.

    1. Reviewer #1 (Public review):

      Summary:

      The study shows that Zizyphi spinosi semen (ZSS), particularly its non-extracted simple crush powder, has significant therapeutic effects on neurodegenerative diseases. It removes Aβ, tau, and α-synuclein oligomers, restores synaptophysin levels, enhances BDNF expression and neurogenesis, and improves cognitive and motor functions in mouse AD, FTD, DLB, and PD models. Additionally, ZSS powder reduces DNA oxidation and cellular senescence in normal-aged mice, increases synaptophysin, BDNF, and neurogenesis, and enhances cognition to levels comparable to young mice.

      Weaknesses:

      (1) While the study demonstrates that ZSS has protective effects across a wide range of animal models, including AD, FTD, DLB, PD, and both young and aged mice, it is broad and lacks a detailed investigation into the underlying mechanisms. This is the most significant concern.

      (2) The authors highlight that the non-extracted simple crush powder of ZSS shows more substantial effects than its hot water extract and extraction residue. However, the manuscript provides very limited data comparing the effects of these three extracts.

      (3) The authors have not provided a rationale for the dosing concentrations used, nor have they tested the effects of the treatment in normal mice to verify its impact under physiological conditions.

      (4) Regarding the assessment of cognitive function in mice, the authors only utilized the Morris Water Maze (MWM) test, which includes a five-day spatial learning training phase followed by a probe trial. The authors focused solely on the learning phase. However, it is relevant to note that data from the learning phase primarily reflects the learning ability of the mice, while the probe trial is more indicative of memory. Therefore, it is essential that probe trial data be included for a more comprehensive analysis. A justification should be included to explain why the latency of 1st is about 50s not 60s.

      (5) The BDNF immunohistochemical staining in the manuscript appears to be non-specific.

      (6) The central pathological regions in PD are the substantia nigra and striatum. Please replace the staining results from the cortex and hippocampus with those from these regions in the PD model.

    1. Reviewer #1 (Public Review):

      In this study, Yang et al. investigated the locations and hierarchies of NFATc1+ and PDGFRα+ cells in dental and periodontal mesenchyme. By combining intersectional and exclusive reporters, they attempted to distinguish among NFATc1+PDGFRα+, NFATc1+PDGFRα-, and NFATc1- PDGFRα+ cells. Using tissue clearing and serial section-based 3D reconstruction, they mapped the distribution atlas of these cell populations. Through DTA-induced ablation of PDGFRα+ cells, they demonstrated the crucial role of PDGFRα+ cells in the formation of the odontoblast cell layer and periodontal components.

      Main issues:

      (1) The authors did not quantify the contribution of PDGFRα+ cells or NFATc1+ cells to dental and periodontal lineages in PDGFRαCreER; Nfatc1DreER;LGRT mice. Zsgreen+ cells represented PDGFRα+ cells and their lineages. Tomato+ cells represented NFATc1+ cells and their lineages. Tomato+Zsgreen+ cells represented NFATc1+PDGFRα+ cells and their lineages. Conducting immunostaining experiments with lineage markers is essential to determine the physiological contributions of these cells to dental and periodontal homeostasis.

      (2) The authors attempted to use PDGFRαCreER; Nfatc1DreER;IR1 mice to illustrate the hierarchies of NFATc1+ and PDGFRα+ cells. According to the principle of the IR1 reporter, it requires sequential induction of PDGFRα-CreER and Nfatc1-DreER to investigate their genetic relationship. Upon induction by tamoxifen, NFATc1+PDGFRα- cells and NFATc1-PDGFRα+ cells were labeled by Tomato and Zsgreen, respectively. However, the reporter expression of NFATc1+PDGFRα+ cells was uncertain, most likely random. Therefore, the hierarchical relationship of NFATc1+ and PDGFRα+ cells cannot be reliably determined from PDGFRαCreER; Nfatc1DreER; IR1 mice.

    1. Reviewer #1 (Public review):

      Summary:

      This paper presents a data processing pipeline to discover causal interactions from time-lapse imaging data, and convicingly illustrates it on a challenging application for the analysis of tumor-on-chip ecosystem data.

      The core of the discovery module is the original tMIIC method of the authors, which is shown in supplementary material to compare favourably to two state-of-the-art methods on synthetic temporal data on a 15 nodes network.

      Strengths:

      This paper tackles the problem of learning causal interactions from temporal data which is an open problem in presence of latent variables.

      The core of the method tMIIC of the authors is nicely presented in connection to Granger-Schreiber causality and to the novel graphical conditions used to infer latent variables and based on a theorem about transfer entropy.

      tMIIC compares favourably to PC and PCMCI+ methods using different kernels on synthetic datasets generated from a network of 15 nodes.

      A full application to tumor-on-chip cellular ecosystems data including cancer cells, immune cells, cancer-associated fibroblasts, endothelial cells and anti cancer drugs, with convincing inference results with respect to both known and novel effects between those components and their contact.

      The code and dataset are available online for the reproducibility of the results.

      Weaknesses:

      The references to "state-of-the-art methods" concerning the inference of causal networks should be more precise by giving citations in the main text, and better discussed in general terms, both in the first section and in the section of presentation of CausalXtract. It is only in the legend of the figures of the supplementary material that we get information.

      Of course, comparison on our own synthetic datasets can always be criticized but this is rather due to the absence of common benchmark and I would recommend the authors to explicitly propose their datasets as benchmark to the community.

    1. Reviewer #1 (Public Review):

      Summary:<br /> Both flies and mammals have D1-like and D2-like dopamine receptors, yet the role of D2-like receptors in Drosophila learning and memory remains underexplored. The paper by Qi et al. investigates the role of the D2-like dopamine receptor D2R in single pairs of dopaminergic neurons (DANs) during single-odor aversive learning in the Drosophila larva. First, they use confocal imaging to screen driver strains with expression in only single pairs of dopaminergic neurons. Next, they use thermogenetic manipulations of one pair of DANs (DAN-c1) to implicate DAN-c1 activity during larval aversive learning. They then use confocal imaging to demonstrate expression of D2R in the DANs and mushroom body of the larval brain. Finally, they show that optogenetic activation during training phenocopies D2R knockdown in these neurons: aversive learning is impaired when DAN-c1 is targeted, while appetitive and aversive learning are impaired when the mushroom body is manipulated. Qi et al. thus propose a model in which D2R limits excessive dopamine release to facilitate successful olfactory learning.

      Strengths:<br /> The paper reproduces prior findings by Qi and Lee (2014), which demonstrated that D2R knockdown in DL1 DANs or the mushroom body impairs aversive olfactory learning in Drosophila larvae. The authors extended this previous work by screening 57 GAL4 drivers to identify tools that drive expression in individual DANs and used one of the tools, the R76F02-AD; R55C10-DBD driver, to manipulate DAN-c1 neurons with greater specificity. They also show that GFP-tagged D2R is expressed in most DANs and the mushroom body. Although the authors only train larvae with a single odor, they demonstrate that driving D2R knockdown in DAN-c1 neurons impairs aversive learning, as do other loss-of-function manipulations of DAN-c1 neurons.

      Weaknesses:<br /> The authors claim to have identified drivers that label single DANs in Figure 1, but their confocal images in Figure S1 suggest that many of those drivers label additional neurons in the larval brain. It is also not clear why only some of the 57 drivers are displayed in Figure S1.<br /> Critically, R76F02-AD; R55C10-DBD labels more than one neuron per hemisphere in Figure S1c, and the authors cite Xie et al. (2018) to note that this driver labels two DANs in adult brains. Therefore, the authors cannot argue that the experiments throughout their paper using this driver exclusively target DAN-c1.<br /> Missing from the screen of 57 drivers is the driver MB320C, which typically labels only PPL1-γ1pedc in the adult and should label DAN-c1 in the larva. If MB320C labels DAN-c1 exclusively in the larva, then the authors should repeat their key experiments with MB320C to provide more evidence for DAN-c1 involvement specifically.<br /> The authors claim that the SS02160 driver used by Eschbach et al. (2020) labels other neurons in addition to DAN-c1. Could the authors use confocal imaging to show how many other neurons SS02160 labels? Given that both Eschbach et al. and Weber et al. (2023) found no evidence that DAN-c1 plays a role in larval aversive learning, it would be informative to see how SS02160 expression compares with the driver the authors use to label DAN-c1.<br /> The claim that DAN-c1 is both necessary and sufficient in larval aversive learning should be reworded. Such a claim would logically exclude any other neuron or even the training stimuli from being involved in aversive learning (see Yoshihara and Yoshihara (2018) for a detailed discussion of the logic), which is presumably not what the authors intended because they describe the possible roles of other DANs during aversive learning in the discussion.<br /> Moreover, if DAN-c1 artificial activation conveyed an aversive teaching signal irrespective of the gustatory stimulus, then it should not impair aversive learning after quinine training (Figure 2k). While the authors interpret Figure 2k (and Figure 5) to indicate that artificial activation causes excessive DAN-c1 dopamine release, an alternative explanation is that artificial activation compromises aversive learning by overriding DAN-c1 activity that could be evoked by quinine.<br /> The authors should not necessarily expect that D2R enhancer driver strains would reflect D2R endogenous expression, since it is known that TH-GAL4 does not label p(PAM) dopaminergic neurons. Their observations of GFP-tagged D2R expression could be strengthened with an anti-D2R antibody such as that used by Lam et al., (1999) or Love et al., (2023).<br /> Finally, the authors could consider the possibility other DANs may also mediate aversive learning via D2R. Knockdown of D2R in DAN-g1 appears to cause a defect in aversive quinine learning compared with its genetic control (Figure S4e). It is unclear why the same genetic control has unexpectedly poor aversive quinine learning after training with propionic acid (Figure S5a). The authors could comment on why RNAi knockdown of D2R in DAN-g1 does not similarly impair aversive quinine learning (Figure S5b).

    1. Reviewer #1 (Public review):

      ⍺-synuclein (syn) is a critical protein involved in many aspects of human health and disease. Previous studies have demonstrated that post-translational modifications (PTMs) play an important role in regulating the structural dynamics of syn. However, how post-translational modifications regulate syn function remains unclear. In this manuscript, Wang et al. reported an exciting discovery that N-acetylation of syn enhances the clustering of synaptic vesicles (SVs) through its interaction with lysophosphatidylcholine (LPC). Using an array of biochemical reconstitution, single vesicle imaging, and structural approaches, the authors uncovered that N-acetylation caused distinct oligomerization of syn in the presence of LPC, which is directly related to the level of SV clustering. This work provides novel insights into the regulation of synaptic transmission by syn and might also shed light on new ways to control neurological disorders caused by syn mutations.

    1. Reviewer #1 (Public review):

      The manuscript introduces a valuable and innovative non-AI computational method for segmenting noisy grayscale images, with a particular focus on identifying immunostained potassium ion channel clusters.

      Strengths:

      (1) Applicability and Usability: The method is exceptionally accessible to biologists and researchers without advanced computational expertise. It offers a highly practical alternative to AI-based methods, which often require significant training data and computational resources, making it an excellent choice for a broader range of laboratories.

      (2) Proof-of-Concept: The manuscript provides compelling evidence through multiple experiments, showcasing the method's superior performance over traditional threshold-based techniques, particularly in noisy environments. The dual immuno-electron microscopy experiments further reinforce the robustness and effectiveness of this approach.

      (3) Clarity and Methodology: The manuscript is exceptionally well-written, with clear and concise descriptions that effectively highlight the method's advantages. The detailed figures and comprehensive references greatly enhance the manuscript's credibility and strongly support the claims made.

      Weaknesses:

      The manuscript does not include comparisons with more advanced segmentation techniques, particularly those based on artificial intelligence. While the authors have provided a rationale for this decision, including such comparisons could have enriched the discussion and offered additional insights. Additionally, there are some concerns about the computational demands of the method, especially when applied to large-scale or 3D image analysis. Although the authors have shared some computational data, further optimization or practical recommendations would enhance the method's utility. Initially, the manuscript lacked a data and code availability statement, which could have limited the method's accessibility. However, this issue has since been resolved, with the code now being made available to the community. Lastly, while the findings related to Kv4.2 in the thalamus are noteworthy, they might achieve even greater impact if presented in a separate paper. Nevertheless, the authors have chosen to retain these results within the current manuscript to strengthen the overall narrative and relevance.

      We appreciate that the authors have provided thorough explanations for their original choices. These justifications offer a clearer understanding of their approach and the reasons behind the presentation of the data.

      Conclusion:

      The revised manuscript successfully addresses the majority of the reviewers' concerns, presenting a strong case for the proposed segmentation method. The method's ease of use for non-experts in AI, combined with its proven effectiveness in proof-of-concept experiments, positions it as a valuable addition to the field. While the manuscript could benefit from incorporating comparisons with more advanced segmentation methods and offering a more detailed discussion of computational requirements, it remains a robust contribution. The decision to include the Kv4.2 findings within the paper is well-justified by the authors, though these results could potentially have an even greater impact if published separately.

    1. Reviewer #2 (Public review):

      Summary:

      In this manuscript, Gonzalez Alam et al. sought to understand how memory interacts with incoming visual information to effectively guide human behavior by using a task that combines spatial contexts (houses) with objects of one or more other semantic categories. Three additional datasets (all from separate participants) were also employed: one that functionally localized regions of interest (ROIs) based on subtractions of different visually presented category types (in this case, scenes, objects, and scrambled objects); another consisting of resting-state functional connectivity scans, and a section of the Human Connectome Project that employed DTI data for structural connectivity analysis. Across multiple analyses, the authors identify dissociations between regions preferentially activated during scene or other object judgments, between the functional connectivity of regions demonstrating such preferences, and in the anatomical connectivity of these same regions. The authors conclude that the processing streams that take in visual information and support semantic or spatial processing are largely parallel and distinct.

      Strengths:

      (1) Recent work has reconceptualized the classic default mode network as parallel and interdigitated systems (e.g., Braga & Buckner, 2017; DiNicola et al., 2021). The current manuscript is timely in that it attempts to describe how information is differentially processed by two streams that appear to begin in visual cortex and connect to different default subnetworks. Even at a group level where neuroanatomy is necessarily blurred across individuals, these results provide clear evidence of stimulus-based processing dissociation.

      (2) The manuscript analyzes data from multiple independent datasets. It is therefore unlikely that a single experimenter choice in any given analysis would spuriously produce the general convergence of the results reported in this manuscript.

      Weaknesses:

      (1) The manuscript makes strong distinctions between spatial processing and other forms of semantic processing. However, it is not clear if scenes are uniquely different from other stimulus categories, such as faces or tools. As is noted by the authors in their revised discussion section, the design of the experiment does not allow for a category-level generalization beyond scenes. The dichotomization of semantic and spatial information invoked throughout the manuscript should be read with this limitation in mind.

      (2) Although the term "objects" is used by the authors to refer to the stimuli placed in scenes, it is a mixture of other stimulus categories, including various types of animals, tools, and other manmade objects. Different regions along the ventral stream are thought to process these different types of stimuli (e.g., Martin, 2007, Ann Rev Psychol), but as they are not being modeled separately, the responses associated with "object" processing in this manuscript are necessarily blurring across known distinctions in functional neuroanatomy.

    1. Reviewer #1 (Public Review):

      Summary:

      Here, the authors, Barber AG et al, developed a new mouse model and investigated an importance of Musashi-2 in lung cancer. Specifically, they found that Musashi-2 is important for lung cancer cells as it controls cancer cell growth, and also regulates several genes that also control cancer cell growth. Development of a new Musashi-2 mouse model is a plus, which confirmed Musashi-2 importance for lung cancer survival, and finding several genes that Musashi controls that are important for lung cancer growth. Additionally, they demonstrated that Musashi-2 overexpression which is tracked by GFP is preferred in lung adenocarcinoma cells. The data is rigorous and only minor revisions are requested.

      Strengths:

      Authors achieved their goals, by developing new Musashi-2 mouse model, confirming Musashi-2 importance for lung cancer survival, and finding several genes that Musashi controls that are important for lung cancer growth.

      Weaknesses:

      The findings of Musashi-2 mouse and human lung cancer growth control are not that novel as prior publication in 2016 showed that already, again, in both human and mouse models (Kudinov et al PNAS, PMID: 27274057), and also the authors missed the point of that paper which did use both miuse and human models to show impact on inbvasion and metastasis- both in vitro and in vivo. Additionally, another publication is currently under revisions recently also generated new Musashi-2 transgenic mouse model which confirmed Musashi-2 support of lung cancer growth (Bychkov I et al, PMID: 37398283; https://www.biorxiv.org/content/10.1101/2023.06.13.544756v1). Another weakness is that Musashi-2 cannot be effectively targeted and the new genes the authors found that Musashi-2 regulates are likely to be also difficult therapeutic targets. Therefore, impact of this new investigation is relatively modest in the field.

      Major suggestions:

      (1) Figure 3: it is unclear what is the efficiency of Msi2 deletion shRNA - could you demonstrate it by at least two independent methods? (QPCR, Western, or IHC?) please quantitate the data.

      (2) In Figure 4, similarly, it is unclear if Msi2 depletion was effective- and what is shRNA efficiency. Please test this by at least two independent methods (QPCR, Western, or IHC) and also please quantitate the data

      (3) the reason for impairment of cell growth demonstrated in Figs 3 and 4 is not clear: is it apoptosis? Necrosis? Cell cycle defects? Autophagy? Senescence? Please probe 2-3 possibilities and provide the data.

      (4) Since Musashi-1 is a Musashi-2 paralogue that could compensate for Musashi-2 loss, please test Msi1 expression levels in matching Fig 3 and Fig 4 sections (in cells/ tumors with Msi2 deletion and in KP cells with Msi2 shRNA). One method could suffice here.

      (5) It is not exactly clear why RNA-seq (as opposed to proteomics) was done to investigate downstream Msi2 targets (since Msi2 is in first place, translational and not transcriptional regulator)- . RNA effects in Fig 5J are quite modest, 2-fold or so. It would be useful (if antibodies available) to test four targets in Fig 5J by Western blot, to see any impact of musashi-2 depletion on those target protein levels. Indeed, several papers - including Kudinov et al PNAS, PMID: 27274057, Makhov P et al PMID: 33723247 and PMID: 37173995 - used proteomics/ RIP approaches and found direct Musashi-2 targets in lung cancer, including EGFR, and others.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors have conducted an exceptionally informative series of studies investigating the neural basis of interoception in transdiagnostic psychiatric symptoms. By comparing differential and overlapping neural activation during 'top-down' and 'bottom-up' interoceptive tasks, they reveal convergent activation largely localised to the ventral dysgranular subregion ('mid-insula'), which differs in extent between patients and controls, replicating and extending previous suggestions of this region as a central locus of disruption in psychiatric disorders. Their work also reveals different extents of divergent activation in the anterior insula during anticipation of interoceptive disruption. This substantially advances our previous knowledge of the anatomy of interoception, and confirms theoretical predictions of the roles of different cytoarchitectural subregions of the insula in interoceptive dysfunction in mental health conditions.

      Strengths:

      The work is exceptional in terms of breadth and depth, making use of multiple imaging and analysis techniques which are non-standard and go well beyond what is known today. The study is statistically well-powered and the tasks are well-validated in the literature. To my knowledge, these functions of the insula in interoception and mental health have never been compared directly before, so the results are novel and informative for both basic science and psychiatry. The work is strongly theory-driven, building on and directly testing results from influential theories and previous studies. It is likely that the results will strengthen our theoretical models of interoception and advance psychiatric studies of the insula.

      Weaknesses:

      The study has three limitations. (1) The interpretation of the resting-state isoproterenol data could potentially represent fluctuations over time rather than following interoception specifically; future studies should investigate test-retest reliability of this measure. Note this does not preclude the strong conclusions which can be drawn from the authors' task-based data. (2) The transdiagnostic patient sample was almost entirely female, and many were currently taking psychotropic medications; future studies should replicate these effects in unmedicated, sex-balanced samples (3) As the authors point out, there may have been task-specific preprocessing/analysis differences that influenced results, for example due to physiological correction in one but not both tasks; however, there are also merits to this analysis approach, such as comparability with previous studies.

    1. Reviewer #2 (Public Review):

      Summary:

      A deletion analysis of the MSL1 gene to assess how different parts of the protein product interact with the MSL2 protein and roX RNA to affect the association of the MSL complex with the male X chromosome of Drosophila was performed.

      Strengths:

      The deletion analysis of the MSL1 protein and the tests of interaction with MSL2 are adequate.

      Weaknesses:

      This reviewer does not adhere to the basic premise of the authors that the MSL complex is the primary mediator of dosage compensation of the X chromosome of Drosophila. Several lines of evidence from various laboratories indicate that it is involved in sequestering the MOF histone acetyltransferase to the X chromosome but there is a constraint on its action there. When the MSL complex is disrupted, there is no overall loss of compensation but there is an increase in autosomal expression. Sun et al (2013, PNAS 110: E808-817) showed that ectopic expression of MSL2 does not increase expression of the X and indeed inhibits the effect of acetylation of H4Lys16 on gene expression. Aleman et al (2021, Cell Reports 35: 109236) showed that dosage compensation of the X chromosome can be robust in the absence of the MSL complex. Together, these results indicate that the MSL complex is not the primary mediator of X chromosome dosage compensation. The authors state that an inverse dosage effect results from a titration of the histone acetylase MOF between the NSL and MSL complexes. This is a misunderstanding of the inverse effect, which is an imbalance of regulatory molecules as described in the citation below. The inverse effect operates in triple X metafemales to produce dosage compensation of the three X chromosomes and a reduced expression of the autosomes (Sun et al 2913 PNAS 110: 7383-7388). There is no MSL complex in metafemales.

      A detailed explanation was provided by Birchler and Veitia (2021, One Hundred Years of Gene Balance: How stoichiometric issues affect gene expression, genome evolution, and quantitative traits. Cytogenetics and Genome Research 161: 529-550). The relevant portions of that article that pertain to Drosophila are quoted below. The cited references can be found in that publication.

      "In Drosophila, the sex chromosomes consist of an X and a Y. The Y in this species contains only a few genes required for male fertility (Zhang et al., 2020). The X consists of approximately 20% of the genome. Thus, females have two X chromosomes and males have one. Muller (1932) found that the expression of genes between the two sexes was similar but when individual genes on the X were varied in dosage they exhibited a proportional dosage effect. Each copy in a male was expressed at about twice the level as each copy in a female. Females with three X chromosomes are highly inviable but when they do survive to the adult stage, Stern (1960) found that they too exhibited dosage compensation in that the expression in the triple X genotype was similar to normal females and males. Studies in triploid flies found that dosage compensation also occurred among X; AAA, XX;AAA, and XXX; AAA genotypes via upregulation of the Xs, where X indicates the dosage of the X and A indicates the triploid nature of the autosomes (see Birchler, 2016 for further discussion). Diploid and triploid females have a similar per gene expression but the other five genotypes each must modulate gene expression by different amounts equivalent to an inverse relationship between the X versus autosomal dosage to achieve a balanced expression between the X and the A (Birchler, 1996).

      Some years ago, mutations were sought in Drosophila that were lethal to males but viable in females. A number of such mutations were found and termed Male Specific Lethal (MSL) loci (Belote and Lucchesi, 1980). Once the products of these genes were identified, they were found to be at high concentrations on the male X chromosome (Kuroda et al., 1991). One of these genes encodes a histone acetyl transferase that acetylates Lysine16 of Histone H4 (Bone et al., 1994; Hilfiker et al., 1997). The recognition of the MSL complex and its association with the male X was an important set of contributions to an understanding of sex chromosome evolution in Drosophila (Kuroda et al., 2016). Thus, the hypothesis arose that the MSL complex accumulated this chromatin modifier on the male X to activate the expression about two-fold to bring about dosage compensation. Other data that contributed to this hypothesis were that when autoradiography of nascent transcription on salivary gland polytene chromosomes was examined in the MSL maleless mutation, the ratio of the number of grains over the X versus an autosomal region was reduced compared to the normal ratio (Belote and Lucchesi, 1980).

      It has been pointed out (Hiebert and Birchler, 1994; Bhadra et al., 1999; Pal Bhadra et al., 2005; Sun et al., 2013a; Birchler, 2016), however, that the grain counts over the X and the autosomes when considered in absolute terms rather than as a ratio show that the X more or less retained dosage compensation and the autosomal numbers are about doubled, i.e. exhibit an inverse dosage effect. The same situation occurs with the msl3 mutation (Okuno et al., 1984), another MSL gene, in that the autoradiographic grain numbers as an absolute measure show retention of X dosage compensation and an autosomal increase. The data treatment to produce an X to A ratio seemed reasonable in the context of the time when all regulation in eukaryotes was considered positive. However, when studies were conducted in such a manner as to assay the absolute effect on gene expression in the maleless mutation, in adults (Hiebert and Birchler, 1994), larvae (Hiebert and Birchler, 1994; Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005), and embryos (Pal Bhadra et al., 2005), the trend was for retention of dosage compensation of X linked genes and an increase in expression of autosomal genes.

      In global studies, if the X to autosomal expression does not change between mutant and normal, one can conclude that dosage compensation is operating. However, a lower X to A ratio could be a loss of compensation or an increased transcriptome size from the increase of the autosomes, as suggested by the absolute data of Belote and Lucchesi (1980) and Okuno et al (1984) and that was visualized directly in embryos (Pal Bhadra et al., 2005). The transcriptome size in aneuploids can change, which cannot be detected in RNA-seq analyses alone (Yang et al., 2021), so it is an important consideration for studies of dosage compensation. It was recently acknowledged that in MSL2 knockdowns the relative X expression is decreased and a moderate autosomal increase is found (Valsecchi et al., 2021b). A similar trend is evident in the microarray data on MSL2 knockdown in SL2 tissue culture cells (Hamada et al., 2005) and in the roX RNA (noncoding RNAs essential for MSL localization on the male X) mutants (Deng and Meller, 2006). This trend is in fact consistent with the absolute data that suggest an increase in the transcriptome size (Figure 7). A global change in transcriptome size can cause a generalized dosage compensation of a single chromosome to appear as a proportional dosage effect (loss of compensation) to some degree (Figure 7).<br /> Examination of expression in triple X metafemales, where there is no MSL complex, found that X-linked genes generally show dosage compensation but there is a generalized inverse effect on the autosomes, which could account for the detrimental effects of metafemales (Birchler et al., 1989; Sun et al., 2013b). An examination in metafemales of alleles of the white eye color gene that do or do not exhibit dosage compensation in males, showed the same response, namely, increased expression if there was no dosage compensation in males and no difference from normal females for the male dosage-compensated alleles (Birchler, 1992). This experiment demonstrated a relationship between the mechanism of dosage compensation in males and metafemales and implicated the inverse dosage effect in both. An involvement of the inverse effect in Drosophila dosage compensation provides an explanation for how the five levels of gene expression can be explained (Birchler, 1996), whereas an all-or-none presence of a complex on the X does not. The stoichiometric relationship of regulatory gene products provides a means to read the relative dosage at multiple doses to produce the appropriate inverse level.

      What then is the function of the MSL complex? It was discovered that the MSL complex will actually constrain the effect of H4 lysine16 acetylation to prevent it from causing an overexpression of genes (Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005; Sun and Birchler 2009; Sun et al., 2013a). Indeed, in the chromatin remodeling Imitation Switch (ISWI) mutants, the male X chromosome was specifically overexpressed suggesting that its normal function is needed for the constraint to occur (Pal Bhadra et al., 2005). Independently, the Mtor nuclear pore component shows a similar specific male X upregulation when Mtor is knocked down and this effect was shown to operate on the transcriptional level (Aleman et al., 2021). Interestingly, the increased expression of the X in the Mtor knockdown is accompanied by an inverse modulation of a substantial subset of autosomal genes, illustrating why the constraining process evolved to counteract male X overexpression. The constraining effect might involve a number of gene products (Birchler, 2016) and is an interesting direction for further study.

      Furthermore, when the H4Lys16 acetylase was individually targeted to reporter genes, there was an increase in expression (Sun et al., 2013a). However, when other members of the MSL complex were present in normal males or ectopically expressed, this increase did not occur (Sun et al., 2013a). It thus appears that the function of the MSL complex is to sequester the acetylase from the autosomes and constrain it on the X (Bhadra et al., 1999; 2000; Pal Bhadra et al., 2005; Sun and Birchler, 2009; Sun et al., 2013a). Indeed, in the Mtor knockdowns, the X linked genes with the greatest upregulation were those with the greatest association with the acetylase and the H4K16ac histone mark (Aleman et al 2021), supporting the idea of a constraining activity that becomes released in the Mtor knockdown. When the MSL complex is disrupted, there is an inverse effect on the autosomes that occurs but in normal circumstances the sequestration mutes this effect. The MSL complex disruption releases the acetylase to be uniformly distributed across all chromosomes as determined cytologically (Bhadra et al., 1999) or via ChIPseq for H4Lys16ac (Valsecchi et al., 2021a). Indeed, the quantity of the H4Lys16ac mark only has a proportional effect on gene expression when the constraining activity is disrupted (Aleman et al., 2021) or when the MSL complex is not present (Sun et al., 2013a). Thus, in normal flies there is a more or less equalized expression of the X and autosomes despite the monosomy for 20% of the genome.

      The component of the complex that is expressed in males and thought to organize the complex to the male X, MSL2, was recently found to also be associated with autosomal dosage sensitive regulatory genes (Valsecchi et al., 2018). MSL2 was found to modulate these autosomal dosage sensitive genes in various directions, which illustrates that MSL2 has a role in dosage balance that goes beyond the X chromosome. This finding is consistent with the evolutionary scenario that the initial attraction of the complex to the X chromosome was to upregulate dosage sensitive genes in hemizygous regions as the progenitor Y became deleted for them, with the constraining activity evolving to prevent an overexpression as the amount of acetylase on the male X increased with time (Birchler, 2016).

      The MSL hypothesis takes an X-centric view that does not accommodate what is now known about dosage effects across the whole genome. The idea that dissolution of the MSL complex would cause reduction in expression of the male X linked genes without any consequences for the autosomes is not consistent with current knowledge of gene regulatory networks and their dosage sensitivity. Indeed, the finding of dosage compensation in large autosomal aneuploids that operates on the transcriptional level (Devlin et al., 1982; 1984; Birchler et al., 1990; Sun et al., 2013c) as well as a predominant inverse effect by the same (Devlin, et al., 1988; Birchler et al., 1990) argues that one must consider the inverse effect for an understanding of the evolution of dosage compensation in Drosophila (and other species). Further discussion of models of Drosophila compensation has been published (Birchler, 2016).

      What is likely to be the most critical issue with sex chromosome evolution is the consequences for dosage sensitive regulatory genes. This fact is nicely illustrated by the retention of these types of genes in different independent vertebrate sex chromosome evolutions (Bellott and Page, 2021). In Drosophila, by contrast, dosage compensation is more of a blanket effect on most but not all X linked genes despite the fact that many genes on the X are unlikely to have dosage detrimental effects, although dosage sensitive genes might have played a role as noted above. The particularly large size of the X in Drosophila compared to the whole genome is potentially a contributing factor because such large genomic imbalance is likely to modulate most genes across the genome. Also, there is no evidence of a WGD in Drosophila as there is in other species for which the inverse effect has been documented (maize, Arabidopsis, yeast, mice, human). These other species have various numbers of retained duplicate dosage sensitive regulatory genes from WGDs. Thus, the relative change of regulatory genes in aneuploids in these species will not be as great compared to some of their interactors in the remainder of the genome, which could result in lesser magnitudes of some trans-acting effects, similarly to how aneuploids in ascending ploidies have fewer effects as described above. The absence of duplicate regulatory genes in Drosophila would predict a stronger inverse effect in general and that could have been capitalized upon to produce dosage compensation of most genes on the X chromosome despite many of them not being dosage critical. While sex chromosome evolution must accommodate dosage sensitive genes for proper development and viability, it could also be capitalized upon to evolve sexual dimorphisms in expression (Sun et al., 2013c)."

      Comments on revised submission:

      The authors did make an effort to address the issue previously raised.

      The authors state that an inverse dosage effect results from a titration of the histone acetylase MOF between the NSL and MSL complexes (lines 87-89). This is a misunderstanding of the inverse effect, which is an imbalance of regulatory molecules. Single regulatory gene dosage series can produce this effect. The inverse effect operates in triple X metafemales to produce dosage compensation of the three X chromosomes and a reduced expression of the autosomes (Sun et al 2913 PNAS 110: 7383-7388). There is no MSL complex in metafemales.

    1. Reviewer #1 (Public review):

      Summary:

      The present paper by Redman et al. investigated the variability of grid cell properties in the MEC by analyzing publicly available large-scale neural recording data. Although previous studies have proposed that grid spacing and orientation are homogeneous within the same grid module, the authors found a small but robust variability in grid spacing and orientation across grid cells in the same module. The authors also showed, through model simulations, that such variability is useful for decoding spatial position.

      Strengths:

      The results of this study provide novel and intriguing insights into how grid cells compose the cognitive map in the axis of the entorhinal cortex and hippocampus. This study analyzes large data sets in an appropriate manner and the results are solid.

      Weaknesses:

      A weakness of this paper is that the scope of the study may be somewhat narrow, as this study focused only on the variability of spacing and orientation across grid cells. I would suggest some additional analysis or discussion that might increase the value of the paper.

      (1) Is the variability in grid spacing and orientation that the authors found intrinsically organized or is it shaped by experience? Previous research has shown that grid representations can be modified through experience (e.g., Boccara et al., Science 2019). To understand the dynamics of the network, it would be important to investigate whether robust variability exists from the beginning of the task period (recording period) or whether variability emerges in an experience-dependent manner within a session.

      (2) It is important to consider the optimal variability size. The larger the variability, the better it is for decoding. On the other hand, as the authors state in the Discussion, it is assumed that variability does not exist in the continuous attractor model. Although this study describes that it does not address how such variability fits the attractor theory, it would be better if more detailed ideas and suggestions were provided as to what direction the study could take to clarify the optimal size of variability.

    1. Reviewer #1 (Public review):

      Summary:

      This very interesting manuscript proposes a general mechanism for how activating signaling proteins respond to species-specific signals arising from a variety of stresses. In brief, the authors propose that the activating signal alters the structure by a universal allosteric mechanism.

      Strengths:

      The unitary mechanism proposed is appealing and testable. They propose that the allosteric module consists of crossed alpha-helical linkers with similar architecture and that their attached regulatory domains connect to phosphatases or other molecules through coiled-coli domains, such that the signal is transduced via rigidifying the alpha helices, permitting downstream enzymatic activity. The authors present genetic and structural prediction data in favor of the model for the system they are studying, and stronger structural data in other systems.

      Weaknesses:

      The evidence is indirect - targeted mutations, structural predictions, and biochemical data. Therefore, these important generalizable conclusions are not buttressed by impeccable data, which would require doing actual structures in B. subtilis, confirming experiments in other organisms, and possibly co-evolutionary coupling. In the absence of such data, it is not possible to rule out variant models.

    1. Reviewer #1 (Public Review):

      Summary:

      SUFU modulates Sonic hedgehog (SHH) signaling and is frequently mutated in the B-subtype of SHH-driven medulloblastoma. The B-subtype occurs mostly in infants, is often metastatic, and lacks specific treatment. Yabut et al. found that Fgf5 was highly expressed in the B-subtype of SHH-driven medulloblastoma by examining a published microarray expression dataset. They then investigated how Fgf5 functions in the cerebellum of mice that have embryonic Sufu loss of function. This loss was induced using the hGFAP-cre transgene, which is expressed in multiple cell types in the developing cerebellum, including granule neuron precursors (GNPs) derived from the rhombic lip. By measuring the area of Pax6+ cells in the external granule cell layer (EGL) of Sufu-cKO mice at postnatal day 0, they find Pax6+ cells occupy a larger area in the posterior lobe adjacent to the secondary fissure, which is poorly defined. They show that Fgf5 RNA and phosphoErk1/2 immunostaining are also higher in the same disrupted region. Some of the phosphoErk1/2+ cells are proliferative in the Sufu-cKO. Western blot analysis of Gli proteins that modulate SHH signaling found reduced expression and absence of Gli1 activity in the region of cerebellar dysgenesis in Sufu-cKO mice. This suggests the GNP expansion in this region is independent of SHH signaling. Amazingly, intraventricular injection of the FGFR1-2 antagonist AZD4547 from P0-4 and examined histologically at P7 found the treatment restored cytoarchitecture in the cerebella of Sufu-cKO mice. This is further supported by NeuN immunostaining in the internal granule cell layer, which labels mature, non-diving neurons, and KI67 immunostaining, indicating dividing cells, and primarily found in the EGL. The mice were treated beginning at a timepoint when cerebellar cytoarchitecture was shown to be disrupted and it is indistinguishable from control following treatment. Figure 3 presents the most convincing and exciting data in this manuscript.

      Sufu-cKO do not readily develop cerebellar tumors. The authors detected phosphorylated H2AX immunostaining, which labels double-strand breaks, in some cells in the EGL in regions of cerebellar dysgenesis in the Sufu-cKO, as was cleaved Caspase 3, a marker of apoptosis. P53, downstream of the double-strand break pathway, the protein was reduced in Sufu-cKO cerebellum. Genetically removing p53 from the Sufu-cKO cerebellum resulted in cerebellar tumors in 2-month old mice. The Sufu;p53-dKO cerebella at P0 lacked clear foliation, and the secondary fissure, even more so than the Sufu-cKO. Fgf5 RNA and signaling (pERK1/2) were also expressed ectopically.

      The conclusions of the paper are largely supported by the data, but some data analysis need to be clarified and extended.

      (1) The rationale for examining Fgf5 in medulloblastoma is not sufficiently convincing. The authors previously reported that Fgf15 was upregulated in neocortical progenitors of mice with conditional loss of Sufu (PMID: 32737167). In Figure 1, the authors report FGF5 expression is higher in SHH-type medulloblastoma, especially the beta and gamma subtypes mostly found in infants. These data were derived from a genome-wide dataset and are shown without correction for multiple testing, including other Fgfs. Showing the expression of other Fgfs with FDR correction would better substantiate their choice or moving this figure to later in the manuscript as support for their mouse investigations would be more convincing.

      (2) The Sufu-cKO cerebellum lacks a clear anchor point at the secondary fissure and foliation is disrupted in the central and posterior lobes. It would be helpful for the authors to review Sudarov & Joyner (PMID: 18053187) for nomenclature specific to the developing cerebellum.

      (3) The metrics used to quantify cerebellar perimeter and immunostaining are not sufficiently described. It is unclear whether the individual points in the bar graph represent a single section from independent mice, or multiple sections from the same mice. For example, in Figures 2B-D. This also applies to Figure 3C-D.

      (4) The data on Fgf5 RNA expression presented in Figure 2E are not sufficiently convincing. The perimeter and cytoarchitecture of the cerebellum are difficult to see and the higher magnification shown in 2F should be indicated in 2E.

      (5) The data presented in Figure 3 are not sufficiently convincing. The number of cells double positive for pErk and KI67 (Figure 3B) are difficult to see and appear to be few, suggesting the quantification may be unreliable.

      (6) The data presented in Figure 4F-J would be more convincing with quantification. The Sufu;p53-dKO appears to have a thickened EGL across the entire vermis perimeter, and very little foliation, relative to control and single cKO cerebella. This is a more widespread effect than the more localized foliation disruption in the Sufu-cKO.

      (7) Figure 5 does not convincingly summarize the results. Blue and purple cells in sagittal cartoon are not defined. Which cells express Fgf5 (or other Fgfs) has not been determined. The yellow cells are not defined in relation to the initial cartoon on the left.

    1. Reviewer #1 (Public review):

      Summary:

      Crosslinking mass spectrometry has become an important tool in structural biology, providing information about protein complex architecture, binding sites and interfaces, and conformational changes. One key challenge of this approach represents the quantitation of crosslinking data to interrogate differential binding states and distributions of conformational states.

      Here, Luo and Ranish present a novel class of isobaric crosslinkers ("Qlinkers"), conduct proof-of-concept benchmarking experiments on known protein complexes, and show example applications on selected target proteins. The data are solid and this could well be an exciting, convincing new approach in the field if the quantitation strategy is made more comprehensive and the quantitative power of isobaric labeling is fully leveraged as outlined below. It's a promising proof-of-concept, and potentially of broad interest for structural biologists.

      Strengths:

      The authors demonstrate the synthesis, application, and quantitation of their "Q2linkers", enabling relative quantitation of two conditions against each other. In benchmarking experiments, the Q2linkers provide accurate quantitation in mixing experiments. Then the authors show applications of Q2linkers on MBP, Calmodulin, selected transcription factors, and polymerase II, investigating protein binding, complex assembly, and conformational dynamics of the respective target proteins. For known interactions, their findings are in line with previous studies, and they show some interesting data for TFIIA/TBP/TFIIB complex formation and conformational changes in pol II upon Rbp4/7 binding.

      Weaknesses:

      This is an elegant approach but the power of isobaric mass tags is not fully leveraged in the current manuscript.

      First, "only" Q2linkers are used. This means only two conditions can be compared. Theoretically, higher-plexed Qlinkers should be accessible and would also be needed to make this a competitive method against other crosslinking quantitation strategies. As it is, two conditions can still be compared relatively easily using LFQ - or stable-isotope-labeling based approaches. A "Q5linker" would be a really useful crosslinker, which would open up comprehensive quantitative XLMS studies.

      Second, the true power of isobaric labeling, accurate quantitation across multiple samples in a single run, is not fully exploited here. The authors only show differential trends for their interaction partners or different conformational states and do not make full quantitative use of their data or conduct statistical analyses. This should be investigated in more detail, e.g. examine Qlinker quantitation of MBP incubated with different concentrations of maltose or Calmodulin incubated with different concentrations of CBPs. Does Qlinker quantitation match ratios predicted using known binding constants or conformational state populations? Is it possible to extract ratios of protein populations in different conformations, assembly, or ligand-bound states?

      With these two points addressed this approach could be an important and convincing tool for structural biologists.

    1. Reviewer #1 (Public review):

      Summary:

      In this manuscript, authors have investigated the effects of JNK inhibition on sucrose-induced metabolic dysfunction in rats. They used multi-tissue network analysis to study the effects of the JNK inhibitor JNK-IN-5A on metabolic dysfunction associated with excessive sucrose consumption. Their results show that JNK inhibition reduces triglyceride accumulation and inflammation in the liver and adipose tissues while promoting metabolic adaptations in skeletal muscle. The study provides new insights into how JNK inhibition can potentially treat metabolic dysfunction-associated fatty liver disease (MAFLD) by modulating inter-tissue communication and metabolic processes.

      Strengths:

      The study has several notable strengths:

      Comprehensive Multi-Tissue Analysis: The research provides a thorough multi-tissue evaluation, examining the effects of JNK inhibition across key metabolically active tissues, including the liver, visceral white adipose tissue, skeletal muscle, and brain. This comprehensive approach offers valuable insights into the systemic effects of JNK inhibition and its potential in treating MAFLD.

      Robust Use of Systems Biology: The study employs advanced systems biology techniques, including transcriptomic analysis and genome-scale metabolic modeling, to uncover the molecular mechanisms underlying JNK inhibition. This integrative approach strengthens the evidence supporting the role of JNK inhibitors in modulating metabolic pathways linked to MAFLD.

      Potential Therapeutic Insights: By demonstrating the effects of JNK inhibition on both hepatic and extrahepatic tissues, the study offers promising therapeutic insights into how JNK inhibitors could be used to mitigate metabolic dysfunction associated with excessive sucrose consumption, a key contributor to MAFLD.

      Behavioral and Metabolic Correlation: The inclusion of behavioral tests alongside metabolic assessments provides a more holistic view of the treatment's effects, allowing for a better understanding of the broader physiological implications of JNK inhibition.

      Weaknesses:

      While the study provides a comprehensive evaluation of JNK inhibitors in mitigating MAFLD conditions, addressing the following points will enhance the manuscript's quality:

      The authors should explicitly mention and provide a detailed list of metabolites affected by sucrose and JNK inhibition treatment that have been previously associated with MAFLD conditions. This will better contextualize the findings within the broader field of metabolic disease research.

      The limitations of the study should be clearly stated, particularly the lack of evidence on the effects of chronic JNK inhibitor treatment and potential off-target effects. Addressing these concerns will offer a more balanced perspective on the therapeutic potential of JNK inhibition.

      The potential risks of using JNK inhibitors in non-MAFLD conditions should be highlighted, with a clear distinction made between the preventive and curative effects of these therapies in mitigating MAFLD conditions. This will ensure the therapeutic implications are properly framed.

      The statistical analysis section could be strengthened by providing a justification for the chosen statistical tests and discussing the study's power. Additionally, a more detailed breakdown of the behavioral test results and their implications would be beneficial for the overall conclusions of the study.

    1. Reviewer #1 (Public review):

      Summary:<br /> The authors create an elegant sensor for TDP -43 loss of function based on cryptic splicing of CFTR and UNC13A. The usefulness of this sensor primarily lies in its use in eventual high throughput screening and eventual in vivo models. The TDP-43 loss of function sensor was also used to express TDP-43 upon reduction of its levels.

      Strengths:<br /> The validation is convincing, the sensor was tested in models of TDP-43 loss of function, knockdown and models of TDP-43 mislocalization and aggregation. The sensor is susceptible to a minimal decrease of TDP-43 and can be used at the protein level unlike most of the tests currently employed,

      Weaknesses:<br /> Although the LOF sensor described in this study may be a primary readout for high-throughput screens, ALS/TDP-43 models typically employ primary readouts such as protein aggregation or mislocalization. The information in the two following points would assist users in making informed choices. 1. Testing the sensor in other cell lines 2. Establishing a correlation between the sensor's readout and the loss of function (LOF) in the physiological genes would be useful given that the LOF sensor is a hybrid structure and doesn't represent any physiological gene. It would be beneficial to determine if a minor decrease (e.g., 2%) in TDP-43 levels is physiologically significant for a subset of exons whose splicing is controlled by TDP-43.

      Considering that most TDP-LOF pathologically occurs due to aggregation and or mislocalization, and in most cases the endogenous TDP-43 gene is functional but the protein becomes non-functional, the use of the loss of function sensor as a switch to produce TDP-43 and its eventual use as gene therapy would have to contend with the fact that the protein produced may also become nonfunctional. This would eventually be easy to test in one of the aggregation modes that were used to test the sensor.. However, as the authors suggest, this is a very interesting system to deliver other genetic modifiers of TDP-43 proteinopathy in a regulated fashion and timely fashion.

  2. Sep 2024
    1. Reviewer #2 (Public Review):

      Summary:

      Hebin et al reported a fascinating story about antibiotic persistence in the biofilms. First, they set up a model to identify the increased persisters in the biofilm status. They found that the adhesion of bacteria to the surface leads to increased c-di-GMP levels, which might lead to the formation of persisters. To figure out the molecular mechanism, they screened the E.coli Keio Knockout Collection and identified the HipH. Finally, the authors used a lot of data to prove that c-di-GMP not only controls HipH over-expression but also inhibits HipH activity, though the inhibition might be weak.

      Strengths:

      They used a lot of state-of-the-art technologies, such as single-cell technologies as well as classical genetic and biochemistry approaches to prove the concept, which makes the conclusions very solid. Overall, it is a very interesting and solid story that might attract diverse readers working with c-di-GMP, persisters, and biofilm.

      Comments on the revised version:

      All my concerns have been addressed.

    1. Reviewer #2 (Public review):

      Summary:<br /> The authors describe five year outcomes of an internship program for graduate students and postdoctoral fellows at their institution spurred by pilot funding from an NIH BEST grant. They hypothesized that such a program would be beneficial to interns, internship hosts, and research advisors. The mixed methods study used surveys and focus groups to gather qualitative and quantitative data from the stakeholder groups, and the authors acknowledge that limitation that the study subjects were self-selected and also had research advisors who agreed to allow them to participate. Thus the generally favorable outcomes may not be applicable to students such as those who are struggling in the lab and/or lack career focus or supportive research advisors. Nonetheless, the overall finding support the hypothesis and also suggest additional benefits, including in some cases positive impact for the lab, improved communication between the intern and their research advisor, and an advantage for recruitment of students to the institution. The data refute one of the principle concerns of research advisors: that by taking students out of the lab, internships reduce individual and overall lab productivity. Students who did internships were significantly less likely to pursue postdoctoral fellowships before entering the biomedical workforce and were more likely to have science-related careers versus research careers than control students who did not do internships, although the study design cannot determine whether this is a causal relationship.

      Strengths:<br /> (1) Sample size is good (123 internships).

      (2) Response rate is high, minimizing potential bias.

      (3) The internship program is well described. Outcomes are clearly defined.

      (4) Methods and statistical analyses appear to be appropriate (although I am not expert in mixed methods).

      (5) "Take-home" lessons for institutions considering implementing internship programs are clearly stated.

      Appraisal:<br /> Overall the authors achieve their aims of describing outcomes of an internship program for graduate career development and offering lessons learned for other institutions seeking to create their own internship programs.

      Impact:<br /> The paper will be very useful for other institutions to dispel some of the concerns of research advisers about internships for PhD students (although not necessarily for postdoctoral fellows). In the long run, wider adoption of internships as part of PhD training will depend not only on faculty buy-in but also on availability of resources and changes to the graduate school funding model so that such programs are not viewed as another "unfunded mandate" in graduate education. Perhaps industry will be motivated to support internships by the positive outcomes for hosts reported in this paper. Additionally, NIH could allow a certain amount of F, T, or even RPG funds to be used to support internships for purposes of career development.

    1. Reviewer #1 (Public review):

      This manuscript by Martinez-Ara et al investigates how combinations of cis-regulatory elements combine to influence gene expression. Using a clever iteration on massively parallel reporter assays (MPRAs), the authors measure the combinatorial effects of pairs of enhancers on specific promoters. Specifically, they assayed the activity of 59x59 different enhancer-enhancer (E-E) combinations on 8 different promoters in mouse embryonic stem cells. The main claims of the paper are that E-E pairs combine nearly additively, and that supra-additive E-E pairs are rare and often promoter-dependent. The data in this study do generally support these claims.

      This paper makes a good contribution to the ongoing discussions about the selectivity of gene regulatory elements. Recent works, such as those by Martinez-Ara et al. and Burgman et al., have indicated limited selectivity between E-P pairs on plasmid-based assays; this paper adds another layer to that by suggesting a similar lack of selectivity between E-E pairs.

      An interesting result in this manuscript is the observation that weak promoters allow more supra-additive E-E interactions than strong promoters (Figure 4b). This nonlinear promoter response to enhancers aligns with the model previously proposed in Hong et al. (from my own group), which posited that core promoter activities are nonlinearly scaled by the genomic environment, and that (similar to the trend observed in Figure 5b) the steepness of the scaling is negatively correlated with promoter strength.

      My only suggestion for the authors is that they include more plots showing how much the intrinsic strengths of the promoters and enhancers they are working with explain the trends in their data.

      Specific Suggestions<br /> Supplementary Figure 4 is presented as evidence for selectivity between single enhancers and promoters. Could the authors inspect the relationship between enhancer/promoter strength and this selectivity? Generating plots similar to Figure 4B and Figure 5B, but for single enhancers, should show if the ability of an enhancer to boost a promoter is inversely correlated to that promoter's intrinsic strength. Also, in Supplementary Figure 4, coloring each point by promoter type would clarify if certain promoters (the weak ones) consistently show higher boost indices across all enhancers. If they do not, the authors may want to speculate how single enhancers can show selectivity for promoters while the effect of adding a second enhancer to an existing E-P has little selectivity. An alternate explanation, based solely on the strength of the elements, would be that when the expression of a gene is low the addition of enhancer(s) have large effects, but when the expression of a gene is high (closer to saturation) the addition of enhancer(s) have small effects.

      Can anything more be said about the enhancers in E-E-P combinations that exhibit supra-additivity? Specifically, it would be interesting to know if certain enhancers, e.g. strong enhancers or enhancers with certain motifs, are more likely to show supra-additivity with a given promoter.

      Comments on revised version:

      The revised manuscript satisfactorily addresses the points I raised in the review. With the addition of the new graphs there is enough data for readers to decide whether the supra-additivity depends only on the strength of the promoter or on some other (undefined) feature of E-P pairs. This manuscript is a solid contribution to the ongoing debate about enhancer-promoter selectivity.

    1. Reviewer #2 (Public Review):

      Rubio et al. study the behavior of the transcription factor Hsf1 under ethanol stress, examining its distribution within the nucleus and the coalescence of heat shock response genes in budding yeast. In comparison to the heat shock response, the response to ethanol stress shows similar gene coalescence and Hsf1 binding. However, there is a notable delay in the transcriptional response to ethanol, and a disconnect between it and the appearance of irreversible Hsf1 condensates/puncta, highlighting important differences in how Hsf1 responds to these two related but distinct environmental stresses.

      The authors have addressed the majority of my previous comments effectively. The Sis1 experiment provides a clear illustration of a distinctive response to ethanol and heat. This work offers a comprehensive perspective on Hsf1 in stress response from multiple angles.

    1. Reviewer #1 (Public review):

      Summary:

      The authors demonstrated that carbon depletion triggers the autophagy-dependent formation of Rubisco Containing Bodies, which contain chloroplast stroma material, but exclude thylakoids. The authors show that RCBs bud directly from the main body of chloroplasts rather than from stromules and that their formation is not dependent on the chloroplast fission factor DRP5. The authors also observed a transient engulfment of the RBCs by the tonoplast during delivery to the vacuolar lumen.

      Strengths:

      The authors demonstrate that autophagy-related protein 8 (ATG8) co-localizes to the chloroplast demarking the place for RCB budding. The authors provide good-quality time-lapse images and co-localization of the markers corroborating previous observations that RCBs contain only stroma material and do not include thylakoid. The text is very well written and easy to follow.

      Weaknesses:

      The study adds more valuable descriptive information about the previously published phenomenon of RCB formation under carbon starvation but does not reveal the putative mechanisms governing formation of RCBs and their release to the vacuole.

      Comments on revised version:

      The authors have done an impressive job revising the manuscript and addressed my comments. The authors clarified previous ambiguities and the new version of the manuscript greatly benefits from the provided quantifications and adjusted discussion.

    1. Reviewer #1 (Public review):

      Summary:

      This manuscript employs yolk sac visceral endoderm cells as a novel model for studying endosomal fusion, observing two distinct fusion behaviors: quick homotypic fusion between late endosomes, and slower heterotypic fusion between late endosomes and lysosomes. The mathematical modeling suggests that vesicle size critically influences the mode of fusion. Further investigations reveal that actin filaments are dynamically associated with late endosomal membranes, and are oriented in the x-y plane and along the apical-basal axis. Actin and Arf2/3 were shown to appear at the rear end of the endosomes along the moving direction suggesting polymerization of actin may provide force for the movement of endosomes. Additionally, the authors found that actin dynamics regulate homotypic and heterotypic fusion events in a different manner. The authors also provide evidence suggesting that Cofilin-dependent actin dynamics are involved in late endosome fusion.

      Strengths:

      The unique feature of this study is that the authors use yolk sac visceral endoderm cells to study endosomal fusion. Yolk sac visceral endoderm cells have huge endocytic vesicles, endosomes and lysosomes, offering an excellent system to explore endosomal fusion dynamics and the assembly of cellular factors on membranes. The manuscript provides a valuable and convincing observation of the modes of endosomal fusion and roles of actin dynamics in this process, and the conclusions of the study is justified by the data.

      Weaknesses:

      While the study offers compelling observations, it falls short in delivering clear mechanistic insights. Key questions remain unaddressed, such as the functional significance of actin filaments that extend apically in positioning late endosomes, the ways in which actin dynamics influence fusion events, and the functional implications of the slower bridge fusion process.

    1. Reviewer #1 (Public review):

      Summary:

      The authors use methylphenidate (MPH) administration after learning a Pavlovian to instrumental transfer (PIT) task to parse decision-making from instrumental influences. While the main effects were null, individual differences in working memory ability moderated the tendency of MPH to boost cognitive control in order to override PIT-biased instrumental learning. Importantly, this working memory moderator had symmetrical effects in appetite and aversive conditions, and these patterns replicated within each valence condition across different values of gain/loss (Fig S1c), suggesting a reliable effect that is generalized across instances of Pavlovian influence.

      Strengths:

      The idea of using pharmacological challenge after learning but prior to transfer is a novel technique that highlights the influence of catecholamines on the expression of learning under Pavlovian bias, and importantly it dissociated this decision feature from the learning of stimulus-outcome or action-outcome pairings.

      Weaknesses:

      While the report is largely straightforward and clearly written, some aspects may be edited to improve the clarity for other readers.

      1) Theoretical clarity. The authors seem to hedge their bets when it comes to placing these findings within a broader theoretical framework.

      2) Analytic clarity: what's c^2?

    1. Reviewer #1 (Public Review):

      Summary:

      One enduring mystery involving the evolution of genomes is the remarkable variation they exhibit with respect to size. Much of that variation is due to differences in the number of transposable elements, which often (but not always) correlates with the overall quantity of DNA. Amplification of TEs is nearly always either selectively neutral or negative with respect to host fitness. Given that larger effective population sizes are more efficient at removing these mutations, it has been hypothesized that TE content, and thus overall genome size, may be a function of effective population size. The authors of this manuscript test this hypothesis by using a uniform approach to analysis of several hundred animal genomes, using the ratio of synonymous to nonsynonymous mutations in coding sequence as a measure of the overall strength of purifying selection, which serves as a proxy for effective population size over time. The data convincingly demonstrates that it is unlikely that effective population size has a strong effect on TE content and, by extension, overall genome size (except for birds).

      Strengths:

      Although this ground has been covered before in many other papers, the strength of this analysis is that it is comprehensive and treats all the genomes with the same pipeline, making comparisons more convincing. Although this is a negative result, it is important because it is relatively comprehensive and indicates that there will be no simple, global hypothesis that can explain the observed variation.

      Weaknesses:

      In several places, I think the authors slip between assertions of correlation and assertions of cause-effect relationships not established in the results. In other places, the arguments end up feeling circular, based, I think, on those inferred causal relationships. It was also puzzling why plants (which show vast differences in DNA content) were ignored altogether.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, the authors utilized human placental samples together with multiple mouse models to explore the mechanisms whereby inflammatory macrophages and T cells are linked to preeclampsia (PE). The authors first undertook CyTOF of placental samples from women with normal pregnancies, PE, gestational diabetes mellitus (GDM), and GDM with superimposed PE (GDM+PE). The authors report an increase of memory-like Th17 cells, memory-like CD8+ T cells, and pro-inflammatory macrophages in PE cases, but not GDM or GDM+PE, together with diminished γδT cells, anti-inflammatory macrophages, and granulocyte myeloid-derived suppressor cells (gMDSC). The authors then undertook several experiments using scRNA-seq, bulk RNA-seq, and flow cytometry in a RUPP model to first show that the transfer of pro-inflammatory macrophages from RUPP mice into normal pregnant mice with depleted macrophages resulted in increased embryo resorption and diminished fetal weight and size. Moreover, pro-inflammatory macrophages induced memory-like Th17 cells in mice. Similarly, injection of T-cells from RUPP mice resulted in increased embryo resorption and diminished fetal weight and size. Such mice that received RUPP-derived T cells displayed similarly worsened outcomes in their second pregnancy in the absence of any additional T cell transfer. The authors identified the IGF1-IGF1R ligand-receptor pair as a factor involved in the macrophage-mediated induction of memory-like Th17 cells, as confirmed by experiments using an IGF1R inhibitor. Finally, the authors transferred IGF1R inhibitor-treated T cells to a pregnant mouse that was administered LPS and depleted of T cells and observed improved outcomes compared to mice that received non-treated T cells. The authors conclude that their study identifies a PE-specific immune cell network regulated by pro-inflammatory macrophages and T cells.

      Strengths:

      Utilization of both human placental samples and multiple mouse models to explore the mechanisms linking inflammatory macrophages and T cells to preeclampsia (PE).<br /> Incorporation of advanced techniques such as CyTOF, scRNA-seq, bulk RNA-seq, and flow cytometry.

      Identification of specific immune cell populations and their roles in PE, including the IGF1-IGF1R ligand-receptor pair in macrophage-mediated Th17 cell differentiation.<br /> Demonstration of the adverse effects of pro-inflammatory macrophages and T cells on pregnancy outcomes through transfer experiments.

      Weaknesses:

      Inconsistent use of uterine and placental cells, which are distinct tissues with different macrophage populations, potentially confounding results.

      Missing observational data for the initial experiment transferring RUPP-derived macrophages to normal pregnant mice.

      Unclear mechanisms of anti-macrophage compounds and their effects on placental/fetal macrophages.

      Difficulty in distinguishing donor cells from recipient cells in murine single-cell data complicates interpretation.

      Limitation of using the LPS model in the final experiments, as it more closely resembles systemic inflammation seen in endotoxemia rather than the specific pathology of PE.

    1. Reviewer #1 (Public review):

      Summary:<br /> Chen et al. identified the role of endocardial id2b expression in cardiac contraction and valve formation through pharmaceutical, genetic, electrophysiology, calcium imaging, and echocardiography analyses. CRISPR/Cas9 generated id2b mutants demonstrated defective AV valve formation, excitation-contraction coupling, reduced endocardial cell proliferation in AV valve, retrograde blood flow, and lethal effects.

      Strengths:<br /> Their methods, data and analyses broadly support their claims.

      Weaknesses:<br /> The molecular mechanism is somewhat preliminary.

    1. Joint Public Review:

      Summary:

      The study by Akita B. Jaykumar et al. explores an interesting and relevant hypothesis whether serine/threonine With-No-lysine (K) kinases (WNK)-1, -2, -3, and -4 engage in insulin-dependent glucose transporter-4 (GLUT4) signaling in the murine central nervous system. The authors especially focused on the hippocampus as this brain region exhibits high expression of insulin and GLUT4. Additionally, disrupted glucose metabolism in the hippocampus has been associated with anxiety disorders, while impaired WNK signaling has been linked to hypertension, learning disabilities, psychiatric disorders, or Alzheimer's disease. The study took advantage of selective pan-WNK inhibitor WNK 643 as the main tool to manipulate WNK 1-4 activity both in vivo by daily, per-oral drug administration to wild-type mice, and in vitro by treating either adult murine brain synaptosomes, hippocampal slices, primary cortical cultures, and human cell lines (HEK293, SH-SY5Y). Using a battery of standard behavior paradigms such as open field test, elevated plus maze test, and fear conditioning, the authors convincingly demonstrate that the inhibition of WNK1-4 results in behavior changes, especially in enhanced learning and memory of WNK643-treated mice. To shed light on the underlying molecular mechanism, the authors implemented multiple biochemical approaches including immunoprecipitation, glucose-uptake assay, surface biotylination assay, immunoblotting, and immunofluorescence. The data suggest that simultaneous insulin stimulation and WNK1-4 inhibition results in increased glucose uptake and the activity of insulin's downstream effectors, phosphorylated Akt and phosphorylated AS160. Moreover, the authors demonstrate that insulin treatment enhances the physical interaction of the WNK effector OSR1/SPAK with Akt substrate AS160. As a result, combined treatment with insulin and the WNK643 inhibitor synergistically increases the targeting of GLUT4 to the plasma membrane. Collectively, these data strongly support the initial hypothesis that neuronal insulin- and WNK-dependent pathways do interact and engage in cognitive functions.

      Strengths:

      The insulin-dependent signaling in the central nervous system is relatively understudied. This explorative study delves into several interesting and clinically relevant possibilities, examining how insulin-dependent signaling and its crosstalk with WNK kinases might affect brain circuits involved in memory formation and/or anxiety. Therefore, these findings might inspire follow-up studies performed in disease models for disorders that exhibit impaired glucose metabolism, deficient memory, or anxiety, such as Diabetes mellitus, Alzheimer's disease, or most psychiatric disorders.

      The graphical presentation of the figures is of high quality, which helps the reader to obtain a good overview and easily understand the experimental design, results, and conclusions.

      The behavioral studies are well conducted and provide valuable insights into the role of WNK kinases in glucose metabolism and their effect on learning and memory. Additionally, the authors evaluate the levels of basal and induced anxiety in Figures 1 and 2, enhancing our understanding of how WNK signaling might engage in cognitive function and anxiety-like behavior, particularly in the context of altered glucose metabolism.

      Weaknesses:

      The study used a WNK643 inhibitor as the only tool to manipulate WNK1-4 activity. This inhibitor seems selective; however, it has been reported that it exhibits different efficiency in inhibiting the individual WNK kinases among each other (e.g. PMID: 31017050, PMID: 36712947). Additionally, the authors do not analyze nor report the expression profiles or activity levels of WNK1, WNK2, WNK3, and WNK4 within the relevant brain regions (i.e. hippocampus, cortex, amygdala). Combined, these weaknesses raise concerns about the direct involvement of WNK kinases within the selected brain regions and behavior circuits. It would be beneficial if the authors provided gene profiling for WNK1, 2, 3, and -4 (e.g. using Allen brain atlas). To confirm the observations, the authors should either add results from using other WNK inhibitors or, preferentially, analyze knock-down or knock-out animals/tissue targeting the single kinases.

      The authors do not report any data on whether the global inhibition of WNKs affects insulin levels. Since the authors wish to demonstrate the synergistic effect of simultaneous insulin treatment and WNK1-4 inhibition, such data are missing.

      The study discovered that the Sortilin receptor binds to OSR1, leading the authors to speculate that Sortilin may be involved in the insulin-dependent GLUT4 surface trafficking. However, the authors do not provide any evidence supporting Sortilin's involvement in insulin- or WNK-dependent GLUT4 trafficking. Thus, this conclusion should be qualified, rephrased, or additional data included.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Ning et al. reported that Bcas2 played an indispensable role in zebrafish primitive hematopoiesis via sequestering β-catenin in the nucleus. The authors showed that loss of Bcas2 caused primitive hematopoietic defects in zebrafish. They unraveled that Bcas2 deficiency promoted β-catenin nuclear export via a CRM1-dependent manner in vivo and in vitro. They further validated that BCAS2 directly interacted with β-catenin in the nucleus and enhanced β-catenin accumulation through its CC domains. They unveil a novel insight into Bcas2, which is critical for zebrafish primitive hematopoiesis via regulating nuclear β-catenin stabilization rather than its canonical pre-mRNA splicing functions. Overall, the study is impressive and well-performed, although there are also some issues to address.

      Strengths:

      The study unveils a novel function of Bcas2, which is critical for zebrafish primitive hematopoiesis by sequestering β-catenin. The authors validated the results in vivo and in vitro. Most of the figures are clear and convincing. This study nicely complements the function of Bcas2 in primitive hematopoiesis.

      Weaknesses:

      A portion of the figures were over-exposed.

    1. Reviewer #1 (Public Review):

      Summary:

      Du et al. report 16 new well-preserved specimens of atiopodan arthropods from the Chengjiang biota, which demonstrate both dosal and vental anatomies of a potential new taxon of atiopodans that are closely related to trolobites. Authors assigned their specimens to Acanthomeridion serratum, and proposed A. anacanthus as a junior subjective synonym of Acanthomeridion serratum. Critically, the presence of ventral plates (interpreted as cephalic liberigenae), together with phylogenic results, lead authors to conclude that the cephalic sutures originated multiple times within the Artiopoda.

      Strengths:

      New specimens are highly qualified and informative. The morphology of dorsal exoskeleton, except for the supposed free cheek, were well illustrated and described in detail, which provides a wealth of information for taxonomic and phylogenic analyses.

    1. Reviewer #1 (Public Review):

      Summary:

      Working memory is imperfect - memories accrue error over time and are biased towards certain identities. For example, previous work has shown memory for orientation is more accurate near the cardinal directions (i.e., variance in responses is smaller for horizontal and vertical stimuli) while being biased towards diagonal orientations (i.e., there is a repulsive bias away from horizontal and vertical stimuli). The magnitude of errors and biases increase the longer an item is held in working memory and when more items are held in working memory (i.e., working memory load is higher). Previous work has argued that biases and errors could be explained by increased perceptual acuity at cardinal directions. However, these models are constrained to sensory perception and do not explain how biases and errors increase over time in memory. The current manuscript builds on this work to show how a two-layer neural network could integrate errors and biases over a memory delay. In brief, the model includes a 'sensory' layer with heterogenous connections that lead to the repulsive bias and decreased error at the cardinal directions. This layer is then reciprocally connected with a classic ring attractor layer. Through their reciprocal interactions, the biases in the sensory layer are constantly integrated into the representation in memory. In this way, the model captures the distribution of biases and errors for different orientations that has been seen in behavior and their increasing magnitude with time. The authors compare the two-layer network to a simpler one-network model, showing that the one model network is harder to tune and shows an attractive bias for memories that have lower error (which is incompatible with empirical results).

      Strengths:

      The manuscript provides a nice review of the dynamics of items in working memory, showing how errors and biases differ across stimulus space. The two-layer neural network model is able to capture the behavioral effects as well as relate to neurophysiological observations that memory representations are distributed across sensory cortex and prefrontal cortex.

      The authors use multiple approaches to understand how the network produces the observed results. For example, analyzing the dynamics of memories in the low-dimensional representational space of the networks provides the reader with an intuition for the observed effects.

      As a point of comparison with the two-layer network, the authors construct a heterogenous one-layer network (analogous to a single memory network with embedded biases). They argue that such a network is incapable of capturing the observed behavioral effects but could potentially explain biases and noise levels in other sensory domains where attractive biases have lower errors (e.g., color).

      The authors show how changes in the strength of Hebbian learning of excitatory and inhibitory synapses can change network behavior. This argues for relatively stronger learning in inhibitory synapses, an interesting prediction.

      The manuscript is well-written. In particular, the figures are well done and nicely schematize the model and the results.

      Weaknesses:

      Despite its strengths, the manuscript does have some weaknesses. These weaknesses are adequately discussed in the manuscript and motivate future research.

      One weakness is that the model is not directly fit to behavioral data, but rather compared to a schematic of behavioral data. As noted above, the model provides insight into the general phenomenon of biases in working memory. However, because the models are not fit directly to data, they may miss some aspects of the data.

      In addition, directly fitting the models to behavioral data could allow for a broader exploration of parameter space for both the one-layer and two-layer models (and their alternatives). Such an approach would provide stronger support for the papers claims (such as "....these evolving errors...require network interaction between two distinct modules."). That being said, the manuscript does explore several alternative models and also acknowledges the limitation of not directly fitting behavior, due to difficulties in fitting complex neural network models to data.

      One important behavioral observation is that both diffusive noise and biases increase with the number of items in working memory. The current model does not capture these effects and it isn't clear how the model architecture could be extended to capture these effects. That being said, the authors note this limitation in the Discussion and present it as a future direction.

      Overall:

      Overall, the manuscript was successful in building a model that captured the biases and noise observed in working memory. This work complements previous studies that have viewed these effects through the lens of optimal coding, extending these models to explain the effects of time in memory. In addition, the two-layer network architecture extends previous work with similar architectures, adding further support to the distributed nature of working memory representations.

    1. Reviewer #2 (Public Review):

      Summary:

      This paper introduces a novel approach for improving personalized cancer immunotherapy by integrating TCR profiling with traditional pHLA binding predictions, addressing the need for more precise neoantigen CRC patients. By analyzing TCR repertoires from tumor-infiltrating lymphocytes and applying machine learning algorithms, the authors developed a predictive model that outperforms conventional methods in specificity and sensitivity. The validation of the model through ELISpot assays confirmed its potential in identifying more effective neoantigens, highlighting the significance of combining TCR and pHLA data for advancing personalized immunotherapy strategies.

      Strengths:

      (1) Comprehensive Patient Data Collection: The study meticulously collected and analyzed clinical data from 27 CRC patients, ensuring a robust foundation for research findings. The detailed documentation of patient demographics, cancer stages, and pathology information enhances the study's credibility and potential applicability to broader patient populations.<br /> (2) The use of machine learning classifiers (RF, LR, XGB) and the combination of pHLA and pHLA-TCR binding predictions significantly enhance the model's accuracy in identifying immunogenic neoantigens, as evidenced by the high AUC values and improved sensitivity, NPV, and PPV.<br /> (3) The use of experimental validation through ELISpot assays adds a practical dimension to the study, confirming the computational predictions with actual immune responses. The calculation of ranking coverage scores and the comparative analysis between the combined model and the conventional NetMHCpan method demonstrate the superior performance of the combined approach in accurately ranking immunogenic neoantigens.<br /> (4) The use of experimental validation through ELISpot assays adds a practical dimension to the study, confirming the computational predictions with actual immune responses.

      Weakness:

      The authors have made comprehensive revisions to the original version of the article, and this version has now addressed my concerns.

    1. Reviewer #1 (Public review):

      Summary:

      UGGTs are involved in the prevention of premature degradation for misfolded glycoproteins, by utilizing UGGT1-KO cells and a number of different ERAD substrates. They proposed a concept by which the fate of glycoproteins can be determined by a tug-of-war between UGGTs and EDEMs.

      Strengths:

      The authors provided a wealth of data to indicate that UGGT1 competes with EDEMs, which promotes the glycoprotein degradation.

      Weaknesses:

      NA

    1. Reviewer #1 (Public review):

      Summary:

      Wang and colleagues conducted a study to determine the neurotransmitter identity of all neurons in C. elegans hermaphrodites and males. They used CRISPR technology to introduce fluorescent gene expression reporters into the genomic loci of NT pathway genes. This approach is expected to better reflect in vivo gene expression compared to other methods like promoter- or fosmid-based transgenes, or available scRNA datasets. The study presents several noteworthy findings, including sexual dimorphisms, patterns of NT co-transmission, neuronal classes that likely use NTs without direct synthesis, and potential identification of unconventional NTs (e.g. betaine releasing neurons). The data is well-described and critically discussed, including a comparison with alternative methods. Although many of the observations and proposals have been previously discussed by the Hobert lab, the current study is particularly valuable due to its comprehensiveness. This NT atlas is the most complete and comprehensive of any nervous system that I am aware of, making it an extremely important tool for the community.

      Strengths:

      Very compelling study presenting the most comprehensive neurotransmitter (NT) map of any model so far, using state-of-the art tools and validations. The work is very important not only as a resource but also for our understanding that (NT) function of neurons is best understood taking into consideration the full set of genes implicated in NT metabolism and transport.

      Weaknesses:

      None, all have been addressed.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors investigate the role of the melanocortin system in puberty onset. They conclude that proopiomelanocortin (POMC) neurons within the arcuate nucleus of the hypothalamus provide important but differing input to kisspeptin neurons in the arcuate or rostral hypothalamus.

      Strengths:

      • innovative and novel
      • technically sound
      • well-designed
      • thorough

      Weaknesses:

      There were no major weaknesses identified.

    1. Reviewer #1 (Public Review):

      Summary:

      This paper applies methods for segmentation, annotation, and visualization of acoustic analysis to zebra finch song. The paper shows that these methods can be used to predict the stage of song development and to quantify acoustic similarity. The methods are solid and are likely to provide a useful tool for scientists aiming to label large datasets of zebra finch vocalizations. The paper has two main parts: 1) establishing a pipeline/ package for analyzing zebra finch birdsong and 2) a method for measuring song imitation.

      Strengths:

      It is useful to see existing methods for syllable segmentation compared to new datasets.

      It is useful, but not surprising, that these methods can be used to predict developmental stage, which is strongly associated with syllable temporal structure.

      It is useful to confirm that these methods can identify abnormalities in deafened and isolated songs.

      Weaknesses:

      For the first part, the implementation seems to be a wrapper on existing techniques. For instance, the first section talks about syllable segmentation; they made a comparison between whisperseg (Gu et al, 2024), tweetynet (Cohen et al, 2022), and amplitude thresholding. They found that whisperseg performed the best, and they included it in the pipeline. They then used whisperseg to analyze syllable duration distributions and rhythm of birds of different ages and confirmed past findings on this developmental process (e.g. Aronov et al, 2011). Next, based on the segmentation, they assign labels by performing UMAP and HDBScan on the spectrogram (nothing new; that's what people have been doing). Then, based on the labels, they claimed they developed a 'new' visualization - syntax raster ( line 180 ). That was done by Sainburg et. al. 2020 in Figure 12E and also in Cohen et al, 2020 - so the claim to have developed 'a new song syntax visualization' is confusing. The rest of the paper is about analyzing the finch data based on AVN features (which are essentially acoustic features already in the classic literature).

      The second part may be something new, but there are opportunities to improve the benchmarking. It is about the pupil-tutor imitation analysis. They introduce a convolutional neural network that takes triplets as an input (each tripled is essentially 3 images stacked together such that you have (anchor, positive, negative), Anchor is a reference spectrogram from, say finch A; positive means a different spectrogram with the same label as anchor from finch A, and negative means a spectrogram not related to A or different syllable label from A. The network is then trained to produce a low-dimensional embedding by ensuring the embedding distance between anchor and positive is less than anchor and negative by a certain margin. Based on the embedding, they then made use of earth mover distance to quantify the similarity in the syllable distribution among finches. They then compared their approach performance with that of sound analysis pro (SAP) and a variant of SAP. A more natural comparison, which they didn't include, is with the VAE approach by Goffinet et al. In this paper (https://doi.org/10.7554/eLife.67855, Fig 7), they also attempted to perform an analysis on the tutor pupil song.

    1. Reviewer #1 (Public Review):

      Summary:

      Zhou and colleagues developed a computational model of replay that heavily builds on cognitive models of memory in context (e.g., the context-maintenance and retrieval model), which have been successfully used to explain memory phenomena in the past. Their model produces results that mirror previous empirical findings in rodents and offers a new computational framework for thinking about replay.

      Strengths:

      The model is compelling and seems to explain a number of findings from the rodent literature. It is commendable that the authors implement commonly used algorithms from wakefulness to model sleep/rest, thereby linking wake and sleep phenomena in a parsimonious way. Additionally, the manuscript's comprehensive perspective on replay, bridging humans and non-human animals, enhanced its theoretical contribution.

      Weaknesses:

      This reviewer is not a computational neuroscientist by training, so some comments may stem from misunderstandings. I hope the authors would see those instances as opportunities to clarify their findings for broader audiences.

      (1) The model predicts that temporally close items will be co-reactivated, yet evidence from humans suggests that temporal context doesn't guide sleep benefits (instead, semantic connections seem to be of more importance; Liu and Ranganath 2021, Schechtman et al 2023). Could these findings be reconciled with the model or is this a limitation of the current framework?

      (2) During replay, the model is set so that the next reactivated item is sampled without replacement (i.e., the model cannot get "stuck" on a single item). I'm not sure what the biological backing behind this is and why the brain can't reactivate the same item consistently. Furthermore, I'm afraid that such a rule may artificially generate sequential reactivation of items regardless of wake training. Could the authors explain this better or show that this isn't the case?

      (3) If I understand correctly, there are two ways in which novelty (i.e., less exposure) is accounted for in the model. The first and more talked about is the suppression mechanism (lines 639-646). The second is a change in learning rates (lines 593-595). It's unclear to me why both procedures are needed, how they differ, and whether these are two different mechanisms that the model implements. Also, since the authors controlled the extent to which each item was experienced during wakefulness, it's not entirely clear to me which of the simulations manipulated novelty on an individual item level, as described in lines 593-595 (if any).

      As to the first mechanism - experience-based suppression - I find it challenging to think of a biological mechanism that would achieve this and is selectively activated immediately before sleep (somehow anticipating its onset). In fact, the prominent synaptic homeostasis hypothesis suggests that such suppression, at least on a synaptic level, is exactly what sleep itself does (i.e., prune or weaken synapses that were enhanced due to learning during the day). This begs the question of whether certain sleep stages (or ultradian cycles) may be involved in pruning, whereas others leverage its results for reactivation (e.g., a sequential hypothesis; Rasch & Born, 2013). That could be a compelling synthesis of this literature. Regardless of whether the authors agree, I believe that this point is a major caveat to the current model. It is addressed in the discussion, but perhaps it would be beneficial to explicitly state to what extent the results rely on the assumption of a pre-sleep suppression mechanism.

      (4) As the manuscript mentions, the only difference between sleep and wake in the model is the initial conditions (a0). This is an obvious simplification, especially given the last author's recent models discussing the very different roles of REM vs NREM. Could the authors suggest how different sleep stages may relate to the model or how it could be developed to interact with other successful models such as the ones the last author has developed (e.g., C-HORSE)? Finally, I wonder how the model would explain findings (including the authors') showing a preference for reactivation of weaker memories. The literature seems to suggest that it isn't just a matter of novelty or exposure, but encoding strength. Can the model explain this? Or would it require additional assumptions or some mechanism for selective endogenous reactivation during sleep and rest?

      (5) Lines 186-200 - Perhaps I'm misunderstanding, but wouldn't it be trivial that an external cue at the end-item of Figure 7a would result in backward replay, simply because there is no potential for forward replay for sequences starting at the last item (there simply aren't any subsequent items)? The opposite is true, of course, for the first-item replay, which can't go backward. More generally, my understanding of the literature on forward vs backward replay is that neither is linked to the rodent's location. Both commonly happen at a resting station that is further away from the track. It seems as though the model's result may not hold if replay occurs away from the track (i.e. if a0 would be equal for both pre- and post-run).

      (6) The manuscript describes a study by Bendor & Wilson (2012) and tightly mimics their results. However, notably, that study did not find triggered replay immediately following sound presentation, but rather a general bias toward reactivation of the cued sequence over longer stretches of time. In other words, it seems that the model's results don't fully mirror the empirical results. One idea that came to mind is that perhaps it is the R/L context - not the first R/L item - that is cued in this study. This is in line with other TMR studies showing what may be seen as contextual reactivation. If the authors think that such a simulation may better mirror the empirical results, I encourage them to try. If not, however, this limitation should be discussed.

      (7) There is some discussion about replay's benefit to memory. One point of interest could be whether this benefit changes between wake and sleep. Relatedly, it would be interesting to see whether the proportion of forward replay, backward replay, or both correlated with memory benefits. I encourage the authors to extend the section on the function of replay and explore these questions.

      (8) Replay has been mostly studied in rodents, with few exceptions, whereas CMR and similar models have mostly been used in humans. Although replay is considered a good model of episodic memory, it is still limited due to limited findings of sequential replay in humans and its reliance on very structured and inherently autocorrelated items (i.e., place fields). I'm wondering if the authors could speak to the implications of those limitations on the generalizability of their model. Relatedly, I wonder if the model could or does lead to generalization to some extent in a way that would align with the complementary learning systems framework.

    1. Reviewer #1 (Public review):

      In this manuscript, the authors address an important issue in Babesia research by repurposing Cipargamin (CIP) as a potential therapeutic against selective Babesia spp. In this study, CIP demonstrated potent in vitro inhibition of B. bovis and B. gibsoni with IC50 values of 20.2 {plus minus} 1.4 nM and 69.4 {plus minus} 2.2 nM, respectively, and the in vivo efficacy against Babesia spp using mouse model. The authors identified two key resistance mutations in the BgATP4 gene (BgATP4L921I and BgATP4L921V) and explored their implications through phenotypic characterization of the parasite using cell biological experiments, complemented by in silico analysis. Overall, the findings are promising and could significantly advance Babesia treatment strategies.

      Strengths:

      In this manuscript, the authors effectively repurpose Cipargamin (CIP) as a potential treatment for Babesia spp. They provide compelling in vitro and in vivo data showing strong efficacy. Key resistance mutations in the BgATP4 gene are identified and analyzed through both phenotypic and in silico methods, offering valuable insights for advancing treatment strategies.

      Weaknesses:

      The manuscript explores important aspects of drug repurposing and rational drug design using Cipargamin (CIP) against Babesia. However, several weaknesses should be addressed. The study lacks novelty as similar research on Cipargamin has been conducted, and the experimental design could be improved. The rationale for choosing CIP over other ATP4-targeting compounds is not well-explained. Validation of mutations relies heavily on in silico predictions without sufficient experimental support. The Ion Transport Assay has limitations and would benefit from additional assays like Radiolabeled Ion Flux and Electrophysiological Assays. Also, the study lacks appropriate control drugs and detailed functional characterization. Further clarity on mutation percentages, additional safety testing, and exploration of cross-resistance would strengthen the findings.

      (1) It is commendable to explore drug repurposing, drug deprescribing, drug repositioning, and rational drug design, especially using established ATP4 inhibitors that are well-studied in Plasmodium and other protozoan parasites. While the study provides some interesting findings, it appears to lack novelty, as similar investigations of Cipargamin on other protozoan parasites have been conducted. The study does not introduce new concepts, and the experimental design could benefit from refinement to strengthen the results. Additionally, the rationale for choosing CIP over other MMV compounds targeting ATP4 is not clearly articulated. Clarifying the specific advantages CIP may offer against Babesia would be beneficial. Finally, the validation of the identified mutations might be strengthened by additional experimental support, as reliance on in silico predictions alone may not fully address the functional impact, particularly given the potential ambiguity of the mutations (BgATP4 L to V and I).

      (2) Conducting an Ion Transport Assay is useful but has limitations. Non-specific binding or transport by other cellular components can lead to inaccurate results, causing false positives or negatives and making data interpretation difficult. Indirect measurements, like changes in fluorescence or electrical potential, can introduce artifacts. To improve accuracy, consider additional assays such as<br /> a. Radiolabeled Ion Flux Assay: tracks the movement of Na^+ using radiolabeled ions, providing direct evidence of ion transport.<br /> b. Electrophysiological Assay: measures ionic currents in real-time with patch-clamp techniques, offering detailed information about ATP4 activity.

      (3) In-silico predictions can provide plausible outcomes, but it is essential to evaluate how the recombinant purified protein and ligand interact and function at physiological levels. This aspect is currently missing and should be included. For example, incorporating immunoprecipitation and ATPase activity assays with both wild-type and mutant proteins, as well as detailed kinetic studies with Cipargamin, would be recommended to validate the findings of the study.

      (4) The study lacks specific suitable control drugs tested both in vitro and in vivo. For accurate drug assessment, especially when evaluating drugs based on a specific phenotype, such as enlarged parasites, it is important to use ATP4 gene-specific inhibitors. Including similar classes of drugs, such as Aminopyrazoles, Dihydroisoquinolines, Pyrazoleamides, Pantothenamides, Imidazolopiperazines (e.g., GNF179), and Bicyclic Azetidine Compounds, would provide more comprehensive validation.

      (5) Functional characterization of CIP through microscopic examination and quantification for assessing parasite size enlargement is not entirely reliable. A Flow Cytometry-Based Assay is recommended instead 9 along with suitable control antiparasitic drugs). To effectively monitor Cipargamin's action, conducting time-course experiments with 6-hour intervals is advisable rather than relying solely on endpoint measurements. Additionally, for accurate assessment of parasite morphology, obtaining representative qualitative images using Scanning Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM) for treated versus untreated samples is recommended for precise measurements.

      (6) A notable contradiction observed is that mutant cells displayed reduced efficacy and affinity but more pronounced phenotypic effects. The BgATP4L921I mutation shows a 2x lower susceptibility (IC50 of 887.9 {plus minus} 61.97 nM) and a predicted binding affinity of -6.26 kcal/mol with CIP. However, the phenotype exhibits significantly lower Na+ concentration in BgATP4L921I (P = 0.0087) (Figure 3E).

      (7) The manuscript does not clarify the percentage of mutations, and the number of sequence iterations performed on the ATP4 gene. It is also unclear whether clonal selection was carried out on the resistant population. If mutations are not present in 100% of the resistant parasites, please indicate the ratio of wild-type to mutant parasites and represent this information in the figure, along with the chromatograms.

      (8) While the compound's toxicity data is well-established, it is advisable to include additional testing in epithelial cells and liver-specific cell lines (e.g., HeLa, HCT, HepG2) if feasible for the authors. This would provide a more comprehensive assessment of the compound's safety profile.

      (9) In the in vivo efficacy study, recrudescent parasites emerged after 8 days of treatment. Did these parasites harbor the same mutation in the ATP4 gene? The authors did not investigate this aspect, which is crucial for understanding the basis of recrudescence.

      (10) The authors should explain their choice of Balb/c mice for evaluating CIP efficacy, as these mice clear the infection and may not fully represent the compound's effectiveness. Investigating CIP efficacy in SCID mice would be valuable, as they provide a more reliable model and eliminate the influence of the immune system. The rationale for not using SCID mice should be clarified.

      (11) Do the in vitro-resistant parasites show any potential for cross-resistance with commonly used antiparasitic drugs? Have the authors considered this possibility, and what are their expectations regarding cross-resistance?

    1. Reviewer #1 (Public review):

      The authors used fluorescence microscopy, image analysis, and mathematical modeling to study the effects of membrane affinity and diffusion rates of MinD monomer and dimer states on MinD gradient formation in B. subtilis. To test these effects, the authors experimentally examined MinD mutants that lock the protein in specific states, including Apo monomer (K16A), ATP-bound monomer (G12V), and ATP-bound dimer (D40A, hydrolysis defective), and compared to wild-type MinD. Overall, the experimental results support the conclusion that reversible membrane binding of MinD is critical for the formation of the MinD gradient, but that the binding affinities between monomers and dimers are similar.

      The modeling part is a new attempt to use the Monte Carlo method to test the conditions for the formation of the MinD gradient in B. subtilis. The modeling results provide good support for the observations and find that the MinD gradient is sensitive to different diffusion rates between monomers and dimers. This simulation is based on several assumptions and predictions, which raises new questions that need to be addressed experimentally in the future. However, the current story is sufficient without testing these assumptions or predictions.

    1. Reviewer #1 (Public review):

      Summary:

      In this work, the authors investigate the molecular dynamics of MinD, a component of the Bacillus subtilis Min system, in vitro and in vivo. In Escherichia coli the Min system is highly dynamic and displays rapid pole-to-pole oscillation whereby a time average minimum of the Min proteins at mid-cell is established. However, in B. subtilis, this is not the case, and there is no MinE present. MinD in B. subtilis dynamically relocalizes from the poles to division sites and binds to MinC and MinJ, which mediates its interaction with DivIVA. This paper reports the biochemical characterization of B. subtilis MinD in vitro and dynamics of MinD variants in vivo, providing mechanistic insight into the mechanism of dynamic localization.

      Strengths:

      In the current study, the authors perform a detailed biochemical characterizion of the in vitro ATPase activity of MinD and demonstrate that rapid hydrolysis is elicited by adding phospholipids. They further show using a collection of substitution mutants of MinD that both monomers and dimers bind to the membrane, and ATP occupancy changes the on and off rates. Identification, quantification, and tracking of discrete Halo-MinD populations were nicely done and showed that mutations in MinD alter dynamic localization, correlating with PL binding on and off rates in vitro.

      Weaknesses:

      While the study shows that MinD in B. subtilis utilizes a different (MinE-independent) activation mechanism, it remains to be determined the extent to which MinJ and/or MinC play a role.

    1. Reviewer #1 (Public Review):

      Summary:

      In previously published work, the authors found that Transforming Growth Factor β Activated Kinase 1 (TAK1) may regulate esophageal squamous cell carcinoma (ESCC) tumor cell proliferation via the RAS/MEK/ERK axis. They explore the mechanisms for TAK1 as a possible tumor suppressor, demonstrating phospholipase C epsilon 1 as an effector of tumor cell migration, invasion and metastatic potential.

      They explore the mechanisms for TAK1 as a possible tumor suppressor, demonstrating phospholipase C epsilon 1 as an effector of tumor cell migration, invasion and metastatic potential.

      Strengths:

      The authors show in vitro that TAK1 overexpression reduces tumor cell migration and invasion while TAK1 knockdown promotes a mesenchymal phenotype (epithelial-mesenchymal transition) and enhances migration and invasion. To explore possible mechanisms of action, the authors focused on phospholipase C epsilon 1 (PLCE1) as a potential effector, having identified this protein in co-immunoprecipitation experiments. Further, they demonstrate that TAK1-mediated phosphorylation of PLCE1 is inhibitory. Each of the observations is supported by different experimental strategies, e.g. use of different approaches for knockdown (pharmacologic, RNA inhibition, CRISPR/Cas). Xenograft experiments showed that suppression/loss of TAK1 is associated with more frequent metastases and conversely that PLCE1 is associated positively with xenograft metastases. A considerable amount of experimental data is presented for review, including supplemental data, that show that TAK1 regulation may be important in ESCC development.

      Weaknesses:

      As noted by the authors, immunoprecipitation (IP) experiments identified a number (24) of proteins as potential targets for the TAK1 ser/thr kinase. Prior work (cited as Shi et al, 2021) focused on a different phosphorylation target for TAK1, Ras association domain family 9 (RASSF9), but a more comprehensive discussion of the co-IP experiments would help place this work in better context.

    1. Reviewer #1 (Public review):

      Summary:

      Soo-Yeon Hwang et al. synthesized and characterized a new set of Chalcone- and Pyrazoline-derived molecules targeting the interaction between ELF3, a transcription factor, and MED23, a coactivator for HER2 transcription. The authors employed biochemical analysis, cell-based assays, and an in vivo xenograft model to demonstrate that the lead compound, Compound 10, inhibits HER2 transcription and protein expression, subsequently inducing anticancer activity in gastric cancer models, particularly in trastuzumab-resistant cell lines. The obtained data is robust and supports the potential anticancer efficacy of Compound 10 for HER2+ gastric cancer.

      Strengths:

      The current manuscript proposes an alternative strategy for targeting HER2-overexpressing cancers by reducing HER2 transcription levels. The study presents compelling evidence that the lead compound, Compound 10, disrupts the binding of ELF3 to MED23, thereby inhibiting HER2 transcription. Notably, cell-based assays and xenograft models demonstrated the compound's significant antitumor activity in gastric cancer models.

    1. Reviewer #1 (Public review):

      Summary:

      In this study, James Lee, Lu Bai, and colleagues use a multifaceted approach to investigate the relationship between transcription factor condensate formation, transcription, and 3D gene clustering of the MET regulon in the model organism S. cerevisiae. This study represents a second clear example of inducible transcriptional condensates in budding yeast, as most evidence for transcriptional condensates arises from studies of mammalian systems. In addition, this study links the genomic location of transcriptional condensates to the potency of transcription of a reporter gene regulated by the master transcription factor contained in the condensate. The strength of evidence supporting these two conclusions is strong. Less strong is evidence supporting the claim that Met4-containing condensates mediate the clustering of genes in the MET regulon.

      Strengths:

      The manuscript is for the most part clearly written, with the overriding model and specific hypothesis being tested clearly explained. Figure legends are particularly well written. An additional strength of the manuscript is that most of the main conclusions are supported by the data. This includes the propensity of Met4 and Met32 to form puncta-like structures under inducing conditions, formation of Met32-containing LLPS-like droplets in vitro (within which Met4 can colocalize), colocalization of Met4-GFP with Met4-target genes under inducing conditions, enhanced transcription of a Met3pr-GFP reporter when targeted within 1.5 - 5 kb of select Met4 target genes, and most impressively, evidence that several MET genes appear to reposition under transcriptionally inducing conditions. The latter is based on a recently reported novel in vivo methylation assay, MTAC, developed by the Bai lab.

      Comments on Revision:

      The authors have adequately addressed most of my concerns. However, the most salient issue - that the work fails to show convincing evidence that nuclear condensates per se drive MET gene clustering - remains. Since the genetic approach led to ambiguous results, another way to link MET gene clustering to TF condensate formation is to perturb the condensates with 1,6-hexanediol. If 1,6-HD treatment dissolves condensates and concomitant MET clustering (while the impact of 2,5-HD is much less) then the conclusion is more solid. Absent such evidence, the authors are left with a correlation, and they should consider toning down the title and abstract (and conclusions stated elsewhere). For example, a more accurate title might be "Transcription Factor Condensates Correlate with MET Gene Clustering and Mediate Enhancement in Gene Expression".

    1. Reviewer #1 (Public Review):

      Summary:

      Wang, Y. et al. used a silicone wire embolus to definitively and acutely clot the pterygopalatine ophthalmic artery in addition to carotid artery ligation to completely block blood supply to the mouse inner retina, which mimic clinical acute retinal artery occlusion. A detailed characterization of this mouse model determined the time course of inner retina degeneration and associated functional deficits, which closely mimic human patients. Whole retina transcriptome profiling and comparison revealed distinct features associated with ischemia, reperfusion, and different model mechanisms. Interestingly and importantly, this team found a sequential event including reperfusion-induced leukocyte infiltration from blood vessels, residual microglial activation, and neuroinflammation that may lead to neuronal cell death.

      Strengths:

      Clear demonstration of the surgery procedure with informative illustrations, images, and superb surgical videos.

      Two time points of ischemia and reperfusion were studied with convincing histological and in vivo data to demonstrate the time course of various changes in retinal neuronal cell survivals, ERG functions, and inner/outer retina thickness.

      The transcriptome comparison among different retinal artery occlusion models provides informative evidence to differentiate these models.

      The potential applications of the in vivo retinal ischemia-reperfusion model and relevant readouts demonstrated by this study will certainly inspire further investigation of the dynamic morphological and functional changes of retinal neurons and glial cell responses during disease progression and before and after treatments.

      Weaknesses:

      The revised manuscript has been significantly improved in clarity and readability. It has addressed all my questions convincingly.

    1. Reviewer #1 (Public Review):

      Summary:

      Building upon their famous tool for the deconvolution of human transcriptomics data (EPIC), Gabriel et al. implemented a new methodology for the quantification of the cellular composition of samples profiled with Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq). To build a signature for ATAC-seq deconvolution, they first created a compendium of ATAC-seq data and derived chromatin accessibility marker peaks and reference profiles for 12 cell types, encompassing immune cells, endothelial cells, and fibroblasts. Then, they coupled this novel signature with the EPIC deconvolution framework based on constrained least-square regression to derive a dedicated tool called EPIC-ATAC. The method was then assessed using real and pseudo-bulk ATAC-seq data from human peripheral blood mononuclear cells (PBMC) and, finally, applied to ATAC-seq data from breast cancer tumors to show it accurately quantifies their immune contexture.

      Strengths:

      Overall, the work is of very high quality. The proposed tool is timely; its implementation, characterization, and validation are based on rigorous methodologies and results in robust estimates. The newly-generated, validation data and the code are publicly available and well-documented. Therefore, I believe this work and the associated resources will greatly benefit the scientific community.

      Weaknesses:

      In the benchmarking analysis, EPIC-ATAC was compared also to deconvolution methods that were originally developed for transcriptomics and not for ATAC-seq data. However, the authors described in detail the specific settings used to analyze this different data modality as robustly as possible, and they discussed possible limitations and ideas for future improvement.

    1. Reviewer #1 (Public review):

      Summary:

      Using multi-region two-photon calcium imaging, the manuscript meticulously explores the structure of noise correlations (NCs) across mouse visual cortex and uses this information to make inferences about the organization of communication channels between primary visual cortex (V1) and higher visual areas (HVAs). Using visual responses to grating stimuli, the manuscript identifies 6 tuning groups of visual cortex neurons, and finds that NCs are highest among neurons belonging to the same tuning group whether or not they are found in the same cortical area. The NCs depend on the similarity of tuning of the neurons (their signal correlations) but are preserved across different stimulus sets - noise correlations recorded using drifting gratings are highly correlated with those measured using naturalistic videos. Based on these findings, the manuscript concludes that populations of neurons with high NCs constitute discrete communication channels that convey visual signals within and across cortical areas.

      Strengths:

      Experiments and analyses are conducted to a high standard and the robustness of noise correlation measurements is carefully validated. To control for potential influences of behaviour-related top-down modulation of noise correlations, the manuscript uses measurements of pupil dynamics as a proxy for behavioural state and shows that this top-down modulation cannot explain the stability of noise correlations across stimuli.

      Weaknesses:

      The interpretation of noise correlation measurements as a proxy from network connectivity is fraught with challenges. While the data clearly indicate the existence of distributed functional ensembles, the notion of communication channels implies the existence of direct anatomical connections between them, which noise correlations cannot measure.

      The traditional view of noise correlations is that they reflect direct connectivity or shared inputs between neurons. While it is valid in a broad sense, noise correlations may reflect shared top-down input as well as local or feedforward connectivity. This is particularly important since mouse cortical neurons are strongly modulated by spontaneous behavior (e.g. Stringer et al, Science, 2019). Therefore, noise correlation between a pair of neurons may reflect whether they are similarly modulated by behavioral state and overt spontaneous behaviors. Consequently, noise correlation alone cannot determine whether neurons belong to discrete communication channels.

    1. Reviewer #1 (Public Review):

      Summary:

      This study uses an online cognitive task to assess how reward and effort are integrated in a motivated decision-making task. In particular the authors were looking to explore how neuropsychiatric symptoms, in particular, apathy and anhedonia, and circadian rhythms affect behavior in this task. Amongst many results, they found that choice bias (the degree to which integrated reward and effort affect decisions) is reduced in individuals with greater neuropsychiatric symptoms, and late chronotypes (being an 'evening person').

      Strengths:

      The authors recruited participants to perform the cognitive task both in and out of sync with their chronotypes, allowing for the important insight that individuals with late chronotypes show a more reduced choice bias when tested in the morning.<br /> Overall, this is a well-designed and controlled online experimental study. The modelling approach is robust, with care being taken to both perform and explain to the readers the various tests used to ensure the models allow the authors to sufficiently test their hypotheses.

      Weaknesses:

      This study was not designed to test the interactions of neuropsychiatric symptoms and chronotypes on decision making, and thus can only make preliminary suggestions regarding how symptoms, chronotypes and time-of-assessment interact.

    1. Reviewer #1 (Public review):

      Summary:

      This important study investigated the role of oxytocin (OT) neurons in the paraventricular nucleus (PVN) and their projections to the medial prefrontal cortex (mPFC) in regulating pup care and infanticide behaviors in mandarin voles. The researchers used techniques like immunofluorescence, optogenetics, OT sensors, and peripheral OT administration. Activating OT neurons in the PVN reduced the time it took pup-caring male voles to approach and retrieve pups, facilitating pup care behavior. However, this activation had no effect on females. Interestingly, this same PVN OT neuron activation also reduced the time for both male and female infanticidal voles to approach and attack pups, suggesting PVN OT neuron activity can promote pup care while inhibiting infanticide behavior. Inhibition of these neurons promoted infanticide. Stimulating PVN->mPFC OT projections facilitated pup care in males and in infanticide prone voles, activation of these terminals prolonged latency to approach and attack. Inhibition of PVN->mPFC OT projections promoted infanticide. Peripheral OT administration increased pup care in males and reduced infanticide in both sexes. However, some results differed in females, suggesting other mechanisms may regulate female pup care.

      Strengths:

      This multi-faceted approach provides converging evidence and strengthens the conclusions drawn from the study and make them very convincing. Additionally, the study examines both pup care and infanticide behaviors, offering insights into the mechanisms underlying these contrasting behaviors. The inclusion of both male and female voles allows for the exploration of potential sex differences in the regulation of pup-directed behaviors. The peripheral OT administration experiments also provide valuable information for potential clinical applications and wildlife management strategies.

      Weaknesses:

      While the study presents exciting findings, there are several weaknesses. The sample sizes used in some experiments, such as the Fos study and optogenetic manipulations, appear to be small, which may limit the statistical power and generalizability of the results.

      There is potential effect of manipulating OT neurons on the release of other neurotransmitters (or the influence of other neurochemicals or brain regions) on pup-directed behaviors, especially in females, are not fully explored. Additionally, it is unclear whether back-propagation of action potentials during optogenetic manipulations causes the same behavioral effect as direct stimulation of PVN OT cells. However, the authors now discuss these possibilities. It is also uncertain whether more OT neurons were manipulated in females compared to males. All other comments have been addressed by the authors.

    1. Reviewer #1 (Public review):

      Summary:

      The authors of this study investigated the development of interoceptive sensitivity in the context of cardiac and respiratory interoception in 3-, 9-, and 18-month-old infants using a combination of both cross-sectional and longitudinal designs. They utilised the cardiac interoception paradigm developed by Maister et al (2017) and also developed a new paradigm to investigate respiratory interoception in infants. The main findings of this research are that 9-month-old infants displayed a preference for stimuli presented synchronously with their own heartbeat and respiration. The authors found less reliable effects in the 18-month-old group, and this was especially true for the respiratory interoceptive data. The authors replicated a visual preference for synchrony over asynchrony for the cardiac domain in 3-month-old infants, while they found inconclusive evidence regarding the respiratory domain. Considering the developmental nature of the study, the authors also investigated the presence of developmental trajectories and associations between the two interoceptive domains. They found evidence for a relationship between cardiac and respiratory interoceptive sensitivity at 18 months only and preliminary evidence for an increase in respiratory interoception between 9 and 18 months.

      Strengths:

      The conclusions of this paper are mostly well supported by data, and the data analysis procedures are rigorous and well justified. The main strengths of the paper are:

      - A first attempt to explore the association between two different interoceptive domains. How different organ-specific axes of interoception relate to each other is still open and exploring this from a developmental lens can help shed light into possible relationships. The authors have to be commended for developing a novel interoceptive tasks aimed at assessing respiratory interoceptive sensitivity in infants and toddlers, and for trying to assess the relationship between cardiac and respiratory interoception across developmental time.<br /> - A thorough justification of the developmental ages selected for the study. The authors provide a rationale behind their choice to examine interoceptive sensitivity at 3, 9, and 18-months of age. These are well justified based on the literature pertaining to self- and social development. Sometimes, I wondered whether explaining the link between these self and social processes and interoception would have been beneficial as a reader not familiar with the topics may miss the point.<br /> - An explanation of direction of looking behaviour using latent curve analysis. I found this additional analysis extremely helpful in providing a better understanding of the data based on previous research and analytical choices. As the authors explain in the manuscript, it is often difficult to interpret the direction of infant looking behaviour as novelty and familiarity preferences can also be driven by hidden confounders (e.g. task difficulty). The authors provide compelling evidence that analytical choices can explain some of these effects. Beyond the field of interoception, these findings will be relevant to development psychologists and will inform future studies using looking time as a measure of infants' ability to discriminate among stimuli.<br /> - The use of simulation analysis to account for small sample size. The authors acknowledge that some of the effects reported in their study could be explained by a small sample size (i.e. the 3-month-olds and 18-month-olds data). Using a simulation approach, the authors try to overcome some of these limitations and provide convincing evidence of interoceptive abilities in infancy and toddlerhood (but see also my next point).

      Weaknesses:

      - While the research question is timely and the methodology is detailed, there is a critical flaw in the experimental design: the lack of randomization of stimuli due to an error in the programming script. The authors very honestly report this error and have performed additional analyses to investigate its potential impact on the study's results. Unfortunately, I am not fully convinced these analyses provide enough reassurance and I believe the technical error still undermines the validity of the findings, making it difficult to draw meaningful conclusions.

    1. Reviewer #1 (Public review):

      Amason et al. investigated the formation of granulomas in response to Chromobacterium violaceum infection, aiming to uncover the cellular mechanisms governing the granuloma response. They identify spatiotemporal gene expression of chemokines and receptors associated with the formation and clearance of granulomas, with a specific focus on those involved in immune trafficking, generating a valuable spatial transcriptomic reference. By analyzing the presence or absence of chemokine/receptor RNA expression, they infer the importance of immune cells in resolving infection. Despite observing increased expression of neutrophil-recruiting chemokines, treatment with reparixin (an inhibitor of CXCR1 and CXCR2) did not inhibit neutrophil recruitment during infection. Focusing on monocyte trafficking, they found that CCR2 knockout mice infected with C. violaceum were unable to form granulomas, ultimately succumbing to infection.

      Readers should note that due to the resolution of the spatial data, it is difficult to associate gene expression differences with individual cell types; the authors focus instead on changes in chemokines and chemokine receptors, and perform experiments to evaluate the importance of CCR2.

      Comments on the revised version:

      The authors have addressed all of my previous comments.

    1. Reviewer #1 (Public review):

      The work by Ginatt et al. uses genome-scale metabolic modeling to identify and characterize trophic interactions between rhizosphere-associated bacteria. Beyond identifying microbial species associated with specific host and soil traits (e.g., disease tolerance), a detailed understanding of the interactions underlying these associations is necessary for developing targeted microbiome-centered interventions for plant health. It has nonetheless remained challenging to define the roles of specific organisms and metabolic species in natural rhizobiomes. Here, the authors combine microbial compositional data obtained through metagenomic sequencing with a new collection of genome-scale models to predict interactions in the native rhizosphere communities of apple rootstocks. To do this, they have established processes to integrate these sources of data and model specific trophic exchanges, which they use to obtain testable hypotheses for targeted modulation of microbiota members in situ.

      The authors carry out a careful model curation process based on metagenomic sequencing data and existing model generation tools, which, together with basing the in silico medium composition on known root exudates, strengthens their predictions of interaction network features. Moreover, its reliance on genome-scale models provides a broader basis for linking sequence-based information to predictions of function on a multispecies level beyond rhizosphere microbiomes.

      Having generated a set of predicted trophic interactions, the authors carried out a detailed analysis linking features of these interactions to organism taxonomy and broader ecosystem properties. Intriguingly, the organisms predicted to grow in the first iteration of their framework (i.e., on only root exudates) broadly correspond to taxonomic groups experimentally shown to benefit from these compounds. Additionally, the simulations predicted some patterns of vitamin and amino acid secretion that are known to form the basis for interactions in the rhizosphere. Together, these outcomes underscore the applicability of this method to help disentangle trophic interaction networks in complex microbiomes.

      The methodology described in this paper represents a useful and promising framework to better understand the complexity of microbial interaction networks in situ. In particular, the authors' simulation of trophic interactions based on cellulose degradation have generated predictions of interactions that can more readily be validated. While a more complete analysis of the method's sensitivity to environmental composition is still needed to fully interpret its conclusions - particularly those predicting the inability of many of the in silico organisms to produce biomass - it represents a valuable addition to the growing toolkit of computational and experimental methods for generating educated hypotheses on complex trophic networks.

    1. Reviewer #1 (Public review):

      Summary:

      The researchers demonstrated that when cytokine priming is combined with exposure to pathogens or pathogen-associated molecular patterns, human alveolar macrophages and monocyte-derived macrophages undergo metabolic adaptations, becoming more glycolytic while reducing oxidative phosphorylation. This metabolic plasticity is more in monocyte derived macrophages as compared to alveolar macrophages.

      Strengths:

      This study presents evidence of metabolic reprogramming in human macrophages, which significantly contributes to our existing understanding of this field primarily derived from murine models.

    1. Reviewer #1 (Public review):

      Summary:

      It is suggested that for each limb, the RG (rhythm generator) can operate in three different regimes: a non-oscillating state-machine regime and a flexor driven and a classical half-center oscillatory regime. This means that the field can move away from the old concept that there is only room for the classic half-center organization

      Strengths:

      A major benefit of the present paper is that a bridge was made between various CPG concepts ( "a potential contradiction between the classical half-center and flexor-driven concepts of spinal RG operation"). Another important step forward is the proposal about the neural control of slow gait ("at slow speeds ({less than or equal to} 0.35 m/s), the spinal network operates in a state regime and requires external inputs for phase transitions, which can come from limb sensory feedback and/or volitional inputs (e.g. from the motor cortex").

      Weaknesses:

      Some references are missing

    1. Reviewer #1 (Public Review):

      Summary:

      In their paper, Zhan et al. have used Pf genetic data from simulated data and Ghanaian field samples to elucidate a relationship between multiplicity of infection (MOI) (the number of distinct parasite clones in a single host infection) and force of infection (FOI). Specifically, they use sequencing data from the var genes of Pf along with Bayesian modeling to estimate MOI individual infections and use these values along with methods from queueing theory that rely on various assumptions to estimate FOI. They compare these estimates to known FOIs in a simulated scenario and describe the relationship between these estimated FOI values and another commonly used metric of transmission EIR (entomological inoculation rate).

      This approach does fill an important gap in malaria epidemiology, namely estimating the force of infection, which is currently complicated by several factors including superinfection, unknown duration of infection, and highly genetically diverse parasite populations. The authors use a new approach borrowing from other fields of statistics and modeling and make extensive efforts to evaluate their approach under a range of realistic sampling scenarios. However, the write-up would greatly benefit from added clarity both in the description of methods and in the presentation of the results. Without these clarifications, rigorously evaluating whether the author's proposed method of estimating FOI is sound remains difficult. Additionally, there are several limitations that call into question the stated generalizability of this method that should at minimum be further discussed by authors and in some cases require a more thorough evaluation.

      Major comments:

      (1) Description and evaluation of FOI estimation procedure.

      a. The methods section describing the two-moment approximation and accompanying appendix is lacking several important details. Equations on lines 891 and 892 are only a small part of the equations in Choi et al. and do not adequately describe the procedure notably several quantities in those equations are never defined some of them are important to understand the method (e.g. A, S as the main random variables for inter-arrival times and service times, aR and bR which are the known time average quantities, and these also rely on the squared coefficient of variation of the random variable which is also never introduced in the paper). Without going back to the Choi paper to understand these quantities, and to understand the assumptions of this method it was not possible to follow how this works in the paper. At a minimum, all variables used in the equations should be clearly defined.

      b. Additionally, the description in the main text of how the queueing procedure can be used to describe malaria infections would benefit from a diagram currently as written it's very difficult to follow.

      c. Just observing the box plots of mean and 95% CI on a plot with the FOI estimate (Figures 1, 2, and 10-14) is not sufficient to adequately assess the performance of this estimator. First, it is not clear whether the authors are displaying the bootstrapped 95%CIs or whether they are just showing the distribution of the mean FOI taken over multiple simulations, and then it seems that they are also estimating mean FOI per host on an annual basis. Showing a distribution of those per-host estimates would also be helpful. Second, a more quantitative assessment of the ability of the estimator to recover the truth across simulations (e.g. proportion of simulations where the truth is captured in the 95% CI or something like this) is important in many cases it seems that the estimator is always underestimating the true FOI and may not even contain the true value in the FOI distribution (e.g. Figure 10, Figure 1 under the mid-IRS panel). But it's not possible to conclude one way or the other based on this visualization. This is a major issue since it calls into question whether there is in fact data to support that these methods give good and consistent FOI estimates.

      d. Furthermore the authors state in the methods that the choice of mean and variance (and thus second moment) parameters for inter-arrival times are varied widely, however, it's not clear what those ranges are there needs to be a clear table or figure caption showing what combinations of values were tested and which results are produced from them, this is an essential component of the method and it's impossible to fully evaluate its performance without this information. This relates to the issue of selecting the mean and variance values that maximize the likelihood of observing a given distribution of MOI estimates, this is very unclear since no likelihoods have been written down in the methods section of the main text, which likelihood are the authors referring to, is this the probability distribution of the steady state queue length distribution? At other places the authors refer to these quantities as Maximum Likelihood estimators, how do they know they have found the MLE? There are no derivations in the manuscript to support this. The authors should specify the likelihood and include in an appendix an explanation of why their estimation procedure is in fact maximizing this likelihood, preferably with evidence of the shape of the likelihood, and how fine the grid of values they tested is for their mean and variance since this could influence the overall quality of the estimation procedure.

      (2) Limitation of FOI estimation procedure.

      a. The authors discuss the importance of the duration of infection to this problem. While I agree that empirically estimating this is not possible, there are other options besides assuming that all 1-5-year-olds have the same duration of infection distribution as naïve adults co-infected with syphilis. E.g. it would be useful to test a wide range of assumed infection duration and assess their impact on the estimation procedure. Furthermore, if the authors are going to stick to the described method for duration of infection, the potentially limited generalizability of this method needs to be further highlighted in both the introduction, and the discussion. In particular, for an estimated mean FOI of about 5 per host per year in the pre-IRS season as estimated in Ghana (Figure 3) it seems that this would not translate to 4-year-old being immune naïve, and certainly this would not necessarily generalize well to a school-aged child population or an adult population.

      b. The evaluation of the capacity parameter c seems to be quite important and is set at 30, however, the authors only describe trying values of 25 and 30, and claim that this does not impact FOI inference, however it is not clear that this is the case. What happens if the carrying capacity is increased substantially? Alternatively, this would be more convincing if the authors provided a mathematical explanation of why the carrying capacity increase will not influence the FOI inference, but absent that, this should be mentioned and discussed as a limitation.

    1. Reviewer #1 (Public Review):

      Summary:

      In this work, Ritchie and colleagues explore functional consequences of neuronal over-expression or deletion of the MAP3K DLK that their labs and others have strongly implicated in both axon degeneration, neuronal cell death, and axon regeneration. Their recent work in eLife (Li, 2021) showed that inducible over-expression of DLK (or the related LZK) induces neuronal death in the cerebellum. Here, they extend this work to show that inducible over-expression in Vglut1+ neurons also kills excitatory neurons in hippocampal CA1, but not CA3. They complement this very interesting finding with translatomics to quantify genes whose mRNAs are differentially translated in the context of DLK over-expression or knockout, the latter manipulation having little to no effect on the phenotypes measured. The authors note that several genes and pathways are differentially regulated according to whether DLK is over-expressed or knocked out. They note DLK-dependent changes in genes related to synaptic function and the cytoskeleton and ultimately relate this in cultured neurons to findings that DLK over-expression negatively impacts synapse number and changes microtubules and neurites, though with a less obvious correlation.

      Strengths:

      This work represents a conceptual advance in defining DLK-dependent changes in translation. Moreover, the finding that DLK may differentially impact neuronal death will become the basis for future studies exploring whether DLK contributes to differential neuronal susceptibility to death, which is a broadly important topic.

      Weaknesses:

      This seems like two works in parallel that the authors have not yet connected. First is that DLK affects the translation of an interesting set of genes, and second, that DLK(OE) kills some neurons, disrupts their synapses, and affects neurite growth in culture.

      Specific questions:

      (1) Is DLK effectively knocked out? The authors reference the floxxed allele in their 2016 work (PMID: 27511108), however, the methods of this paper say that the mouse will be characterized in a future publication. Has this ever been published? The major concern is that here the authors show that Cre-mediated deletion results in a smaller molecular weight protein and the maintenance of mRNA levels.

      (2) Why does DLK(OE) not kill CA3 neurons? The phenomenon is clear but there is no link to gene expression changes. In fact, the highlighted transcript in this work, Stmn4, changes in a DLK-dependent manner in CA3.

      (3) Why are whole hippocampi analyzed to IP ribosome-associated mRNAs? The authors nicely show a differential effect of DLK on CA1 vs CA3, but then - at least according to their methods ¬- lyse whole hippocampi to perform IP/sequencing. Their data are therefore a mix of cells where DLK does and does not change cell death. The key issue is whether DLK does/does not have an effect based on the expression changes it drives.

      (4) Is the subtle decrease in synapse number (Basson/Homer co-loc.) in the DLK (OE) simply a function of neurons (and their synapses, presumably) having died? At the P15 time point that the authors choose because cell death is minimal, there is still a ~25% reduction in CA1 thickness (Figure 2B), which is larger than the ~15% change in synapses (Figure 5H) they describe.

    1. Reviewer #1 (Public Review):

      This report contains two parts. In the first part, several experiments were carried out to show that CsoR binds to CheA, inhibits CheA phosphorylation, and impairs P. putida chemotaxis. The second part provides some evidence that CsoR is a copper-binding protein, binds to CheA in a copper-dependent manner, and regulates P. putida response to copper, a chemorepellent. Based on these results, a working model is proposed to describe how CsoR coordinates chemotaxis and resistance to copper in P. putida. While the second part of the study is relatively solid, there are some major concerns about the first part.

      Critiques:

      (1) The rigor from prior research is not clear. In addition to talking about other bacterial chemotaxis, the Introduction should briefly summarize previous work on P. putida chemotaxis and copper resistance.

      (2) The rationale for identifying those CheA-binding proteins is vague. CheA has been extensively studied and its functional domains (P1 to P5) have been well characterized. Compared to its counterparts from other bacteria, does P. putida CheA contain a unique motif or domain? Does CsoR bind to other bacterial CheAs or only to P. putida CheA?

      (3) Line 133-136, "Collectively, using pull-down, BTH, and BiFC assays, we identified 16 new CheA-interacting proteins in P. putida." It is surprising that so many proteins were identified but none of them were chemotaxis proteins, in particular those known to interact with CheA, such as CheW, CheY and CheZ, which raises a concern about the specificity of these methods. BTH and BiFC often give false-positive results and thus should be substantiated by other approaches such as co-IP, surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC) along with mutagenesis studies.

      (4) Line 147-149, "Fig. 2a, five strains (WT+pcsoR, WT+pispG, WT+pnfuA, WT+pphaD, and WT+pPP_1644) displayed smaller colony than the control strain (WT+pVec), indicating a weaker chemotaxis ability in these five strains." If copper is a chemorepellent, these strains should swim away from high concentrations of copper; thus, the sizes of colonies couldn't be used to measure this response. In the cited reference (reference 29), bacterial response to phenol was measured using a response index (RI).

      (5) Figures 2 and 3 show both CsoR and PhaD bind to CheA and inhibit CheA autophosphorylation. Do these two proteins share any sequence or structural similarity? Does PhaD also bind to copper? Otherwise, it is difficult to understand these results.

      (6) Line 195-196, "CsoR/PhaD had no apparent influence on the phosphate transfer between CheA and CheY". CheA controls bacterial chemotaxis through CheY phosphorylation. If this is true, how do CsoR and PhaD affect chemotaxis?

      (7) Figure 3 shows that CsoR/PhaD bind to CheA through P1, P3, and P4. This result is intriguing. All CheA proteins contain these three domains. If this is true, CsoR/PhaD should bind to other bacterial CheAs too. That said, this experiment is premature and needs to be confirmed by other approaches.

      (8) Figure 5, does PhaD contain these three residues (C40, H65, and C69)? If not, how does PhaD inhibit CheA autophosphorylation and chemotactic response to copper?

      (9) Does deletion of cosR or cheA have any impact on P. putida resistance to high concentrations of copper?

    1. Reviewer #1 (Public Review):

      Summary:

      This work uses transgenic reporter lines to isolate entpd5a+ cells representing classical osteoblasts in the head and non-classical (osterix-) notochordal sheath cells. The authors also include entpd5a- cells, col2a1a+ cells to represent the closely associated cartilage cells. In a combination of ATAC and RNA-Seq analysis, the genome-wide transcriptomic and chromatin status of each cell population is characterized, validating their methodology and providing fundamental insights into the nature of each cell type, especially the less well-studied notochordal sheath cells. Using these data, the authors then turn to a thorough and convincing analysis of the regulatory regions that control the expression of the entpd5a gene in each cell population. Determination of transcriptional activities in developing zebrafish, again combined with ATAC data and expression data of putative regulators, results in a compelling and detailed picture of the regulatory mechanisms governing the expression of this crucial gene.

      Strengths:

      The major strength of this paper is the clever combination of RNA-Seq and ATAC analysis, further combined with functional transcriptional analysis of the regulatory elements of one crucial gene. This results in a very compelling story.

      Weaknesses:

      No major weaknesses were identified, except for all the follow-up experiments that one can think of, but that would be outside of the scope of this paper.

    1. Reviewer #1 (Public review):

      Summary:

      In this manuscript, Chen et al. used cryo-ET and in vitro reconstituted system to demonstrate that the autoinhibited form of LRRK2 can also assemble into filaments that wrap around the microtubule, although the filaments are typically shorter and less regular compared to the previously reported active-LRRK2 filaments. The structure revealed a new interface involving the N-terminal repeats that were disordered in the previous active-LRRK2 filament structure. The autoinhibited-LRRK2 filament also has different helical parameters compared to the active form.

      Strengths:

      The structure obtained in this study is the highest resolution of LRRK2 filaments done by subtomogram averaging, representing a major technical advance compared to the previous Cell paper from the same group. Overall, I think the data are well presented with beautiful graphic rendering, and valuable insights can be gained from this structural study.

      Weaknesses:

      (1) There are only three main figures, together with 9 supplemental figures. The authors may consider breaking the currently overwhelming Figures 1 and 3 into smaller figures and moving some of the supplemental figures to the main figure, e.g., Figure S7.

      (2) The key analysis of this manuscript is to compare the current structure with the previous active-LRRK2 filament structure. Currently, such a comparison is buried in Figure 3H. It should be part of Figure 1.

    1. Reviewer #1 (Public Review):

      Summary:

      This research sheds light on the nuanced role of ABHD6 in the regulation of AMPARs, highlighting its interaction with TARP γ-2 as a critical factor in modulating receptor-gating kinetics. It is crucial to understand that while ABHD6 alone does not alter AMPAR kinetics, its presence alongside TARP γ-2 leads to accelerated deactivation and desensitization of AMPARs, impacting synaptic transmission dynamics.

      Strengths:

      Important findings in the research include:<br /> - ABHD6 does not affect the gating kinetics of GluA1 and GluA2(Q) homomeric receptors independently.<br /> - In the presence of TARP γ-2, ABHD6 accelerates deactivation and desensitization of these receptors, regardless of their splicing or editing isoforms.<br /> - The effect is consistent for both homomeric GluA1 and GluA2(Q) receptors and heteromeric GluA1i/GluA2(R)i-G receptors.<br /> - The recovery from desensitization of GluA1 with the flip splicing isoform is slowed by ABHD6 in the presence of TARP γ-2.

      Weaknesses:

      However, the study focuses on specific receptor subunits and isoforms, which may not fully represent the diversity of AMPAR compositions found in vivo (e.g. though the authors have claimed that TARP γ-2 failed to increase GluA3-induced currents significantly, the effect on GluA4 or the explanation was missing). Further research is needed to explore the implications of these findings in more complex neuronal environments.

    1. Reviewer #1 (Public review):

      Summary:

      Opioids and related drugs are powerful analgesics that reduce suffering from pain. Unfortunately, their use often leads to addiction and there is an opioid-abuse epidemic that affects people worldwide. This study represents an ongoing effort to develop non-opioid analgesics for pain management. The findings point to an alternative approach to control post-surgical pain in lieu of opioid medications.

      Strengths:

      (1) The study responds to the urgent need for the development of non-opioid analgesics.

      (2) The study demonstrates the efficacy of Clarix Flo (FLO) and HC-HA/PTX3 from the human amniotic membrane (AM) in reducing pain in a mouse model without the adverse effects of opioids.

      (3) The study further explored the underlying mechanisms of how HC-HA/PTX3 produces its effects on neurons, suggesting the molecules/pathways involved in pain relief.

      (4) The potential use of naturally derived biologics from human birth tissues (AM) is safe and sustainable, compared to synthetic pharmaceuticals.

      (5) The study was conducted with scientific rigor, involving purification of active components, comparative analysis with multiple controls, and mechanistic explorations.

      Weaknesses:

      (1) It should be cautioned that while the preclinical findings are promising, these results still need to be translated into clinical settings that are complex and often unpredictable.

      (2) The study shows the efficacy of FLO and HC-HA/PTX3 in one preclinical model of post-surgical pain. The observed effect may be variable in other pain conditions.

    1. Reviewer #1 (Public review):

      This paper examines the role of MLCK (myosin light chain kinase) and MLCP (myosin light chain phosphatase) in axon regeneration. Using loss-of-function approaches based on small molecule inhibitors and siRNA knockdown, the authors explore axon regeneration in cell culture and in animal models. Their evidence shows that MLCK activity facilitates axon extension/regeneration, while MLCP prevents it.

      Major concern:

      A global inconsistency in the conclusions of the authors is evident when trying to understand the role of NMII in axon growth and to understand the present results in light of previous reports by the authors and many others on the role of NMII in axon extension. The discussion of the matter fails to acknowledge a vast literature on how NMII activity is regulated. The authors study enzymes responsible for the phosphorylation and dephosphorylation of NMII, referring to something that is strongly proven elsewhere, that phosphorylation activates NMII and dephosphorylation deactivates it. The authors mention their own previous evidence using inhibitors of NMII ATPase activity (blebbistatin, Bleb for short) and inhibitors of a kinase that phosphorylates NMII (ROCK), highlighting that Bleb increases axon growth. Since Bleb inhibits the ATPase activity of NMII, it follows that NMII is in itself an inhibitor of axon growth, and hence when NMII is inhibited, the inhibition on axon growth is relieved, and axonal growth takes place (REF1). It is known that NMII exists in an inactive folded state, and ser19 phosphorylation (by MLCK or ROCK) extends the protein, allowing NMII filament formation, ATPase activity, and force generation on actin filaments (REF2). From this, it is derived that if MLCK is inhibited, then there is no NMII phosphorylation, and hence no NMII activity, and, according to their previous work, this should promote axon growth. On the contrary, the authors show the opposite effect: in the lack of phospho-MLC, authors show axon growth inhibition.

      Reporting evidence challenging previous conclusions is common business in scientific endeavors, but the problem with the current manuscript is that it fails to point to and appropriately discuss this contradiction. Instead, the authors refer to the fact that MLCK and Bleb inhibit NMII in different steps of the activation process. While this is true, this explanation does not solve the contradiction. There are many options to accommodate the information, but it is not the purpose of this revision to provide them. Since the manuscript is focused solely on phosphorylation states of MLC and axon extension, the claims are simply at odds with the current literature, and this important finding, if true, is not properly discussed.

      What follows is a discussion of the merits and limitations of different claims of the manuscript in light of the evidence presented.

      (1) Using western blot and immunohistochemical analyses, authors first show that MLCK expression is increased in DRG sensory neurons following peripheral axotomy, concomitant to an increase in MLC phosphorylation, suggesting a causal effect (Figure 1). The authors claim that it is common that axon growth-promoting genes are upregulated. It would have been interesting at this point to study in this scenario the regulation of MLCP, which is a main subject in this work, and expect its downregulation.

      (2) Using DRG cultures and sciatic nerve crush in the context of MLCK inhibition and down-regulation, authors conclude that MLCK activity is required for mammalian peripheral axon regeneration both in vitro and in vivo (Figure 2).

      The in vitro evidence is of standard methods and convincing. However, here, as well as in all other experiments using siRNAs, it is not clear what the control is about (the identity of the plasmids and sequences, if any).

      Related to this, it is not helpful to show the same exact picture as a control example in Figures 2 and 3 (panels J and E, respectively). Either because they should not have received the same control treatment, or simply because it raises concern that there are no other control examples worth showing. In these images, it is not also clear where and how the crush site is determined in the GFP channel. This is of major importance since the axonal length is measured from the presumed crush site. Apart from providing further details in the text, the authors should include convincing images.

      (3) The authors then examined the role of the phosphatase MLCP in axon growth during regeneration. The authors first use a known MLCP blocker, phorbol 12,13-dibutyrate (PDBu), to show that is able to increase the levels of p-MLC, with a concomitant increase in the extent of axon regrowth of DRG neurons, both in permissive as well as non-permissive. The authors repeat the experiments using the knockdown of MYPT1, a key component of the MLC-phosphatase, and again can observe a growth-promoting effect (Figure 3).

      The authors further show evidence for the growth-enhancing effect in vivo, in nerve crush experiments. The evidence in vivo deserves more evidence and experimental details (see comment 2). Some key weaknesses of the data were mentioned previously (unclear RNAi controls and duplication of shown images), but in this case, it is also not clear if there is a change only in the extent of growth, or also in the number of axons that are able to regenerate.

      (4) In the next set of experiments (presented in Figure 4) authors extend the previous observations in primary cultures from the CNS. For that, they use cortical and hippocampal cultures, and pharmacological and genetic loss-of-function using the above-mentioned strategies. The expected results were obtained in both CNS neurons: inhibition or knockdown of the kinase decreases axon growth, whereas inhibition or knockdown of the phosphatase increases growth. A main weakness in this set is that it is not indicated when (at what day in vitro, DIV) the treatments are performed. This is important to correctly interpret the results, since in the first days in vitro these neurons follow well-characterized stages of development, with characteristic cellular events with relevance to what is being evaluated. Importantly, this would be of value to understand whether the treatments affect axonal specification and/or axonal extension. Although these events are correlated, they imply a different set of molecular events.

      The title of this section is misleading: line 241 "MLCK/MLCP activity regulated axon growth in the embryonic CNS"... the title (and the conclusion) implies that the experiments were performed in situ, looking at axons in the developing brain. The most accurate title and conclusion should mention that the evidence was collected in CNS primary cultures derived from embryos.

      (5) Performing nerve crush injury in CNS nerves (optic nerve and spinal cord), and the local application of PBDu, the author shows contrasting results (Figure 5). In the ON nerve, they can see axons extending beyond the lesion site due to PBDu. On the contrary, the authors fail to observe so in the corticospinal tract present in the spinal cord. The authors fail to discuss this matter in detail. Also, they accommodate the interpretation of the evidence in light of a process known as axon retraction, and its prevention by MLCP inhibition. Since the whole paper is on axon extension, and it is known that mechanistically axon retraction is not merely the opposite of axon extension, the claim needs far more evidence.

      In panel 5F and the supplementary data, the authors mention the occurrence of retraction bulbs, but the images are too small to support the claim, and it is not clear how these numbers were normalized to the number of axons labeled in each condition.

      (6) The author combines MLCK and MLCP inhibitors with Bleb, trying to verify if both pairs of inhibitors act on the same target/pathway (Figure 6). The rationale is wrong for at least two reasons.<br /> a- Because both lines of evidence point to contrasting actions of NMII on axon growth, one approach could never "rescue" the other.<br /> b- Because the approaches target different steps on NMII activation, one could never "prevent" or rescue the other. For example, for Bleb to provide a phenotype, it should find any p-MLC, because it is only that form of MLC that is capable of inhibiting its ATPase site. In light of this, it is not surprising that Bleb is unable to exert any action in a situation where there is no p-MLC (ML-7, which by inhibiting the kinase drives the levels of p-MLC to zero, Figure 4A). Hence, the results are not possible to validate in the current general interpretation of the authors. (See 'major concern').

      (7) In Figure 7, the authors argue that the scheme of replating and using ML7 before or after replating is evidence for a local cytoskeletal action of the drug. However, an alternative simpler explanation is that the drug acts acutely on its target, and that, as such, does not "survive" the replating procedure. Hence, the conclusion raised by the evidence shown is not supported.

      (8) In Figure 8, the authors show that the inhibitory treatments on MLCK and MLCP (ML7 and PRBu) alter the morphology of growth cones. However, it is not clear how this is correlated with axon growth. The authors also mention in various parts of the text that a local change in the growth cone is evidence for a local action/activity of the drug or enzyme. However the local change<->local action is not a logical truth. It can well be that MLCK and MLCP activity trigger molecular events that ultimately have an effect elsewhere, and by looking at "elsewhere" one observes of course a local effect, but is not because the direct action of MLCK or MLCP are localized. To prove true localized effects there are numerous efforts that can be made, starting from live imaging, fluorescent sensors, and compartmentalized cultures, just to mention a few.

      References:

      (1) Eun-Mi Hur 1, In Hong Yang, Deok-Ho Kim, Justin Byun, Saijilafu, Wen-Lin Xu, Philip R Nicovich, Raymond Cheong, Andre Levchenko, Nitish Thakor, Feng-Quan Zhou. 2011. Engineering neuronal growth cones to promote axon regeneration over inhibitory molecules. Proc Natl Acad Sci U S A. 2011 Mar 22;108(12):5057-62. doi: 10.1073/pnas.1011258108.

      (2) Garrido-Casado M, Asensio-Juárez G, Talayero VC, Vicente-Manzanares M. 2024. Engines of change: Nonmuscle myosin II in mechanobiology. Curr Opin Cell Biol. 2024 Apr;87:102344. doi: 10.1016/j.ceb.2024.102344.

      (3) Karen A Newell-Litwa 1, Rick Horwitz 2, Marcelo L Lamers. 2015. Non-muscle myosin II in disease: mechanisms and therapeutic opportunities. Dis Model Mech. 2015 Dec;8(12):1495-515. doi: 10.1242/dmm.022103.

    1. Reviewer #1 (Public Review):

      Summary of what the authors were trying to achieve:

      In this manuscript, the authors investigated the role of β-CTF on synaptic function and memory. They report that β-CTF can trigger the loss of synapses in neurons that were transiently transfected in cultured hippocampal slices and that this synapse loss occurs independently of Aβ. They confirmed previous research (Kim et al, Molecular Psychiatry, 2016) that β-CTF-induced cellular toxicity occurs through a mechanism involving a hexapeptide domain (YENPTY) in β-CTF that induces endosomal dysfunction. Although the current study also explores the role of β-CTF in synaptic and memory function in the brain using mice chronically expressing β-CTF, the studies are inconclusive because potential effects of Aβ generated by γ-secretase cleavage of β-CTF were not considered. Based on their findings, the authors suggest developing therapies to treat Alzheimer's disease by targeting β-CTF, but did not address the lack of clinical improvement in trials of several different BACE1 inhibitors, which target β-CTF by preventing its formation.

      Major strengths and weaknesses of the methods and results:

      The conclusions of the in vitro experiments using cultured hippocampal slices were well supported by the data, but aspects of the in vivo experiments and proteomic studies need additional clarification.

      (1) In contrast to the in vitro experiments in which a γ-secretase inhibitor was used to exclude possible effects of Aβ, this possibility was not examined in in-vivo experiments assessing synapse loss and function (Figure 3) and cognitive function (Figure 4). The absence of plaque formation (Figure 4B) is not sufficient to exclude the possibility that Aβ is involved. The potential involvement of Aβ is an important consideration given the 4-month duration of protein expression in the in vivo studies.

      (2) The possibility that the results of the proteomic studies conducted in primary cultured hippocampal neurons depend in part on Aβ was also not taken into consideration.

      Likely impact of the work on the field, and the utility of the methods and data to the community:

      The authors' use of sparse expression to examine the role of β-CTF on spine loss could be a useful general tool for examining synapses in brain tissue.

      Additional context that might help readers interpret or understand the significance of the work:

      The discovery of BACE1 stimulated an international effort to develop BACE1 inhibitors to treat Alzheimer's disease. BACE1 inhibitors block the formation of β-CTF which, in turn, prevents the formation of Aβ and other fragments. Unfortunately, BACE1 inhibitors not only did not improve cognition in patients with Alzheimer's disease, they appeared to worsen it, suggesting that producing β-CTF actually facilitates learning and memory. Therefore, it seems unlikely that the disruptive effects of β-CTF on endosomes plays a significant role in human disease. Insights from the authors that shed further light on this issue would be welcome.

    1. Reviewer #1 (Public review):

      Summary:

      The present study addresses whether physiological signals influence aperiodic brain activity with a focus on age-related changes. The authors report age effects on aperiodic cardiac activity derived from ECG in low and high-frequency ranges in roughly 2300 participants from four different sites. Slopes of the ECGs were associated with common heart variability measures, which, according to the authors, shows that ECG, even at higher frequencies, conveys meaningful information. Using temporal response functions on concurrent ECG and M/EEG time series, the authors demonstrate that cardiac activity is instantaneously reflected in neural recordings, even after applying ICA analysis to remove cardiac activity. This was more strongly the case for EEG than MEG data. Finally, spectral parameterization was done in large-scale resting-state MEG and ECG data in individuals between 18 and 88 years, and age effects were tested. A steepening of spectral slopes with age was observed particularly for ECG and, to a lesser extent, in cleaned MEG data in most frequency ranges and sensors investigated. The authors conclude that commonly observed age effects on neural aperiodic activity can mainly be explained by cardiac activity.

      Strengths:

      Compared to previous investigations, the authors demonstrate the effects of aging on the spectral slope in the currently largest MEG dataset with equal age distribution available. Their efforts of replicating observed effects in another large MEG dataset and considering potential confounding by ocular activity, head movements, or preprocessing methods are commendable and valuable to the community. This study also employs a wide range of fitting ranges and two commonly used algorithms for spectral parameterization of neural and cardiac activity, hence providing a comprehensive overview of the impact of methodological choices. Based on their findings, the authors give recommendations for the separation of physiological and neural sources of aperiodic activity.

      Weaknesses:

      While the aim of the study is well-motivated and analyses rigorously conducted, the overall structure of the manuscript, as it stands now, is partially misleading. Some of the described results are not well-embedded and lack discussion.

    1. Reviewer #1 (Public Review):

      Summary:

      Previous studies have shown that treatment with 17α-estradiol (a stereoisomer of the 17β-estradiol) extends lifespan in male mice but not in females. The current study by Li et al, aimed to identify cell-specific clusters and populations in the hypothalamus of aged male rats treated with 17α-estradiol (treated for 6 months). This study identifies genes and pathways affected by 17α-estradiol in the aged hypothalamus.

      Strengths:

      Using single-nucleus transcriptomic sequencing (snRNA-seq) on the hypothalamus from aged male rats treated with 17α-estradiol they show that 17α-estradiol significantly attenuated age-related increases in cellular metabolism, stress, and decreased synaptic activity in neurons.

      Moreover, sc-analysis identified GnRH as one of the key mediators of 17α-estradiol's effects on energy homeostasis. Furthermore, they show that CRH neurons exhibited a senescent phenotype, suggesting a potential side effect of the 17α-estradiol. These conclusions are supported by supervised clustering by neuropeptides, hormones, and their receptors.

      Weaknesses:

      However, the study has several limitations that reduce the strength of the key claims in the manuscript. In particular:

      (1) The study focused only on males and did not include comparisons with females. However, previous studies have shown that 17α-estradiol extends lifespan in a sex-specific manner in mice, affecting males but not females. Without the comparison with the female data, it's difficult to assess its relevance to the lifespan.

      (2) It is not known whether 17α-estradiol leads to lifespan extension in male rats similar to male mice. Therefore, it is not possible to conclude that the observed effects in the hypothalamus, are linked to the lifespan extension.

      (3) The effect of 17α-estradiol on non-neuronal cells such as microglia and astrocytes is not well-described (Figure 1). Previous studies demonstrated that 17α-estradiol reduces microgliosis and astrogliosis in the hypothalamus of aged male mice. Current data suggest that the proportion of oligo, and microglia were increased by the drug treatment, while the proportions of astrocytes were decreased. These data might suggest possible species differences, differences in the treatment regimen, or differences in drug efficiency. This has to be discussed.

      (4) A more detailed analysis of glial cell types within the hypothalamus in response to drugs should be provided.

      (5) The conclusion that CRH neurons are going into senescence is not clearly supported by the data. A more detailed analysis of the hypothalamus such as histological examination to assess cellular senescence markers in CRH neurons, is needed to support this claim.

    1. GLP-1 as idea for the Wolf/Bear cross in my Fiction Worldbuilding...

      Makes creatures more resilient to food scarcity (might even be useful for the scarcity in summer due to fire rains).

      ( ~4:55)

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Shao et al. investigate the contribution of different cortical areas to working memory maintenance and control processes, an important topic involving different ideas about how the human brain represents and uses information when it is no longer available to sensory systems. In two fMRI experiments, they demonstrate that the human frontal cortex (area sPCS) represents stimulus (orientation) information both during typical maintenance, but even more so when a categorical response demand is present. That is, when participants have to apply an added level of decision control to the WM stimulus, sPCS areas encode stimulus information more than conditions without this added demand. These effects are then expanded upon using multi-area neural network models, recapitulating the empirical gradient of memory vs control effects from visual to parietal and frontal cortices. In general, the experiments and analyses provide solid support for the authors' conclusions, and control experiments and analyses are provided to help interpret and isolate the frontal cortex effect of interest. However, I suggest some alternative explanations and important additional analyses that would help ensure an even stronger level of support for these results and interpretations.

      Strengths:

      - The authors use an interesting and clever task design across two fMRI experiments that is able to parse out contributions of WM maintenance alone along with categorical, rule-based decisions. Importantly, the second experiment only uses one fixed rule, providing both an internal replication of Experiment 1's effects and extending them to a different situation when rule-switching effects are not involved across mini-blocks.

      - The reported analyses using both inverted encoding models (IEM) and decoders (SVM) demonstrate the stimulus reconstruction effects across different methods, which may be sensitive to different aspects of the relationship between patterns of brain activity and the experimental stimuli.

      - Linking the multivariate activity patterns to memory behavior is critical in thinking about the potential differential roles of cortical areas in sub-serving successful working memory. Figure 3 nicely shows a similar interaction to that of Figure 2 in the role of sPCS in the categorization vs. maintenance tasks.

      - The cross-decoding analysis in Figure 4 is a clever and interesting way to parse out how stimulus and rule/category information may be intertwined, which would have been one of the foremost potential questions or analyses requested by careful readers. However, I think more additional text in the Methods and Results to lay out the exact logic of this abstract category metric will help readers better interpret the potential importance of this analysis and result.

      Weaknesses:

      - Selection and presentation of regions of interest: I appreciate the authors' care in separating the sPCS region as "frontal cortex", which is not necessarily part of the prefrontal cortex, on which many ideas of working memory maintenance activity are based. However, to help myself and readers interpret these findings, at a minimum the boundaries of each ROI should be provided as part of the main text or extended data figures. Relatedly, the authors use a probabilistic visual atlas to define ROIs in the visual, parietal, and frontal cortices. But other regions of both lateral frontal and parietal cortices show retinotopic responses (Mackey and Curtis, eLife, 2017: https://elifesciences.org/articles/22974) and are perhaps worth considering. Do the inferior PCS regions or inferior frontal sulcus show a similar pattern of effects across tasks? And what about the middle frontal gyrus areas of the prefrontal cortex, which are most analogous to the findings in NHP studies that the authors mention in their discussion, but do not show retinotopic responses? Reporting the effects (or lack thereof) in other areas of the frontal cortex will be critical for readers to interpret the role of the frontal cortex in guiding WM behavior and supporting the strongly worded conclusions of broad frontal cortex functioning in the paper. For example, to what extent can sPCS results be explained by visual retinotopic responses? (Mackey and Curtis, eLife, 2017: https://elifesciences.org/articles/22974).

      - When looking at the time course of effects in Figure 2, for example, the sPCS maintenance vs categorization effects occur very late into the WM delay period. More information is needed to help separate this potential effect from that of the response period and potential premotor/motor-related influences. For example, are the timecourses shifted to account for hemodynamic lag, and if so, by how much? Do the sPCS effects blend into the response period? This is critical, too, for a task that does not use a jittered delay period, and potential response timing and planning can be conducted by participants near the end of the WM delay. Regardless, parsing out the timing and relationship to response planning is important, and an ROI for M1 or premotor cortex could also help as a control comparison point, as in reference (24).

      - Interpreting effect sizes of IEM and decoding analysis in different ROIs. Here, the authors are interested in the interaction effects across maintenance and categorization tasks (bar plots in Figure 2), but the effect sizes in even the categorization task (y-axes) are always larger in EVC and IPS than in the sPCS region... To what extent do the authors think this representational fidelity result can or cannot be compared across regions? For example, a reader may wonder how much the sPCS representation matters for the task, perhaps, if memory access is always there in EVC and IPS? Or perhaps late sPCS representations are borrowing/accessing these earlier representations? Giving the reader some more intuition for the effect sizes of representational fidelity will be important. Even in Figure 3 for the behavior, all effects are also seen in IPS as well. More detail or context at minimum is needed about the representational fidelity metric, which is cited in ref (35) but not given in detail. These considerations are important given the claims of the frontal cortex serving such an important for flexible control, here.

    1. Reviewer #1 (Public review):

      Induction of beta cell regeneration is a promising approach for the treatment of diabetes. In this study, Massoz et.al., identified calcineurin (CaN) as a new potential modulator of beta cell regeneration by using zebrafish as model. They also showed that calcineurin (CaN) works together with Notch signaling calcineurin (CaN) to promote the beta cell regeneration. Overall, the paper is well organized, and technically sound. However, some evidences seem weak to get the conclusion.

    1. Reviewer #1 (Public Review):

      Dong Liu et al. successfully established a short-term zebrafish model by treating the embryos with high concentrations of monosaccharides, resembling the hyperangiogenic characteristics observed in proliferative diabetic retinopathy. The authors found that excessive angiogenesis induced by glucose and noncaloric monosaccharides can be achieved by activating the quiescent endothelial cells into proliferating tip cells. Importantly, the authors further confirmed the effects of monosaccharides on inducing excessive angiogenesis were mediated by the foxo1a-marcksl1a pathway. These results demonstrate the potentially detrimental effects of the noncaloric monosaccharides on blood vessel function and provided novel insights into the underlying mechanisms.

    1. Reviewer #1 (Public Review):

      Summary:

      The manuscript reports that expression of the E. coli operon topAI/yjhQ/yjhP is controlled by the translation status of a small open reading frame, that authors have discovered and named toiL, located in the leader region of the operon. The authors propose the following model for topAI activation: Under normal conditions, toiL is translated but topAI is not expressed because of Rho-dependent transcription termination within the topAI ORF and because its ribosome binding site and start codon are trapped in an mRNA hairpin. Ribosome stalling at various codons of the toiL ORF, caused by the presence of some ribosome-targeting antibiotics, triggers an mRNA conformational switch which allows translation of topAI and, in addition, activation of the operon's transcription because the presence of translating ribosomes at the topAI ORF blocks Rho from terminating transcription. Even though the model is appealing and several of the experimental data support some aspects of it, several inconsistencies remain to be solved. In addition, even though TopAI was shown to be an inhibitor of topoisomerase I (Yamaguchi & Inouye, 2015, NAR 43:10387), the authors suggest, without offering any experimental support, that, because ribosome-targeting antibiotics act as inducers, expression of the topAI/yjhQ/yjhP operon may confer resistance to these drugs.

      Strengths:

      - There is good experimental support of the transcriptional repression/activation switch aspect of the model, derived from well-designed transcriptional reporters and ChIP-qPCR approaches.

      - There is a clever use of the topAI-lacZ reporter to find the 23S rRNA mutants where expression topAI was upregulated. This eventually led the authors to identify that translation events occurring at toiL are important to regulate the topAI/yjhQ/yjhP operon. This section can be strengthened if the authors suggest an explanation for how mutant ribosomes translating toiL increased topAI expression. Is there any published evidence that ribosomes with the identified mutations translate slowly (decreased fidelity does not necessarily mean slow translation, does it?)?

      - Authors incorporate relevant links to the antibiotic-mediated expression regulation of bacterial resistance genes. Authors can also mention the tryptophan-mediated ribosome stalling at the tnaC leader ORF that activates the expression of tryptophan metabolism genes through blockage of Rho-mediated transcriptional attenuation.

      Weaknesses:

      The main weaknesses of the work are related to several experimental results that are not consistent with the model, or related to a lack of data that needs to be included to support the model.

      The following are a few examples:

      - It is surprising that authors do not mention that several published Ribo-seq data from E. coli cells show active translation of toiL (for example Li et al., 2014, Cell 157: 624). Therefore, it is hard to reconcile with the model that starts codon/Shine-Dalgarno mutations in the toiL-lux reporter have no effect on luciferase expression (Figure 2C, bar graphs of the no antibiotic control samples).

      - The SHAPE reactivity data shown in Figure 5A are not consistent with the toiL ORF being translated. In addition, it is difficult to visualize the effect of tetracycline on mRNA conformation with the representation used in Figure 5B. It would be better to show SHAPE reactivity without/with Tet (as shown in panel A of the figure).

      - The "increased coverage" of topAI/yjhP/yjhQ in the presence of tetracycline from the Ribo-seq data shown in Figure 6A can be due to activation of translation, transcription, or both. For readers to know which of these possibilities apply, authors need to provide RNA-seq data and show the profiles of the topAI/yjhQ/yjhP genes in control/Tet-treated cells.

      - Similarly, to support the data of increased ribosomal footprints at the toiL start codon in the presence of Tet (Figure 6B), authors should show the profile of the toiL gene from control and Tet-treated cells.

      - Representation of the mRNA structures in the model shown in Figure 5, does not help with visualizing 1) how ribosomes translate toiL since the ORF is trapped in double-stranded mRNA, and 2) how ribosome stalling on toiL would lead to the release of the initiation region of topAI to achieve expression activation.

      - The authors speculate that, because ribosome-targeting antibiotics act as expression inducers [by the way, authors should mention and comment that, more than a decade ago, it had been reported that kanamycin (PMID: 12736533) and gentamycin (PMID: 19013277) are inducers of topAI and yjhQ], the genes of the topAI/yjhQ/yjhP operon may confer resistance to these antibiotics. Such a suggestion can be experimentally checked by simply testing whether strains lacking these genes have increased sensitivity to the antibiotic inducers.

    1. Reviewer #1 (Public Review):

      The question of whether eyespots mimic eyes has certainly been around for a very long time and led to a good deal of debate and contention. This isn't purely an issue of how eyespots work either, but more widely an example of the potential pitfalls of adopting 'just-so-stories' in biology before conducting the appropriate experiments. Recent years have seen a range of studies testing eye mimicry, often purporting to find evidence for or against it, and not always entirely objectively. Thus, the current study is very welcome, rigorously analysing the findings across a suite of papers based on evidence/effect sizes in a meta-analysis.

      The work is very well conducted, robust, objective, and makes a range of valuable contributions and conclusions, with an extensive use of literature for the research. I have no issues with the analysis undertaken. The results and conclusions are compelling. It's probably fair to say that the topic needs more experiments to really reach firm conclusions but the authors do a good job of acknowledging this and highlighting where that future work would be best placed.

    1. Reviewer #1 (Public Review):

      Summary:

      In this study, Bonnifet et al. profile the presence of L1 ORF1p in the mouse and human brain. They claim that ORF1p is expressed in the human and mouse brain at a steady state and that there is an age-dependent increase in expression. This is a timely report as two recent papers have extensively documented the presence of full-length L1 transcripts in the mouse and human brain (PMID: 38773348 & PMID: 37910626). Thus, the finding that L1 ORF1p is consistently expressed in the brain is not surprising, but important to document.

      Strengths:

      Several parts of this manuscript appear to be well done and include the necessary controls. In particular, the evidence for steady-state expression of ORF1p in the mouse brain appears robust.

      Weaknesses:

      Several parts of the manuscript appear to be more preliminary and need further experiments to validate their claims. In particular, the data suggesting expression of L1 ORF1p in the human brain and the data suggesting increased expression in the aged brain need further validation. Detailed comments:

      (1) The expression of ORF1p in the human brain shown in Figure 1j is not convincing. Why are there two strong bands in the WB? How can the authors be sure that this signal represents ORF1p expression and not non-specific labelling? Additional validations and controls are needed to verify the specificity of this signal.

      (2) The data shown in Figure 2g are not convincing. How can the authors be sure that this signal represents ORF1p expression and not non-specific labelling? Extensive additional validations and controls are needed to verify the specificity of this signal.

      (3) The data showing a reduction in ORF1p expression in the aged mouse brain is confusing and maybe even misleading. Although there is an increase in the intensity of the ORF1p signal in ORF1p+ cells, the data clearly shows that fewer cells express ORF1p in the aged brain. If these changes indicate an overall loss or gain of ORF1p, expression in the aged brain is not resolved. Thus, conclusions should be more carefully phrased in this section. It is important to show the quantification of NeuN+ and NeuN- cells in young vs aged (not only the proportions as shown in Figure 3b) to determine if the difference in the number of ORF1p+ cells is due to loss of neurons or perhaps a sampling issue. More so, it would be essential to perform WB and/or proteomics experiments to complement the IHC data for the aged mouse samples.

      (4) The transcriptomic data presented in Figure 4 and Figure 5 are not convincing. Quantification of transposon expression on short read sequencing has important limitations. Longer reads and complementary approaches are needed to study the expression of evolutionarily young L1s (see PMID: 38773348 & PMID: 37910626 for examples of the current state of the art). Given the read length and the unstranded sequencing approach, I would at least ask the authors to add genome browser tracks of the upregulated loci so that we can properly assess the clarity of the results. I would also suggest adding the mappability profile of the elements in question. In addition, since this manuscript focuses on ORF1p, it would be essential to document changes in protein levels (and not just transcripts) in the ageing human brain.

      (5) More information is needed on RNAseq of microdissections of dopaminergic neurons from 'healthy' post-mortem samples of different ages. No further information on these samples is provided. I would suggest adding a table with the clinical information of these samples (especially age, sex, and cause of death). The authors should also discuss whether this experiment has sufficient power. The human ageing cohort seems very small to me.

      (6) The findings in this manuscript apply to both human and mouse brains. However, the landscape of the evolutionarily young L1 subfamilies between these two species is very different and should be part of the discussion. For example, the regulatory sequences that drive L1 expression are quite different in human and mouse L1s. This should be discussed.

      (7) On page 3 the authors write: "generally accepted that TE activation can be both, a cause and consequence of aging". This statement does not reflect the current state of the field. On the contrary, this is still an area of extensive investigation and many of the findings supporting this hypothesis need to be confirmed in independent studies. This statement should be revised to reflect this reality.

    1. Reviewer #1 (Public review):

      This study by Wu et al. provides valuable computational insights into PROTAC-related protein complexes, focusing on linker roles, protein-protein interaction stability, and lysine residue accessibility. The findings are significant for PROTAC development in cancer treatment, particularly breast and prostate cancers.

      The authors' claims about the role of PROTAC linkers and protein-protein interaction stability are generally supported by their computational data. However, the conclusions regarding lysine accessibility could be strengthened with more in-depth analysis. The use of the term "protein functional dynamics" is not fully justified by the presented work, which focuses primarily on structural dynamics rather than functional aspects.

      Strengths:

      (1) Comprehensive computational analysis of PROTAC-related protein complexes.

      (2) Focus on critical aspects: linker role, protein-protein interaction stability, and lysine accessibility.

      Weaknesses:

      (1) Limited examination of lysine accessibility despite its stated importance.

      (2) Use of RMSD as the primary metric for conformational assessment, which may overlook important local structural changes.

    1. Reviewer #1 (Public review):

      Summary:

      This work made a lot of efforts to explore the multifaceted roles of the inferior colliculus (IC) in auditory processing, extending beyond traditional sensory encoding. The authors recorded neuronal activitity from the IC at single unit level when monkeys were passively exposed or actively engaged in behavioral task. They concluded that 1)IC neurons showed sustained firing patterns related to sound duration, indicating their roles in temporal perception, 2) IC neuronal firing rates increased as sound sequences progress, reflecting modulation by behavioral context rather than reward anticipation, 3) IC neurons encode reward prediction error and their capability of adjusting responses based on reward predictability, 4) IC neural activity correlates with decision-making. In summary, this study tried to provide a new perspective on IC functions by exploring its roles in sensory prediction and reward processing, which are not traditionally associated with this structure.

      Strengths:

      The major strength of this work is that the authors performed electrophysiological recordings from the IC of behaving monkeys. Compared with the auditory cortex and thalamus, the IC in monkeys has not been adequately explored.

      Weaknesses:

      (1) The authors cited several papers focusing on dopaminergic inputs in the IC to suggest the involvement of this brain region in cognitive functions. However, all those cited work were done in rodents. Whether monkey's IC shares similar inputs is not clear.<br /> (2) The authors confused the two terms, novelty and deviation. According to their behavioral paradigm, deviation rather than novelty should be used in the paper because all the stimuli have been presented to the monkeys during training. Therefore, there is actually no novel stimuli but only deviant stimuli. This reflects that the author has misunderstood the basic concept.<br /> (3) Most of the conclusions were made based on correlational analysis or speculation without providing causal evidences.<br /> (4) Results are presented in a very "straightforward" manner with too many detailed descriptions of phenomena but lack of summary and information synthesis. For example, the first section of Results is very long but did not convey clear information.<br /> (5) The logic between different sections of Results is not clear.<br /> (6) In the Discussion, there is excessive repetition of results, and further comparison with and discussion of potentially related work are very insufficient. For example, Metzger, R.R., et al. (J Neurosc, 2006) have shown similar firing patterns of IC neurons and correlated their findings with reward.

    1. Reviewer #1 (Public review):

      Summary:

      In the manuscript "Intergenerational transport of double-stranded RNA limits heritable epigenetic changes" Shugarts and colleagues investigate intergenerational dsRNA transport in the nematode C. elegans. They induce oxidative damage in worms, blocking dsRNA import into cells (and potentially affecting the worms in other ways). Oxidative stress inhibits dsRNA import and the associated heritable regulation of gene expression in the adult germline (Fig. 2). The authors identify a novel gene, sid-1-dependent gene-1 (sdg-1), which is induced upon inhibition of SID-1 (Fig. 3). Both transient inhibition and genetic depletion of SID-1 lead to the upregulation of sdg-1 and a second gene, sdg-2 (Fig. 5). The expression of SDG-1 is variable, potentially indicating buffering regulation. While the expression of Sdg-1 could be consistent with a role in intergenerational transport of dsRNA, neither its overexpression nor loss-of-function impacts dsRNA-mediated silencing (Fig. 7) in the germline. It would be interesting to test if sdg-2 functions redundantly.

      In summary, the authors have identified a novel worm-specific protein (sdg-1) that is induced upon loss of dsRNA import via SID-1, but is not required to mediate SID-1 RNA regulatory effects.

      Remaining Questions:

      • The authors use an experimental system that induces oxidative damage specifically in neurons to release dsRNAs into the circulation. Would the same effect be observed if oxidative damage were induced in other cell types?

      • Besides dsRNA, which other RNAs and cellular products (macromolecules and small signalling molecules) are released into the circulation that could affect the observed changes in germ cells?

      • SID-1 modifies RNA regulation within the germline (Fig. 7) and upregulates sdg-1 and sdg-2 (Fig. 5). However, SID-1's effects do not appear to be mediated via sdg-1. Testing the role of sdg-2 would be intriguing.

      • Are sdg-1 or sdg-2 conserved in other nematodes or potentially in other species? Sdg-1 appears to be encoded or captured by a retro-element in the C. elegans genome and exhibits stochastic expression in different isolates. Is this a recent adaptation in the C. elegans genome, or is it present in other nematodes? Does loss-of-function of sdg-1 or sdg-2 have any observable effect?

      Clarification for Readability:

      To enhance readability and avoid misunderstandings, it is crucial to specify the model organism and its specific dsRNA pathways that are not conserved in vertebrates:

      • In the first sentence of the paragraph "Here, we dissect the intergenerational transport of extracellular dsRNA ...", the authors should specify "in the nematode C. elegans". Unlike vertebrates, which recognise dsRNA as a foreign threat, worms and other invertebrates pervasively use dsRNA for signalling. Additionally, worms, unlike vertebrates and insects, encode RNA-dependent RNA polymerases that generate dsRNA from ssRNA substrates, enabling amplification of small RNA production. Especially in dsRNA biology, specifying the model organism is essential to avoid confusion about potential effects in humans.

      • Similarly, the authors should specify "in C. elegans" in the sentence "Therefore, we propose that the import of extracellular dsRNA into the germline tunes intracellular pathways that cause heritable RNA silencing." This is important because C. elegans small RNA pathways differ significantly from those in other organisms, particularly in the PIWI-interacting RNA (piRNA) pathways, which depend on dsRNA in C. elegans but uses ssRNA in vertebrates. Specification is crucial to prevent misinterpretation by the reader. It is well understood that mechanisms of transgenerational inheritance that operate in nematodes or plants are not conserved in mammals.

      • The first sentence of the discussion, "Our analyses suggest a model for ...", would also benefit from specifying "in C. elegans". The same applies to the figure captions. Clarification of the model organism should be added to the first sentence, especially in Figure 1.

    1. Reviewer #1 (Public Review):

      This work makes several contributions: (1) a method for the self-supervised segmentation of cells in 3D microscopy images, (2) an cell-segmented dataset comprising six volumes from a mesoSPIM sample of a mouse brain, and (3) a napari plugin to apply and train the proposed method.

      (1) Method

      This work presents itself as a generalizable method contribution with a wide scope: self-supervised 3D cell segmentation in microscopy images. My main critique is that there is almost no evidence for the proposed method to have that wide of a scope. Instead, the paper is more akin to a case report that shows that a particular self-supervised method is good enough to segment cells in two datasets with specific properties.

      To support the claim that their method "address[es] the inherent complexity of quantifying cells in 3D volumes", the method should be evaluated in a comprehensive study including different kinds of light and electron microscopy images, different markers, and resolutions to cover the diversity of microscopy images that both title and abstract are alluding to.

      The main dataset used here (a mesoSPIM dataset of a whole mouse brain) features well-isolated cells that are easily distinguishable from the background. Otsu thresholding followed by a connected component analysis already segments most of those cells correctly. The proposed method relies on an intensity-based segmentation method (a soft version of a normalized cut) and has at least five free parameters (radius, intensity, and spatial sigma for SoftNCut, as well as a morphological closing radius, and a merge threshold for touching cells in the post-processing). Given the benefit of tweaking parameters (like thresholds, morphological operation radii, and expected object sizes), it would be illuminating to know how other non-learning-based methods will compare on this dataset, especially if given the same treatment of segmentation post-processing that the proposed method receives. After inspecting the WNet3D predictions (using the napari plugin) on the used datasets I find them almost identical to the raw intensity values, casting doubt as to whether the high segmentation accuracy is really due to the self-supervised learning or instead a function of the post-processing pipeline after thresholding.

      I suggest the following baselines be included to better understand how much of the segmentation accuracy is due to parameter tweaking on the considered datasets versus a novel method contribution:<br /> * comparison to thresholding (with the same post-processing as the proposed method)<br /> * comparison to a normalized cut segmentation (with the same post-processing as the proposed method)<br /> * comparison to references 8 and 9.

      I further strongly encourage the authors to discuss the limitations of their method. From what I understand, the proposed method works only on well-separated objects (due to the semantic segmentation bottleneck), is based on contrastive FG/BG intensity values (due to the SoftNCut loss), and requires tuning of a few parameters (which might be challenging if no ground-truth is available).

      (2) Dataset

      I commend the authors for providing ground-truth labels for more than 2500 cells. I would appreciate it if the Methods section could mention how exactly the cells were labelled. I found a good overlap between the ground truth and Otsu thresholding of the intensity images. Was the ground truth generated by proofreading an initial automatic segmentation, or entirely done by hand? If the former, which method was used to generate the initial segmentation, and are there any concerns that the ground truth might be biased towards a given segmentation method?

      (3) Napari plugin

      The plugin is well-documented and works by following the installation instructions. However, I was not able to recreate the segmentations reported in the paper with the default settings for the pre-trained WNet3D: segments are generally too large and there are a lot of false positives. Both the prediction and the final instance segmentation also show substantial border artifacts, possibly due to a block-wise processing scheme.

    1. Reviewer #1 (Public review):

      Summary:

      Insulin is crucial for maintaining metabolic homeostasis, and its release is regulated by various pathways, including blood glucose levels and neuromodulatory systems. The authors investigated the role of neuromodulators in regulating the dynamics of the adult Drosophila IPC population. They showed that IPCs express various receptors for monoaminergic and peptidergic neuromodulators, as well as synaptic neurotransmitters with highly heterogeneous profiles across the IPC population. Activating specific modulatory inputs, e.g. dopaminergic, octopaminergic or peptidergic (Leucokinin) using an optogenetic approach coupled with in vivo electrophysiology unveiled heterogeneous responses of individual IPCs resulting in excitatory, inhibitory or no responses. Interestingly, calcium imaging of the entire IPC population with or without simultaneous electrophysiological recording of individual cells showed highly specific and stable responses of individual IPCs suggesting their intrinsic properties are determined by the expressed receptor repertoire. Using the adult fly connectome they further corroborate the synaptic input of excitatory and inhibitory neuronal subsets of IPCs. The authors conclude that the heterogeneous modulation of individual IPC activity is more likely to allow for flexible control of insulin release to adapt to changes in metabolic demand and environmental cues.

      Strengths:

      This study provides a comprehensive, multi-level analysis of IPC properties utilizing single-nucleus RNA sequencing, anatomical receptor expression mapping, connectomics, electrophysiological recordings, calcium-imaging and an optogenetics-based 'intrinsic pharmacology' approach. It highlights the heterogeneous receptor profiles of IPCs, demonstrating complex and differential modulation within the IPC population. The authors convincingly showed that different neuromodulatory inputs exhibit varied effects on IPC activity and simultaneous occurrence of heterogeneous responses in IPCs with some populations exciting a subset of IPCs while inhibiting others, showcasing the intricate nature of IPC modulation and diverse roles of IPC subgroups. The temporal dynamic of IPC modulation showed that polysynaptic and neuromodulatory connections play a major role in IPC response. The authors demonstrated that certain neuromodulatory inputs, e.g. dopamine, can shift the overall IPC population activity towards either an excited or inhibited state. The study thus provides a fundamental entry point to understanding the complex influence of neuromodulatory inputs on the insulinergic system of Drosophila.

      Weakness:

      GPCRs are typically expressed at low levels and while the transcriptomic and reporter expression analysis was comprehensive, both approaches have the caveat that they do not allow validating protein level expression. Thus, some receptors might have been missed while others might be false positives. The authors acknowledged the challenges in accurately accessing receptor expression in complex modulatory systems indicating there are limitations in full understanding of the receptor profiles of IPCs.

      While this study provides valuable insights into the heterogeneity of IPC responses and receptor expression, it will require future studies to elucidate how these modulatory inputs affect insulin release and transcriptional long-term changes.<br /> The authors further analyzed male and female snRNAseq data and claimed that the differences in receptor expression were minimal. The experimental analyses used mated females only and while the study is very complete in this respect, it would have been extremely interesting to compare male flies in terms of their response profiles.<br /> Lastly as also pointed out by the authors, their approach of using optogenetically driven excitation of modulatory neuronal subsets limits the interpretation of the results due to the possibly confounding direct or indirect effect of fast synaptic transmission on IPC excitation/inhibition, and the broad expression of some neuromodulatory lines used in this analysis.

      Overall, however, the conclusions of this study are well supported by the data provided by the authors. Moreover, their detailed and thorough analysis of IPC modulation will have a significant impact on the field of metabolic regulation to understand the complex regulatory mechanism of insulin release, which can now be studied further to provide insight about metabolic homeostasis and neural control of metabolic processes.

    1. Reviewer #1 (Public Review):

      Kreeger and colleagues have explored the balance of excitation and inhibition in the cochlear nucleus octopus cells of mice using morphological, electrophysiological, and computational methods. On the surface, the conclusion, that synaptic inhibition is present, does not seem like a leap. However, the octopus cells have been in the past portrayed as devoid of inhibition. This view was supported by the seeming lack of glycinergic fibers in the octopus cell area and the lack of apparent IPSPs. Here, Kreeger et al. used beautiful immunohistochemical and mouse genetic methods to quantify the inhibitory and excitatory boutons over the complete surface of individual octopus cells and further analysed the proportions of the different subtypes of spiral ganglion cell inputs. I think the analysis stands as one of the most complete descriptions of any neuron, leaving little doubt about the presence of glycinergic boutons.

      Kreeger et al then examined inhibition physiologically, but here I felt that the study was incomplete. Specifically, no attempt was made to assess the actual, biological values of synaptic conductance for AMPAR and GlyR. Thus, we don't really know how potent the GlyR could be in mediating inhibition. Here are some numbered comments:

      (1) "EPSPs" were evoked either optogenetically or with electrical stimulation. The resulting depolarizations are interpreted to be EPSPs. However previous studies from Oertel show that octopus cells have tiny spikes, and distinguishing them from EPSPs is tricky. No mention is made here about how or whether that was done. Thus, the analysis of EPSP amplitude is ambiguous.

      (2) For this and later analysis, a voltage clamp of synaptic inputs would have been a simple alternative to avoid contaminating spikes or shunts by background or voltage-gated conductances. Yet only the current clamp was employed. I can understand that the authors might feel that the voltage clamp is 'flawed' because of the failure to clamp dendrites. But that may have been a good price to pay in this case. The authors should have at least justified their choice of method and detailed its caveats.

      (3) The modeling raised several concerns. First, there is little presentation of assumptions, and of course, a model is entirely about its assumptions. For example, what excitatory conductance amplitudes were used? The same for inhibitory conductance? How were these values arrived at? The authors note that EPSGs and IPSGs had peaks at 0.3 and 3 ms. On what basis were these numbers obtained? The model's conclusions entirely depend on these values, and no measurements were made here that could have provided them. Parenthetical reference is made to Figure S5 where a range of values are tested, but with little explanation or justification.

      (4) In experiments that combined E and I stimulation, what exactly were time timecourses of the conductance changes, and how 'synchronous' were they, given the different methods to evoke them? (had the authors done voltage clamp they would know the answers).

      (5) Figure 4G is confusing to me. Its point, according to the text, is to show that changes in membrane properties induced by a block of Kv and HCN channels would not be expected to alter the amplitudes of EPSCs and IPSCs across the dendritic expanse. Now we are talking about currents (not shunting effects), and the presumption is that the blockers would alter the resting potential and thus the driving force for the currents. But what was the measured membrane potential change in the blockers? Surely that was documented. To me, the bigger concern (stated in the text) is whether the blockers altered exocytosis, and thus the increase in IPSP amplitude in blockers is due BOTH to loss of shunting and increase in presynaptic spike width. Added to this is that 4AP will reduce the spike threshold, thus allowing more ChR2-expressing axons to reach the threshold. Figure 4G does not address this point.

      (6) Figure 5F is striking as the key piece of biological data that shows that inhibition does reduce the amplitude of "EPSPs" in octopus cells. Given the other uncertainties mentioned, I wondered if it makes sense as an example of shunting inhibition. Specifically, what are the relative synaptic conductances, and would you predict a 25% reduction given the actual (not modeled) values?

      (7) Some of the supplemental figures, like 4 and 5, are hardly mentioned. Few will glean anything from them unless the authors direct attention to them and explain them better. In general, the readers would benefit from more complete explanations of what was done.

    1. Reviewer #1 (Public Review):

      Summary:

      This paper conducted a GWAS meta-analysis for COVID-19 hospitalization among admixed American populations. The authors identified four genome-wide significant associations, including two novel loci (BAZ2B and DDIAS), and an additional risk locus near CREBBP using cross-ancestry meta-analysis. They utilized multiple strategies to prioritize risk variants and target genes. Finally, they constructed and assessed a polygenic risk score model with 49 variants associated with critical COVID-19 conditions.

      Strengths:

      Given that most of the previous studies were done in European ancestries, this study provides unique findings about the genetics of COVID-19 in admixed American populations. The GWAS data would be a valuable resource for the community. The authors conducted comprehensive analyses using multiple different strategies, including Bayesian fine mapping, colocalization, TWAS, etc., to prioritize risk variants and target genes. The polygenic risk score (PGS) result demonstrated the ability of cross-population PGS model for COVID-19 risk stratification.

      Weaknesses:

      (1) One of the major limitations of this study is that the GWAS sample size is relatively small, which limits its power.<br /> (2) Lack of replication cohort.<br /> (3) Colocalization and TWAS used eQTL data from GTEx data, which are mainly from European ancestries.

      Comments on latest version:

      The authors addressed most of my concerns.

    1. Reviewer #1 (Public review):

      Summary:

      Rößling et al., report in this study that the perception of RALF1 by the FER receptor is mediated by the association of RALF1 with deesterified pectin, contributing to the regulation of the cell wall matrix and plasma membrane dynamics. In addition, they report that this mode of action is independent from the previously reported cell wall sensing mechanism mediated by the FER-LRX complex.

      This manuscript reproduces and aligns with the results from a recently published study (Liu et al., Cell) where they also report that RALF1 can interact with deesterified pectin, forming coacervates and promoting the recruitment of LLG-FER at the membrane.

    1. Reviewer #1 (Public review):

      Summary:

      The authors investigate the mechanism behind the widely observed but poorly understood phenomenon of reversible vimentin disassembly upon hypotonic challenge. Using permeabilized COS-7 cells expressing vimentin-mEos3.2, the authors demonstrate that vimentin disassembly is not due to lower osmotic pressure but rather due to decreased intracellular ionic strength. They propose a model in which vimentin filament stability is predicted by the protein's net charge and support this idea through approaches that involve (i) manipulating buffer ionic strength, (ii) manipulating buffer pH, or (iii) introducing charged amino acids into the linker of the exogenously expressed vimentin-mEos3.2.

      Strengths & Weaknesses:

      While the discovery is intriguing and presents an interesting concept, significant shortcomings in experimental design and numerous inconsistencies prevent it from reaching the high standards expected. The lack of reproducibility, inadequate controls, and insufficient quantification make the findings feel very preliminary. Additionally, the authors need to address the apparent discrepancies between their current results and their previous work implicating calpains and altered calcium levels in vimentin disassembly upon hypotonic challenge (which has led to much confusion in the field). This discrepancy should be thoroughly addressed in the discussion with the authors citing their prior work and explaining why it was incorrect.

      An additional concern is the relevance of the findings to vimentin biology inside cells. The most important insight in this work is the observation that an isotonic buffer balanced with non-electrolytes (glucose or sorbitol) is sufficient to drive vimentin disassembly. The authors show that vimentin disassembly is not due to changes in osmotic pressure but rather due to a change in the concentration of critical dissolved ions, specifically the number of charged states on vimentin. What is missing is when and how this is controlled within cells under physiological conditions - not just when cells are permeabilized with detergents (conditions that cells rarely survive). Without this deeper dive into vimentin states within cells and how it is controlled, the paper seems very narrow in its focus.

    1. Reviewer #2 (Public review):

      This study uses single-unit recordings in the monkey STN to examine the evidence for three theoretical models that propose distinct roles for the STN in perceptual decision-making. Importantly, the proposed functional roles are predictive of unique patterns of neural activity. Using k-means clustering with seeds informed by each model's predictions, the current study identified three neural clusters with activity dynamics that resembled those predicted by the described theoretical models. The authors are thorough and transparent in reporting the analyses used to validate the clustering procedure and the stability of the clustering results. To further establish a causal role for the STN in decision-making, the researchers applied microsimulation to the STN and found effects on response times, choice preferences, and latent decision parameters estimated with a drift-diffusion model. Overall, the study provides strong evidence for a functionally diverse population of STN neurons that could indeed support multiple roles involved in perceptual decision-making. The manuscript would benefit from stronger evidence linking each neural cluster to specific decision roles in order to strengthen the overall conclusions.

      The interpretation of the results, and specifically, the degree to which the identified clusters support each model, is largely dependent on whether the artificial vectors used as model-based clustering seeds adequately capture the expected behavior under each theoretical model. The manuscript would benefit from providing further justification for the specific model predictions summarized in Figure 1B. Further, although each cluster's activity can be described in the context of the discussed models, these same neural dynamics could also reflect other processes not specific to the models. That is, while a model attributing the STN's role to assessing evidence accumulation may predict a ramping up of neural activity, activity ramping is not a selective correlate of evidence accumulation and could be indicative of a number of processes, e.g., uncertainty, the passage of time, etc.. This lack of specificity makes it challenging to infer the functional relevance of cluster activity and should be acknowledged in the discussion.

      Additionally, although the effects of STN microstimulation on behavior provide important causal evidence linking the STN to decision processes, the stimulation results are highly variable and difficult to interpret. The authors provide a reasonable explanation for the variability, showing that neurons from unique clusters are anatomically intermingled such that stimulation likely affects neurons across several clusters. It is worth noting, however, that a substantial body of literature suggests that neural populations in the STN are topographically organized in a manner that is crucial for its role in action selection, providing "channels" that guide action execution. The authors should comment on how the current results, indicative of little anatomical clustering amongst the functional clusters, relates to other reports showing topographical organization.

      Overall, the association between the identified clusters and the function ascribed to the STN by each of the models is largely descriptive and should be interpreted accordingly. For example, Figure 3 is referenced when describing which cluster activity is choice/coherence dependent, yet it is unclear what specific criteria and measures are being used to determine whether activity is choice/coherence "dependent." Visually, coherence activity seems to largely overlap in panel B (top row). Is there a statistically significant distinction between low and high coherence in this plot? The interpretation of these plots and the methods used to determine choice/coherence "dependence" needs further explanation.

      In general, the association between cluster activity and each model could be more directly tested. At least two of the models assume coordination with other brain regions. Does the current dataset include recordings from any of these regions (e.g., mPFC or GPe) that could be used to bolster claims about the functional relevance of specific subpopulations? For example, one would expect coordinated activity between neural activity in mPFC and Cluster 2 according to the Ratcliff and Frank model. Additionally, the reported drift-diffusion model (DDM) results are difficult to interpret as microsimulation appears to have broad and varied effects across almost all the DDM model parameters. The DDM framework could, however, be used to more specifically test the relationships between each neural cluster and specific decision functions described in each model. Several studies have successfully shown that neural activity tracks specific latent decision parameters estimated by the DDM by including neural activity as a predictor in the model. Using this approach, the current study could examine whether each cluster's activity is predictive of specific decision parameters (e.g., evidence accumulation, decision thresholds, etc.). For example, according to the Ratcliff and Frank model, activity in cluster 2 might track decisions thresholds.

      Review of revision

      The authors have sufficiently addressed the concerns raised in the initial reviews and have revised their manuscript accordingly. We commend the authors for these efforts and feel that the revisions have strengthened the major claims of the manuscript.

    1. Reviewer #1 (Public Review):

      In this manuscript by Wu et al., the authors present the high resolution cryoEM structures of the WT Kv1.2 voltage-gated potassium channel. Along with this structure, the authors have solved several structures of mutants or experimental conditions relevant to the slow inactivation process that these channels undergo and which is not yet completely understood.

      One of the main findings is the determination of the structure of a mutant (W366F) that is thought to correspond to the slow inactivated state. These experiments confirm results in similar mutants in different channels from Kv1.2 that indicate that inactivation is associated with an enlarged selectivity filter.

      Another interesting structure is the complex of Kv1.2 with the pore blocking toxin Dendrotoxin 1. The results shown in the revised version indicate that the mechanism of block is similar to that of related blocking-toxins, in which a lysine residue penetrates in the pore. Surprisingly, in these new structures, the bound toxin results in a pore with empty external potassium binding sites.

      The quality of the structural data presented in this revised manuscript is very high and allows for unambiguous assignment of side chains. The conclusions are supported by the data. This is an important contribution that should further our understanding of voltage-dependent potassium channel gating. In the revised version, the authors have addressed my previous specific comments.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this paper, authors investigated the role of RUNT-related transcription factor 2 (RUNX2) in oral squamous carcinoma (OSCC) growth and resistance to ferroptosis. They found that RUNX2 suppresses ferroptosis through transcriptional regulation of peroxiredoxin-2. They further explored the upstream positive regulator of RUNX2, HOXA10 and found that HOXA10/RUNX2/PRDX2 axis protects OSCC from ferroptosis.

      Strengths:<br /> The study is well designed and provides a novel mechanism of HOXA10/RUNX2/PRDX2 control of ferroptosis in OSCC.

      Weaknesses:

      According to the data presented in (Figure 2F, Figure 3Fand G, Figure 5D and Figure 6E and F), apoptosis seems to be affected in the same amount as ferroptosis by HOXA10/RUNX2/PRDX2 axis, which raises questions on the authors' specific focus on ferroptosis in this study. Reasonably, authors should adapt the title and the abstract in a way that recapitulates the whole data, which is HOXA10/RUNX2/PRDX2 axis control of cell death, including ferroptosis and apoptosis in OSCC.

      Comments:

      - In the description of the result section related to Figure 3E, the author wrote "In addition, we found that isoform II-knockdown induced shrunken mitochondria with vanished cristae with transmission electron microscopy (Figure 3E). These results suggest that RUNX2 isoform II may suppress ferroptosis." The interpretation provided here is not clear to the reviewer. How shrunken mitochondria and vanished cristae can be linked to ferroptosis?<br /> - The electron microscopy images show more elongated mitochondria in the RUNX2 isoform II-KO cells than in RUNX2 isoform II positive cells, which might result from the fusion of mitochondria. These images should completed with a fluorescent mitochondria staining of these cells.<br /> - What is the oxygen consumption rate in RUNX2 KO cells?<br /> - The increase in cell proliferation after RUNX2 overexpression in Figure 2A is not convincing, is there any differences in their migration or invasion capacity?<br /> - The in vivo study shows 50% reduction in primary tumor growth after RUNX2 inhibition by shRNA in CAL 27 xenografts, but only one shRNA is shown. Is this one shRNA clone? At least 2 shRNA clones should be used.<br /> - Apoptosis and necroptosis seem to be affected in the same amount as ferroptosis by HOXA10/RUNX2/PRDX2 axis. This is evident from experiments in Figure 3E, F and from Figure 6E, F and Figure 3G. Either Fer-1, Z-VAD,or Nec-1 used alone, were not able to fully restore cell proliferation to control cell level, which implies an additive effect of ferroptosis, apoptosis and necrosis. The author should verify potential additive or synergistic effect of the combination of Fer-1 and Z-VAD in these assays after si-RUNX2 in Figure 3 F and G and after si-HOX assays.<br /> - What is the effect of PRDX2 or HOXA10 depletion on tumor growth?<br /> - What is the clinical relevance of HOXA10 in OSCC patients?

    1. Reviewer #1 (Public review):

      Summary:

      This study develops and validates a neural subspace similarity analysis for testing whether neural representations of graph structures generalize across graph size and stimulus sets. The authors show the method works in rat grid and place cell data, finding that grid but not place cells generalize across different environments, as expected. The authors then perform additional analyses and simulations to show that this method should also work on fMRI data. Finally, the authors test their method on fMRI responses from the entorhinal cortex (EC) in a task that involves graphs that vary in size (and stimulus set) and statistical structure (hexagonal and community). They find neural representations of stimulus sets in lateral occipital complex (LOC) generalize across statistical structure and that EC activity generalizes across stimulus sets/graph size, but only for the hexagonal structures.

      Strengths:

      (1) The overall topic is very interesting and timely and the manuscript is well-written.

      (2) The method is clever and powerful. It could be important for future research testing whether neural representations are aligned across problems with different state manifestations.

      (3) The findings provide new insights into generalizable neural representations of abstract task states in the entorhinal cortex.

      Weaknesses:

      (1) The manuscript would benefit from improving the figures. Moreover, the clarity could be strengthened by including conceptual/schematic figures illustrating the logic and steps of the method early in the paper. This could be combined with an illustration of the remapping properties of grid and place cells and how the method captures these properties.

      (2) Hexagonal and community structures appear to be confounded by training order. All subjects learned the hexagonal graph always before the community graph. As such, any differences between the two graphs could thus be explained (in theory) by order effects (although this is practically unlikely). However, given community and hexagonal structures shared the same stimuli, it is possible that subjects had to find ways to represent the community structures separately from the hexagonal structures. This could potentially explain why the authors did not find generalizations across graph sizes for community structures.

      (3) The authors include the results from a searchlight analysis to show the specificity of the effects of EC. A better way to show specificity would be to test for a double dissociation between the visual and structural contrast in two independently defined regions (e.g., anatomical ROIs of LOC and EC).

      (4) Subjects had more experience with the hexagonal and community structures before and during fMRI scanning. This is another confound, and possible reason why there was no generalization across stimulus sets for the community structure.

    1. Reviewer #1 (Public review):

      Summary:

      Insects and their relatives are commonly infected with microbes that are transmitted from mothers to their offspring. A number of these microbes have independently evolved the ability to kill the sons of infected females very early in their development; this male killing strategy has evolved because males are transmission dead-ends for the microbe. A major question in the field has been to identify the genes that cause male killing and to understand how they work. This has been especially challenging because most male-killing microbes cannot be genetically manipulated. This study focuses on a male-killing bacterium called Wolbachia. Different Wolbachia strains kill male embryos in beetles, flies, moths, and other arthropods. This is remarkable because how sex is determined differs widely in these hosts. Two Wolbachia genes have been previously implicated in male-killing by Wolbachia: oscar (in moth male-killing) and wmk (in fly male-killing). The genomes of some male-killing Wolbachia contain both of these genes, so it is a challenge to disentangle the two.

      This paper provides strong evidence that oscar is responsible for male-killing in moths. Here, the authors study a strain of Wolbachia that kills males in a pest of tea, Homona magnanima. Overexpressing oscar, but not wmk, kills male moth embryos. This is because oscar interferes with masculinizer, the master gene that controls sex determination in moths and butterflies. Interfering with the masculinizer gene in this way leads the (male) embryo down a path of female development, which causes problems in regulating the expression of genes that are found on the sex chromosomes.

      Strengths:

      The authors use a broad number of approaches to implicate oscar, and to dissect its mechanism of male lethality. These approaches include:<br /> (1) Overexpressing oscar (and wmk) by injecting RNA into moth eggs.<br /> (2) Determining the sex of embryos by staining female sex chromosomes.<br /> (3) Determining the consequences of oscar expression by assaying sex-specific splice variants of doublesex, a key sex determination gene, and by quantifying gene expression and dosage of sex chromosomes, using RNASeq.<br /> (4) Expressing oscar along with masculinizer from various moth and butterfly species, in a silkmoth cell line.

      This extends recently published studies implicating oscar in male-killing by Wolbachia in Ostrinia corn borer moths, although the Homona and Ostrinia oscar proteins are quite divergent. Combined with other studies, there is now broad support for oscar as the male-killing gene in moths and butterflies (i.e. order Lepidoptera). So an outstanding question is to understand the role of wmk. Is it the master male-killing gene in insects other than Lepidoptera and if so, how does it operate?

      Weaknesses:

      I found the transfection assays of oscar and masculinizer in the silkworm cell line (Figure 4) to be difficult to follow. There are also places in the text where more explanation would be helpful for non-experts (see recommendations).

    1. Reviewer #1 (Public Review):

      Summary:

      Machii et al. reported a possible molecular mechanism underlying the parallel evolution of lip hypertrophy in African cichlids. The multifaceted approach taken in this manuscript is highly valued, as it uses histology, proteomics, and transcriptomics to reveal how phylogenetically distinct thick-lips have evolved in parallel. Findings from histology and proteomics connected to wnt signaling through the transcriptome are very exciting.

      Strengths:

      There is consistency between the results and it is possible to make a strong argument from the results.

      Weaknesses:

      The authors do not discuss based on genomic information; the genomes of the cichlids from the three lakes have been decoded and are therefore available. However, indeed, the species in Lake Tanganyika and Lake Malawi/Victoria are genetically distant from each other, so a comparative genome analysis would not have yielded the results presented here. I recommend adding such a discussion to the Discussion.

    1. Reviewer #1 (Public review):

      The central finding of the current manuscript is that embryonic ablation of PRMT1 results in a craniofacial phenotype that is primarily linked to downstream intron splicing defects. This manuscript is one of several to underscore the relative importance of intron splicing to gene expression regulation during development, and moreover, to recapitulate splicing-related craniofacial defects. Specifically, authors introduce a regulatory axis consisting of PRMT1-SFPQ that directs mechanisms of long intron retention. This finding represents a significant contribution to our understanding of splicing regulation, in the sense that it highlights the regulatory impact that post-translational modification of splicing-related proteins can have on intron processing. Further, it emphasizes the importance of extending the study of splicing regulation beyond core components of the spliceosome, to include their upstream regulators as well.

      The significance of neural crest cells in the development of craniofacial structures has long been considered a major contributor to developmental phenotypes. This specific symptomology is heavily associated with spliceosomopathies, wherein disruption of spliceosome components is the primary mechanism of disease pathogenesis. Thus, the PRMT1 associated phenotype is noteworthy. The role of PRMT1 in methylating downstream splicing factors introduces a new avenue of research focused on the mechanisms of spliceosome component activation and their effects on splicing. The strength of the current study lies in their establishing the molecular mechanism through which PRMT1 could alter craniofacial development through regulation of the transcriptome, but the data presented to support the claim that a PRMT1-SFPQ axis directly regulates intron retention of the relevant gene networks should be robust and with multiple forms of clear validation. For example, elevated intron retention findings are based on the intron retention index, and according to the manuscript, are assessed considering the relative expression of exons and introns from a given transcript. However, delineating between intron retention and other forms of alternative splicing (i.e., cryptic splice site recognition) requires a more comprehensive consideration of the intron splicing defects that could be represented in data. A certain threshold of intron read coverage (i.e., the percent of an intron that is covered by mapped reads) is needed to ascertain if those that are proximal to exons could represent alternative introns ends rather than full intron retention events. In other words, intron retention is a type of alternative splicing that can be difficult to analyze in isolation given the confounding influence of cryptic splicing and cryptic exon inclusion. If other forms of alternative splicing were assessed and not detected, more confident retention calls can be made.

      While data presented to support the PRMT1-SFPQ activation axis is quite compelling, that this is directly responsible for the elevated intron retention remains enigmatic. First, in characterizing their PRMT1 knockout model, it is unclear whether the elevated intron retention events directly correspond to downregulated genes. Moreover, intron splicing is a well-documented node for gene regulation during embryogenesis and in other proliferation models, and craniofacial defects are known to be associated with 'spliceosomopathies'. However, reproduction of this phenotype does not suggest that the targets of interest are inherently splicing factors, and a more robust assessment is needed to determine the exact nature of alternative splicing in this system. Because there are several known splicing factors downstream of PRMT1 and presented in the supplemental data, the specific attribution of retention to SFPQ would be additionally served by separating its splicing footprint from that of other factors that are primed to cause alternative splicing.

      Clarifying the relationship between SFPQ and splicing regulation is important given that the observed splicing defects are incongruous with published data presented by Takeuchi et al., (2018) regarding SFPQ control of neuronal apoptosis in mice. In this system, SFPQ was more specifically attributed to the regulation of transcription elongation over long introns and its knockout did not result in significant splicing changes. Thus, to establish the specificity for the SFPQ in regulating these retention events, authors would need to show that the same phenotype is not achieved by mis-regulation of other splicing factors. That the authors chose SFPQ based on its binding profile is understandable but potentially confounding given its mechanism of action in transcription of long introns (Takeuchi 2018). Because mechanisms and rates of transcription can influence splicing and exon definition interactions, the role of SFPQ as a transcription elongation factor versus a splicing factor is inadequately disentangled by authors.

    1. Reviewer #2 (Public review):

      Summary:

      Hall et al describe the superiority of ONT sequencing and deep learning-based variant callers to deliver higher SNP and Indel accuracy compared to previous gold-standard Illumina short-read sequencing. Furthermore, they provide recommendations for read sequencing depth and computational requirements when performing variant calling.

      Strengths:

      The study describes compelling data showing ONT superiority when using deep learning-based variant callers, such as Clair3, compared to Illumina sequencing. This challenges the paradigm that Illumina sequencing is the gold standard for variant calling in bacterial genomes. The authors provide evidence that homopolymeric regions, a systematic and problematic issue with ONT data, are no longer a concern in ONT sequencing.

      Weaknesses:

      The study is limited in the number of samples included, even though it covers different species with divergent genome sequences, likely covering major evolutionary changes. The methods section could be more detailed. A structural variation analysis would be an interesting next step.

    1. Reviewer #1 (Public Review):

      Summary:

      In this manuscript, Rohde et al. discuss how single cells isolated from the presomitic mesoderm of the zebrafish embryo follow a cell-autonomous differentiation "programme", which is dependent on the initial anteroposterior position in the embryo.

      Strengths:

      This work and, in particular, the comparison to cellular behaviour in vivo presents a detailed description of the oscillatory system that brings the developmental biology forward in their understanding of somitogenesis.<br /> The main novelty lies in the direct comparison of these isolated single cells to single cells tracked within the developing embryo. This allows them to show that isolated cells follow a similar path of differentiation without direct contact to neighbours or the presence of external morphogen gradients. Based on this, the authors propose an internal timer that starts ticking as cells traverse the presomitic mesoderm, while external signals modify this behaviour.

      There are a few direct questions that follow up from this study, for instance, intercellular synchronization influences the variability of the timer. However, I agree with the authors that such experiments are out of the scope of this study.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors aimed to identify potential biomarkers for acute myocardial infarction (AMI) through blood metabolomics and fecal microbiome analysis. They found that long chain fatty acids (LCFAs) could serve as biomarkers for AMI and demonstrated a correlation between LCFAs and the gut microbiome. Additionally, in silico molecular docking and in vitro thrombogenic assays showed that these LCFAs can induce platelet aggregation.

      Strengths:

      The study utilized a comprehensive approach combining blood metabolomics and fecal microbiome analysis.

      The findings suggest a novel use of LCFAs as biomarkers for AMI.

      The correlation between LCFAs and the gut microbiome is a significant contribution to understanding the interplay between gut health and heart disease.

      The use of in silico and in vitro assays provides mechanistic insights into how LCFAs may influence platelet aggregation.

      Weaknesses:

      The evidence is incomplete as it does not definitively prove that gut dysbiosis contributes to fatty acid dysmetabolism.

      The study primarily shows an association between the gut microbiome and fatty acid metabolism without establishing causation.

    1. Joint Public Review

      The molecular mechanisms that mediate the regulated exocytosis of neuropeptides and neurotrophins from neurons via large dense-core vesicles (LDCVs) are still incompletely understood. Motivated by their earlier discovery that the Rab3-RIM1 pathway is essential for neuronal LDCV exocytosis, the authors now examined the role of the Rab3 effector Rabphilin-3A in neuronal LDCV secretion. Based on live, confocal, and super-resolution imaging approaches, the authors provide evidence for a synaptic enrichment of Rabphilin-3A and for independent trafficking of Rabphilin-3A and LDCVs. Using an elegant NPY-pHluorin imaging approach, they show that genetic deletion of Rabphilin-3A causes an increase in electrically triggered LDCV fusion events and increased neurite length. Finally, knock-out-replacement studies, involving Rabphilin-3A mutants deficient in either Rab3- or SNAP25-binding, indicate that the synaptic enrichment of Rabphilin-3A depends on its Rab3 binding ability, while its ability to bind to SNAP25 is required for its effects on LDCV secretion and neurite development. The authors conclude that Rabphilin-3A negatively regulates LDCV exocytosis and propose that this mechanism also affects neurite growth, e.g. by limiting neurotrophin secretion. These are important findings that advance our mechanistic understanding of neuronal large dense-core vesicle (LDCV) secretion.

      The major strengths of the present paper:

      (i) The use of a powerful Rabphilin-3A KO mouse model.<br /> (ii) Stringent lentiviral expression and rescue approaches as a strong genetic foundation of the study.<br /> (iii) An elegant FRAP imaging approach.<br /> (iv) A cutting-edge NPY-pHluorin-based imaging approach to detect LDCV fusion events.

      Weaknesses of the present paper:

      (i) It remains unclear why a process that affects a general synaptic SNARE fusion protein - SNAP25 - would specifically affect LDCV but not synaptic vesicle fusion.<br /> (iii) The mechanistic links between Rabphilin-3A function, LDCV density in neurites, neurite outgrowth, and the proposed underlying mechanisms involving trophic factor release remain unresolved.

    1. Reviewer #1 (Public Review):

      O'Leary and colleagues sought to understand the factors that underlie memory processes, including formation, retrieval, and forgetting. The present data identify time, environmental enrichment, Rac-1, context reexposure, and brief reminders of the familiar object as factors that alter discrimination between novel and familiar objects. This is complimented with an engram approach to quantify cells that are active during learning to examine how their activation is impacted with each of the above factors at test. There are many strengths in the manuscript, including systematic testing of several factors that contribute to poor discrimination between novel and familiar objects. These results are interesting and outline essential boundaries of incidental, nonaversive memory. With this behavioral data, authors apply a modeling approach to understand the factors that contribute to good and poor object memory recall.

    1. Reviewer #1 (Public Review):

      The authors present a model for multisensory correlation detection that is based on the neurobiologically plausible Hassenstein Reichardt detector (Parise & Ernst, 2016). They demonstrate that this model can account for human behaviour in synchrony or temporal order judgements and related temporal tasks in two new data sets (acquired in this study) and a range of previous data sets. While the current study is limited to the model assessment for relatively simple audiovisual signals, in future communications, the authors demonstrate that the model can also account for audiovisual integration of complex naturalistic signals such as speech and music.

      The significance of this work lies in its ability to explain multisensory perception using fundamental neural mechanisms previously identified in insect motion processing.

      Strengths:

      (1) The model goes beyond descriptive models such as cumulative Gaussians for TOJ and differences in cumulative Gaussians for SJ tasks by providing a mechanism that builds on the neurobiologically plausible Hassenstein-Reichardt detector.<br /> (2) This model can account for results from two new experiments that focus on the detection of correlated transients and frequency doubling. The model also accounts for several behavioural results from experiments including stochastic sequences of A/V events and sine wave modulations (and naturalistic Av signals such as speech and music as shown in future communications).

    1. Reviewer #1 (Public Review):

      Summary:

      "Neural noise", here operationalized as an imbalance between excitatory and inhibitory neural activity, has been posited as a core cause of developmental dyslexia, a prevalent learning disability that impacts reading accuracy and fluency. This study is the first to systematically evaluate the neural noise hypothesis of dyslexia. Neural noise was measured using neurophysiological (electroencephalography [EEG]) and neurochemical (magnetic resonance spectroscopy [MRS]) in adolescents and young adults with and without dyslexia. The authors did not find evidence of elevated neural noise in the dyslexia group from EEG or MRS measures, and Bayes factors generally informed against including the grouping factor in the models. Although the comparisons between groups with and without dyslexia did not support the neural noise hypothesis, a mediation model that quantified phonological processing and reading abilities continuously revealed that EEG beta power in the left superior temporal sulcus was positively associated with reading ability via phonological awareness. This finding lends support for analysis of associations between neural excitatory/inhibitory factors and reading ability along a continuum, rather than as with a case/control approach, and indicates the relevance of phonological awareness as an intermediate trait that may provide a more proximal link between neurobiology and reading ability. Further research is needed across developmental stages and over a broader set of brain regions to more comprehensively assess the neural noise hypothesis of dyslexia, and alternative neurobiological mechanisms of this disorder should be explored.

      Strengths:

      The inclusion of multiple methods of assessing neural noise (neurophysiological and neurochemical) is a major advantage of this paper. MRS at 7T confers an advantage of more accurately distinguishing and quantifying glutamate, which is a primary target of this study. In addition, the subject-specific functional localization of the MRS acquisition is an innovative approach. MRS acquisition and processing details are noted in the supplementary materials according to the experts' consensus-recommended checklist (https://doi.org/10.1002/nbm.4484). Commenting on the rigor, the EEG methods is beyond my expertise as a reviewer.

      Participants recruited for this study included those with a clinical diagnosis of dyslexia, which strengthens confidence in the accuracy of the diagnosis. The assessment of reading and language abilities during the study further confirms the persistently poorer performance of the dyslexia group compared to the control group.

      The correlational analysis and mediation analysis provide complementary information to the main case-control analyses, and the examination of associations between EEG and MRS measures of neural noise is novel and interesting.

      The authors follow good practice for open science, including data and code sharing. They also apply statistical rigor, using Bayes Factors to support conclusions of null evidence rather than relying only on non-significant findings. In the discussion, they acknowledge the limitations and generalizability of the evidence and provide directions for future research on this topic.

      Weaknesses:

      Though the methods employed in the paper are generally strong, there are certain aspects that are not clearly described in the Materials & Methods section, such as a description of the statistical analyses used for hypothesis testing.

      With regard to metabolite quantification, it is unclear why the authors chose to analyze and report metabolite values in terms of creatine ratios rather than quantification based on a water reference given that the MRS acquisition appears to support using a water reference. GABA is typically quantified using J-editing sequences as lower field strengths (~3T), and there is some evidence that the GABA signal can be reliably measured at 7T without editing, however, the authors should discuss potential limitations, such as reliability of Glu and GABA measurements with short-TE semi-laser at 7T. In addition, MRS measurements of GABA are known to be influenced by macromolecules, and GABA is often denoted as GABA+ to indicate that other compounds contribute to the measured signal, especially at a short TE and in the absence of symmetric spectral editing. A general discussion of the strengths and limitations of unedited Glu and GABA quantification at 7T is warranted given the interest of this work to researchers who may not be experts in MRS.

      Further, the single MRS voxel location is a limitation of the study as neurochemistry can vary regionally within individuals, and the putative excitatory/inhibitory imbalance in dyslexia may appear in regions outside the left temporal cortex (e.g., network-wide or in frontal regions involved in top-down executive processes). While the functional localization of the MRS voxel is a novelty and a potential advantage, it is unclear whether voxel placement based on left-lateralized reading-related neural activity may bias the experiment to be more sensitive to small, activity-related fluctuations in neurotransmitters in the CON group vs. the DYS group who may have developed an altered, compensatory reading strategy.

      As the authors note in the discussion, sex could serve as a moderator of associations between neural noise and reading abilities and should be considered in future studies.

      Appraisal:

      The authors present a thorough evaluation of the neural noise hypothesis of developmental dyslexia in a sample of adolescents and young adults using multiple methods of measuring excitatory/inhibitory imbalances as an indicator of neural noise. The authors concluded that there was no support for the neural noise hypothesis of dyslexia in their study based on null significance and Bayes factors. This conclusion is justified, and further research is called for to more broadly evaluate the neural noise hypothesis in developmental dyslexia.

      Impact:

      This study provides an exemplary foundation for the evaluation of the neural noise hypothesis of dyslexia. Other researchers may adopt the model applied in this paper to examine neural noise in various populations with/without dyslexia, or across a continuum of reading abilities, to more thoroughly examine the evidence (or lack thereof) for this hypothesis. Notably, the lack of evidence here does not rule out the possibility of a role for neural noise in dyslexia, and the authors point out that presentation with co-occurring conditions, such as ADHD, may contribute to neural noise in dyslexia. Dyslexia remains a multi-faceted and heterogenous neurodevelopmental condition, and many genetic, neurobiological, and environmental factors play a role. This study demonstrates one step toward evaluating neurobiological mechanisms that may contribute to reading difficulties.

    1. Reviewer #1 (Public Review):

      Summary:

      This study analyzed biomarker data from 28 subjects with geographic atrophy (GA) in a Phase I/II clinical trial of PPY988, a subretinal AAV2 complement factor I (CFI) gene therapy, to evaluate pharmacokinetics and pharmacodynamics. Post-treatment, a 2-fold increase in the vitreous humor (VH) FI was observed, correlating with a reduction in FB breakdown product Ba but minimal changes in other complement factors. The aqueous humor (AH) was found to be an unreliable proxy for VH in assessing complement activation. In vitro assays showed that the increase in FI had a minor effect on the complement amplification loop compared to the more potent C3 inhibitor pegcetacoplan. These findings suggest that PPY988 may not provide enough FI protein to effectively modulate complement activation and slow GA progression, highlighting the need for a thorough biomarker review to determine optimal dosing in future studies.

      Strengths:

      This manuscript provides critical data on the efficacy of gene therapy for the eye, specifically introducing complement FI expression. It presents the results from a halted clinical trial, making sharing this data essential for understanding the outcomes of this gene therapy approach. The findings offer valuable insights and lessons for future gene therapy attempts in similar contexts.

      Weaknesses:

      No particular weaknesses. The study was carefully performed and limitations are discussed.

      I have just some concerns about the methodology used. The authors use the MILLIPLEX assays, which allow for multiplexed detection of complement proteins and they mention extensive validation. How are the measurements with this assay correlating with gold standard methods? Is the specificity and the expected normal ranges preserved with this assay? This also stands for the Olink assay. Some of the proteins are measured by both assay and/or by standard ELISA. How do these measurements correlate?

    1. Reviewer #1 (Public Review):

      Summary:

      Englert et al. proposed a functional connectome-based Hopfield artificial neural network (fcHNN) architecture to reveal attractor states and activity flows across various conditions, including resting state, task-evoked, and pathological conditions. The fcHNN can reconstruct characteristics of resting-state and task-evoked brain activities. Additionally, the fcHNN demonstrates differences in attractor states between individuals with autism and typically developing individuals.

      Strengths:

      (1) The study used seven datasets, which somewhat ensures robust replication and validation of generalization across various conditions.

      (2) The proposed fcHNN improves upon existing activity flow models by mimicking artificial neural networks, thereby enhancing the representational ability of the model. This advancement enables the model to more accurately reconstruct the dynamic characteristics of brain activity.

      (3) The fcHNN projection offers an interesting visualization, allowing researchers to observe attractor states and activity flow patterns directly.

      Weaknesses:

      (1) The fcHNN projection can offer low-dimensional dynamic visualizations, but its interpretability is limited, making it difficult to make strong claims based on these projections. The interpretability should be enhanced in the results and discussion.

      (2) The presentation of results is not clear enough, including figures, wording, and statistical analysis, which contributes to the overall difficulty in understanding the manuscript. This lack of clarity in presenting key findings can obscure the insights that the study aims to convey, making it challenging for readers to fully grasp the implications and significance of the research.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors aimed to investigate the oscillatory activity of GnRH neurones in freely behaving mice. By utilising GCaMP fiber photometry, they sought to record real-time neuronal activity to understand the patterns and dynamics of GnRH neuron firing and their implications for reproductive physiology.

      Strengths:

      (1) The use of GCaMP fiber photometry allows for high temporal resolution recordings of neuronal activity, providing real-time data on the dynamics of GnRH neurones.

      (2) Recording in freely behaving animals ensures that the findings are physiologically relevant and not artifacts of a controlled laboratory environment.

      (3) The authors used statistical methods to characterise the oscillatory patterns, ensuring the reliability of their findings.

      Weaknesses:

      (1) While the study identifies distinct oscillatory patterns in GnRH neurones' calcium dynamics, it falls short in exploring the functional implications of these patterns for GnRH pulsatility and overall reproductive physiology.

      (2) The study lacks a broader discussion to include comparisons with existing studies on GnRH neurone activity and pulsatility and highlight how the findings of this study align with or differ from previous research and what novel contributions are made.

      (3) The authors aimed to characterise the oscillatory activity of GnRH neurons and successfully identified distinct oscillatory patterns. The results support the conclusion that GnRH neurons exhibit complex oscillatory behaviours, which are critical for understanding their role in reproductive physiology. However, it has not been made clear what exactly the authors mean by "multi-dimensional oscillatory patterns" and how has this been shown.

    1. Reviewer #1 (Public Review):

      Summary:

      In this series of studies, Locantore et al. investigated the role of SST-expressing neurons in the entopeduncular nucleus (EPNSst+) in probabilistic switching tasks, a paradigm that requires continued learning to guide future actions. In prior work, this group had demonstrated EPNSst+ neurons co-release both glutamate and GABA and project to the lateral habenula (LHb), and LHb activity is also necessary for outcome evaluation necessary for performance in probabilistic decision-making tasks. Previous slice physiology works have shown that the balance of glutamate/GABA co-release is plastic, altering the net effect of EPN on downstream brain areas and neural circuit function. The authors used a combination of in vivo calcium monitoring with fiber photometry and computational modeling to demonstrate that EPNSst+ neural activity represents movement, choice direction, and reward outcomes in their behavioral task. However, viral-genetic manipulations to synaptically silence these neurons or selectively eliminate glutamate release had no effect on behavioral performance in well-trained animals. The authors conclude that despite their representation of task variables, EPN Sst+ neuron synaptic output is dispensable for task performance.

      Strengths and Weaknesses:

      Overall, the manuscript is exceptionally scholarly, with a clear articulation of the scientific question and a discussion of the findings and their limitations. The analyses and interpretations are careful and rigorous. This review appreciates the thorough explanation of the behavioral modeling and GLM for deconvolving the photometry signal around behavioral events, and the transparency and thoroughness of the analyses in the supplemental figures. This extra care has the result of increasing the accessibility for non-experts, and bolsters confidence in the results. To bolster a reader's understanding of results, we suggest it would be interesting to see the same mouse represented across panels (i.e. Figures 1 F-J, Supplementary Figures 1 F, K, etc i.e via the inclusion of faint hash lines connecting individual data points across variables. Additionally, Figure 3E demonstrates that eliminating the 'reward' and 'choice and reward' terms from the GLM significantly worsens model performance; to demonstrate the magnitude of this effect, it would be interesting to include a reconstruction of the photometry signal after holding out of both or one of these terms, alongside the 'original' and 'reconstructed' photometry traces in panel D. This would help give context for how the model performance degrades by exclusion of those key terms. Finally, the authors claimed calcium activity increased following ipsilateral movements. However, Figure 3C clearly shows that both SXcontra and SXipsi increase beta coefficients. Instead, the choice direction may be represented in these neurons, given that beta coefficients increase following CXipsi and before SEipsi, presumably when animals make executive decisions. Could the authors clarify their interpretation on this point? Also, it is not clear if there is a photometry response related to motor parameters (i.e. head direction or locomotion, licking), which could change the interpretation of the reward outcome if it is related to a motor response; could the authors show photometry signal from representative 'high licking' or 'low licking' reward trials, or from spontaneous periods of high vs. low locomotor speeds (if the sessions are recorded) to otherwise clarify this point?

      There are a few limitations with the design and timing of the synaptic manipulations that would improve the manuscript if discussed or clarified. The authors take care to validate the intersectional genetic strategies: Tetanus Toxin virus (which eliminates synaptic vesicle fusion) or CRISPR editing of Slc17a6, which prevents glutamate loading into synaptic vesicles. The magnitude of effect in the slice physiology results is striking. However, this relies on the co-infection of a second AAV to express channelrhodopsin for the purposes of validation, and it is surely the case that there will not be 100% overlap between the proportion of cells infected. Alternative means of glutamate packaging (other VGluT isoforms, other transporters, etc) could also compensate for the partial absence of VGluT2, which should be discussed. The authors do not perform a complimentary experiment to delete GABA release (i.e. via VGAT editing), which is understandable, given the absence of an effect with the pan-synaptic manipulation. A more significant concern is the timing of these manipulations as the authors acknowledge. The manipulations are all done in well-trained animals, who continue to perform during the length of viral expression. Moreover, after carefully showing that mice use different strategies on the 70/30 version vs the 90/10 version of the task, only performance on the 90/10 version is assessed after the manipulation. Together, the observation that EPNsst activity does not alter performance on a well-learned, 90/10 switching task decreases the impact of the findings, as this population may play a larger role during task acquisition or under more dynamic task conditions. Additional experiments could be done to strengthen the current evidence, although the limitation is transparently discussed by the authors.

      Finally, intersectional strategies target LHb-projecting neurons, although in the original characterization, it is not entirely clear that the LHb is the only projection target of EPNsst neurons. A projection map would help clarify this point.

      Overall, the authors used a pertinent experimental paradigm and common cell-specific approaches to address a major gap in the field, which is the functional role of glutamate/GABA co-release from the major basal ganglia output nucleus in action selection and evaluation. The study is carefully conducted, their analyses are thorough, and the data are often convincing and thought-provoking. However, the limitations of their synaptic manipulations with respect to the behavioral assays reduce generalizability and to some extent the impact of their findings.

    1. Reviewer #1 (Public review):

      Using the UK Biobank, this study assessed the value of nuclear magnetic resonance measured metabolites as predictors of progression to diabetes. The authors identified a panel of 9 circulating metabolites that improved the ability in risk prediction of progression from prediabetes to diabetes. In general, this is a well-performed study, and the findings may provide a new approach to identifying those at high risk of developing diabetes.

      Comments on the revised version:

      Thanks so much for carefully addressing my comments.

    1. Reviewer #1 (Public review):

      Summary:

      The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.

      Strengths:

      (1) 7T high-resolution MRS is used<br /> (2) This study combines the behavioral tests, MRS, and fMRI.

      Major<br /> I have no further comments. The approach and experiment are sound. The only overall drawback is the relatively low sample size.

      Weaknesses:<br /> (1) Line 138, "This finding supports the hypothesis that motion perception is associated with neural activity in MT+ area". This sentence is strange because it is a well-established finding in numerous human fMRI papers. I think the authors should be more specific about what this finding implies.

      Response: We thank reviewer for pointing this out. We have revised it to:" This finding is in line with prior results, which indicates that motion perception is associated with neural activity in hMT+ area, but not in EVC (primarily in V1)" (lines 156-158)

      Reply: This argument should be refined. Numerous studies have shown the key role of V1 in motion perception. V1 contains a vast proportion of direction selective neurons. I am asking how your results here are related to existing literature. This argument is incorrect and too rough. Can you please revise this?

      (9) Line 213, as far as I know, the study (Melnick et al., 2013) is a psychophysical study and did not provide evidence that the spatial suppression effect is associated with MT+.

      Response: We thank reviewer for pointing this out. It was a mistake to use this reference, and we have revised it accordingly. (line 242)

      Reply: Thanks. New citation is good. But that paper is a modeling study. The direct empirical evidence on humans should be as follow:

      Tadin, D., Silvanto, J., Pascual-Leone, A. & Battelli, L. (2011) Improved motion perception and impaired spatial suppression following disruption of cortical area MT/V5. Journal of Neuroscience, 31, 1279-1283.

    1. Reviewer #1 (Public Review):

      Summary:

      In this work, Qiu and colleagues examined the effects of preovulatory (i.e., proestrous or late follicular phase) levels of circulating estradiol on multiple calcium and potassium channel conductances in arcuate nucleus kisspeptin neurons. Although these cells are strongly linked to a role as the "GnRH pulse generator," the goal here was to examine the physiological properties of these cells in a hormonal milieu mimicking late proestrus, the time of the preovulatory GnRH-LH surge. Computational modeling is used to manipulate multiple conductances simultaneously and support a role for certain calcium channels in facilitating a switch in firing mode from tonic to bursting. CRISPR knockdown of the TRPC5 channel reduced overall excitability, but this was only examined in cells from ovariectomized mice without estradiol treatment. The manuscript has been substantially improved from the initial version by the addition of new experiments and clarification of important figures. Importantly, the overlap of data with previous reports from the same group has been corrected.

      Strengths:

      (1) Examination of multiple types of calcium and potassium currents, both through electrophysiology and molecular biology.

      (2) Focus on arcuate kisspeptin neurons during the surge is relatively conceptually novel as the anteroventral periventricular nucleus (AVPV) kisspeptin neurons have received much more attention as the "surge generator" population.

      (3) The modeling studies allow for direct examination of manipulation of single and multiple conductances, whereas the electrophysiology studies necessarily require examination of each current in isolation. Construction of an arcuate kisspeptin neuron model promises to be of value to the reproductive neuroendocrinology field.

      Weaknesses:

      A remaining weakness in this revised version of the manuscript is that the relevance of the CRISPR experiments is still rather tenuous given that the goal is to understand what happens in the estrogen-treatment condition, and these experiments were performed only in OVX mice. Similar concerns reflect that the computational model examining the effect of E2 infers multiple conductances based on qPCR data and an assumption that the conductances are directionally proportional to the level of gene expression, and then tunes these to the current recordings obtained from OVX mice, without a direct confirmation in OVX+E2 conditions that the model parameters accurately reflect the properties of these currents in the presence of estrogen.

    1. Reviewer #1 (Public Review):

      Summary:

      An online database called MRAD has been developed to identify the risk or protective factors for AD.

      Strengths:

      This study is a very intriguing study of great clinical and scientific significance that provided a thorough and comprehensive evaluation with regard to risk or protective factors for AD. It also provided physicians and scientists with a very convenient, free as well as user-friendly tool for further scientific investigation.

      Comments on revised version:

      The authors have resolved all of my previous comments. It's a decent paper worth to be published in this field.

    1. Reviewer #1 (Public Review):

      Summary:

      This is a reviewed manuscript submission to better understand mechanisms for why HIV individuals have diastolic dysfunction. Due to a lack of robust animal models, the team developed iPS-CM models to study HFpEF. The revised manuscript has toned down claims regarding diastolic function given the lack of mechanical testing. The team has focused on the altered Ca2+ phenotype, which improves the precision of the claims of the team. There remain questions on the functional relevance of the altered calcium handling given the lack of physiological assays. There also remain some questions about whether SGLT2 protein is expressed in these models without testing it, and whether the effects of SGLT2i could be off-target.

      Overall, the revised manuscript is improved. I have no major remaining concerns except that the lack of biomechanical assessments diminishes the significance of the study as altered calcium alone would not be considered sufficient evidence for diastolic dysfunction, which was major task set out to answer by the group.

    1. Reviewer #1 (Public Review):

      Summary:

      This study, titled "Enhancing Bone Regeneration and Osseointegration using rhPTH(1-34) and Dimeric R25CPTH(1-34) in an Osteoporotic Beagle Model," provides valuable insights into the therapeutic effects of two parathyroid hormone (PTH) analogs on bone regeneration and osseointegration. The research is methodologically sound, employing a robust animal model and a comprehensive array of analytical techniques, including micro-CT, histological/histomorphometric analyses, and serum biochemical analysis.

      Strengths:

      The use of a large animal model, which closely mimics postmenopausal osteoporosis in humans, enhances the study's relevance to clinical applications. The study is well-structured, with clear objectives, detailed methods, and a logical flow from introduction to conclusion. The findings are significant, demonstrating the potential of rhPTH(1-34) and dimeric R25CPTH(1-34) in enhancing bone regeneration, particularly in the context of osteoporosis.

      Weaknesses: There are no major weaknesses.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors performed a systematic review and meta-analysis to investigate whether the frequency of emergence of resistance is different if combination antibiotic therapy is used compared to fewer antibiotics. The review shows that there is currently insufficient evidence to reach a conclusion due to the limited sample size. High-quality studies evaluating appropriate antimicrobial resistance endpoints are needed.

      Strengths:

      The strength of the manuscript is that the article addresses a relevant research question which is often debated. The article is well-written and the methodology used is valid. The review shows that there is currently insufficient evidence to reach a conclusion due to the limited sample size. High-quality studies evaluating appropriate antimicrobial resistance endpoints are needed. I have several comments and suggestions for the manuscript.

      Weaknesses:

      Weaknesses of the manuscript are the large clinical and statistical heterogeneity and the lack of clear definitions of acquisition of resistance. Both these weaknesses complicate the interpretation of the study results.

      Comments on latest version:

      The authors adressed all the comments that were shared in the previous peer review. I still believe that both clinical and statistical heterogeneity remains a problem with the interpretation of the meta-analysis. However, as the authors state, this is in line with the original research question as formulated on Prospero.

    1. Reviewer #1 (Public Review):

      Summary:

      In this paper the authors provide a thorough demonstration of the role that one particular type of voltage-gated potassium channel, Kv1.8, plays in a low voltage activated conductance found in type I vestibular hair cells. Along the way, they find that this same channel protein appears to function in type II vestibular hair cells as well, contributing to other macroscopic conductances. Overall, Kv1.8 may provide especially low input resistance and short time constants to facilitate encoding of more rapid head movements in animals that have necks. Combination with other channel proteins, in different ratios, may contribute to the diversified excitability of vestibular hair cells.

      Strengths:

      The experiments are comprehensive and clearly described, both in text and in the figures. Statistical analyses are provided throughout.

      Weaknesses:

      None.

    1. Reviewer #1 (Public Review):

      The study investigates Cancer Driving Nucleotides (CDNs) using the TCGA database, finding that these recurring point mutations could greatly enhance our understanding of cancer genomics and improve personalized treatment strategies. Despite identifying 50-150 CDNs per cancer type, the research reveals that a significant number remain undiscovered, limiting current therapeutic applications, and underscoring the need for further larger-scale research.

      Strengths:

      The study provides a detailed examination of cancer-driving mutations at the nucleotide level, offering a more precise understanding than traditional gene-level analyses. The authors found a significant number of CDNs remain undiscovered, with only 0-2 identified per patient out of an expected 5-8, indicating that many important mutations are still missing. The study indicated that identifying more CDNs could potentially significantly impact the development of personalized cancer therapies, improving patient outcomes.

      Weaknesses:

      The study is constrained by relatively small sample sizes for each cancer type, which reduces the statistical power and robustness of the findings. ICGC and other large-scale WGS datasets are publicly available but were not included in this study.

      To be able to identify rare driver mutations, more samples are needed to improve the statistical power, which is well-known in cancer research.

      The challenges in direct functional testing of CDNs due to the complexity of tumor evolution and unknown mutation combinations limit the practical applicability of the findings.

      The QC of the TCGA data was not very strict, i.e, "patients with more than 3000 coding region point mutations were filtered out as potential hypermutator phenotypes", it would be better to remove patients beyond +/- 3*S.D from the mean number of mutations for each cancer type. Given some point mutations with >3 hits in the TCGA dataset, they were just false positive mutation callings, particularly in the large repeat regions in the human genome.

      The codes for the statistical calculation (i.e., calculation of Ai_e, et al) are not publicly available, which makes the findings hard to be replicated.

    1. Reviewer #1 (Public Review):

      Calcium channels are key regulators of synaptic strength and plasticity, yet how these channels are differentially utilized to enable synaptic diversity is not clear. In this manuscript, the authors use new endogenous tagging of the Drosophila CaV2 channel Cac and three auxiliary subunits to investigate distinct calcium channel functions at two motor neuron subtypes at the fly NMJ, Is and Ib. Although it is clear from previous studies that Pr is higher at Is over Ib, it is not clear why. The authors confirm these differences using postsynaptic calcium imaging combined with post-hoc Cac-TdTomato imaging. Then, through a series of confocal and super resolution imaging studies, the authors describe differences in calcium channel and active zone structure between Is and Ib motor neuron terminals, and the role of Brp and homeostatic plasticity in regulating channel abundance. Finally, the authors show that while the CaBeta subunit is present at similar levels at Is and Ib active zones, there is an interesting reduction in Stj at Is active zones. The authors conclude that these differences in active zone structure and architecture contribute to the generation of the observed heterogeneity in synaptic strength.

      Overall the manuscript is well written, and the successful generation of the new endogenous Cac tags (Td-Tomato, Halo) and CaBeta, stj, and stolid genes with V5 tags will be powerful reagents for the field to enable new studies on calcium channels in synaptic structure, function, and plasticity. There are also some interesting, though not entirely unexpected, findings regarding how Brp and homeostatic plasticity modulate calcium channel abundance. The key factors generating diversity in synaptic strength beyond simple Ca2+ influx are well articulated in framing this study. Beyond the particularly useful new reagents for the field presented, the new data demonstrating a concerted and coupled increase in Cac, Stj, and CaB together after plasticity provides an interesting new dimension to the study and a foundation for new work moving forward.

      Comments on revision:

      This is a much improved revised manuscript, where the authors have done an excellent job of responding to my initial concerns. In particular, the key factors generating diversity in synaptic strength beyond simple Ca2+ influx are better articulated in framing this study. Beyond the particularly useful new reagents for the field presented, the new data demonstrating a concerted and coupled increase in Cac, Stj, and CaB together after plasticity provides an interesting new dimension to the study and a foundation for new work moving forward.

      Upon reflection, I think my initial review came across as a bit harsh, and I am happy to now update my original evaluation to better reflect the importance and impact of this very nice study. I commend the authors on an outstanding study.

    1. Reviewer #1 (Public Review):

      Greter et al. provide an interesting and creative use of lactulose as a "microbial metabolism" inducer, combined with tracking of H2 and other fermentation end products. The topic is timely and will likely be of broad interest to researchers studying nutrition, circadian rhythm, and gut microbiota. However, a couple of moderate to major concerns were noted that may impact the interpretation of the current data:

      (1) Much of the data relies on housing gnotobiotic mice in metabolic cages, but I couldn't find any details of methods to assess contamination during multiple days of housing outside of gnotobiotic isolators/cages. Given the complexity of the metabolic cage system used, sterility would likely be incredibly challenging to achieve. More details needed to be included about how potential contamination of the mice was assessed, ideally with 16S rRNA gene sequencing data of the endpoint samples and/or qPCR for total colonization levels relative to the more targeted data shown.

      (2) The language could be softened to provide a more nuanced discussion of the results. While lactulose does seem to induce microbial metabolism it also could have direct effects on the host due to its osmotic activity or other off-target effects. Thus, it seems more precise to just refer to lactulose specifically in the figure titles and relevant text. Additionally, the degree to which lactulose "disrupts the diurnal rhythm" isn't clear from the data shown, especially given that the markers of circadian rhythm rapidly recover from the perturbation. It is probably more precise to instead state that lactulose transiently induces fermentation during the light phase or something to that effect. The discussion could also be expanded to address what methods are available or could be developed to build upon the concepts here; for example, the use of genetic inducers of metabolism which may avoid the more complex responses to lactulose.

      Despite these concerns, this was still an intriguing and valuable addition to the growing literature on the interface of the microbiome and circadian fields.

    1. Reviewer #1 (Public Review):

      Summary:

      Recordings were made from the dentate nucleus of two monkeys during a decision-making task. Correlates of stimulus position and stimulus information were found to varying degrees in the neuronal activities.

      Strengths:

      A difficult decision-making task was examined in two monkeys.

      Weaknesses:

      One of the monkeys did not fully learn the task. The manuscript lacked a coherent hypothesis to be tested, and no attempt was made to consider the possibility that this part of the brain may have little to do with the task that was being studied.

    1. Reviewer #1 (Public Review):

      Summary:<br /> In this manuscript, Singh, Wu and colleagues explore functional links between septins and the exocyst complex. The exocyst in a conserved octameric complex that mediates the tethering of secretory vesicles for exocytosis in eukaryotes. In fission yeast cells, the exocyst is necessary for cell division, where it localizes mostly at the rim of the division plane, but septins, which localize in a similar manner, are non-essential. The main findings of the work are that septins are required for the specific localization of the exocyst to the rim of the division plane, and the likely consequent localization of the glucanase Eng1 at this same location, where it is known to promote cell separation. In the absence of septins, the exocyst still localizes to the division plane but is not restricted to the rim. They also show some defects in the localization of secretory vesicles and glucan synthase cargo. They further propose that interactions between septins and exocysts are direct, as shown through Alphafold2 predictions (of unclear strength) and clean coIP experiments.

      Strengths:<br /> The septin, exocyst and Eng1 localization data are well supported, showing that the septin rim recruits the exocyst and (likely consequently) the Eng1 glucanase at this location. One major finding of the manuscript is that of a physical interaction between septins and exocyst subunits. Indeed, many of the coIPs supporting this discovery are very clear.

      Weaknesses:<br /> I am less convinced by the strength of the physical interaction of septins with the exocyst complex. Notably, one important open question is whether septins interact with the intact exocyst complex, as claimed in the text, or whether the interactions occur only with individual subunits. The two-hybrid and coIP data only show weak interactions with individual subunits, and some coIPs (for instance Sec3 and Exo70 with Spn1 and Spn4) are negative, suggesting that the exocyst complex does not remain intact in these experiments. Given the known structure of the full exocyst complex and septin filaments (at least in S. cerevisiae), the Alphafold2 predicted structure could be used to probe whether the proposed interaction sites are compatible with full complex formation.

      The effect of spn1∆ on Eng1 localization is very clear, but the effect on secretory vesicles (Ypt3, Syb1) and glucan synthase Bgs1 is less convincing. The effect is small, and it is not clear how the cells are matched for the stage of cytokinesis.

    1. Reviewer #1 (Public Review):

      Summary:

      This study was to examine the associations of a healthy lifestyle with comprehensive and organ-specific biological ages. It emphasized the importance of lifestyle factors in biological ages, which were defined using common blood biomarkers and body measures.

      Strengths:

      The data were from a large cohort study and defined comprehensive and six-specified biological ages.

      Weaknesses:

      (1) Since only 8.5% of participants from the CMEC (China Multi-Ethnic Cohort Study) were included in the study, has any section bias happened?

      (2) The authors should specify the efficiency of FFQ. How can FFQ genuinely reflect the actual intake? Moreover, how was the aMED calculated?

      (3) HLI (range) and HLI (category) should be clearly defined.

      (4) The comprehensive rationale and each specific BA construction should be clearly defined and discussed. For example, can cardiopulmonary BA be reflected only by using cardiopulmonary status? I do not think so.

      (5) The lifestyle index is defined based on an equal-weight approach, but this does not reflect reality and cannot fully answer the research questions it raises.

    1. Reviewer #1 (Public Review):

      Summary:

      The authors set out to understand the role played by a key global metabolic regulator called Crp/cAMP in the formation of persister Escherichia coli that survive antibiotic treatment without acquiring genetic mutations.

      In order to achieve this aim, the authors employ an interdisciplinary approach exquisitely integrating standard microbiology assays with cutting-edge genomic, metabolomic, and proteomics screening.

      The data presented by the authors convincingly demonstrate that the deletion of two key genes that are part of the Crp/cAMP complex (i.e. crp and cyaA) leads to a significant decrease in the number of persisters, thus pointing towards a key role played by the Crp/cAMP complex in the formation of persisters in E. coli.

      The data presented also demonstrate that deletion of the crp gene leads to an overall decrease in energy metabolism and an overall increase in anabolic metabolism at the population level. It is not clear either what the contribution of the cyaA gene is in this respect, or why the deletion of cyaA has an opposite effect on cAMP concentration compared to crp deletion, although the authors present two reasonable untested hypotheses in the discussion. The authors might also want to explicitly acknowledge that these key data are obtained at the whole population level rather than at the level of the persister subpopulation.

      Finally, the authors convincingly show that the persisters they investigated are non-growing and have a higher redox activity and that the deletion of key genes involved in energy metabolism leads to a decrease in the number of persisters.

      These data will be key for future investigations on the biochemical mechanisms that allow bacteria to adapt to stressors such as nutrient depletion or exposure to antibiotics. As such this work will likely have an impact in a variety of fields such as bacterial biochemistry, antimicrobial resistance research, and environmental microbiology.

      Strengths:

      Interdisciplinary approach.<br /> Excellent use of replication and ensuring reproducibility.<br /> Excellent understanding and presentation of the biochemical mechanisms underpinning bacterial physiology via an integrated genomic, metabolomic, and proteomic screening.

      Weaknesses:

      Two genes from the Crp/cAMP complex (crp and cyaA) are hypothesised to be key for persistence but key metabolomics and proteomics data are obtained from only one deletion mutant in the crp gene.

      The deletion of crp and cyaA have opposite effects on the concentration of cAMP, a comparison of metabolomics and proteomics data obtained using both mutants might aid in understanding this difference.

      Metabolomics, proteomics, and metabolic activity data are obtained at the whole population level rather than at the level of the persister sub-population.

    1. Joint Public Review:

      Solitary Fibrous Tumors (SFTs) are a rare malignancy defined by NAB2-STAT6 fusions. Because the molecular understanding of the disease is largely lacking, there are currently no targeted treatment approaches. Using primary tumor and adjacent normal tissue samples and cells inducibly expressing NAB2-STAT6, Hill et al. perform a detailed characterization of the transcriptomic and epigenomic NAB2-STAT6 SFT signatures. They identify enrichment or EGR1/NAB2 (but not STAT6) sites bound by the fusion protein and increased expression of EGR1 targets. Their studies indicate that NAB2-STAT6 fusion may direct the nuclear translocation of NAB2 and EGR1 proteins and potentially NAB1. Transcriptionally, NAB2-STAT6 SFTs most closely resemble neuroendocrine tumors.

      This pioneering study provides critical insight into the molecular pathogenesis of SFTs, pivotal for the future development of mechanistically informed treatment approaches. The study is rigorously executed and well-written. This new knowledge is an important addition to the field. Recommendations for minor improvements can be made.

    1. Reviewer #1 (Public Review):

      Summary:

      The overall goal of the manuscript is to delineate pathways that are conditionally essential with the Bam complex and associated chaperones. The Bam complex is made of several proteins, including BamA and BamD, which are essential. The protein complex works to insert proteins in the asymmetric outer membrane. Substrates are translated in the cytoplasm prior to transport across the cell envelope to the Bam complex. Transport includes non-essential periplasmic chaperones, SurA, Skp, and DegP. According to the authors, the pathways were assumed to be redundant. The Bam complex also includes non-essential components, BamBCE. These were thought to be accessory components that interact with BamA and BamD to coordinate optimal activity. While some roles have been assigned to BamE and BamB, a detailed understanding of the role of each accessory Bam protein is lacking. In this study, more specific roles for each non-essential Bam component are proposed.

      Strengths:

      The overall findings are intriguing and could advance our understanding as to how the Gram-negative cell envelope is assembled. These studies could provide new targets for antimicrobial treatment. In general, the manuscript was well-written.

      Weaknesses:

      While the overall findings are interesting, I had some concerns with the data analysis, presentation, and conclusions. Not all the conclusions are supported by data. The proposed revisions include experimental and editorial work. The manuscript is generally well-written and could provide impactful data to advance the field if the concerns are addressed.

      Major concerns:

      Overall Comments:

      (1) The cutoffs the authors used to define "conditionally essential" mutants are not reported. The results also lack validation for lethality using a titratable system. It would be ideal to validate several genes in each dataset to determine cutoffs (i.e. 5-fold decrease in insertion mutants) for conditional lethality. It was not done (or described) here.

      (2) Also, two mutations that both make the cells sick could provide an additive effect (i.e. dapF and BamB), which doesn't necessarily mean the pathways are linked. The authors should revise their wording. They have not shown genetic linkage in some cases.

      (3) Mutations throughout the manuscript are not complemented. It would be ideal to add complementation data to show the gene-phenotype relationship is specific.

      (4) Also, I would argue the term "conditionally essential genes" should be replaced with "synthetically lethal". Strains were compared in the same conditions but with different genetic backgrounds.

    1. Reviewer #1 (Public Review):

      Summary:

      This study explores the roles of dact1 and dact2 in zebrafish embryonic axis formation and craniofacial morphogenesis. The researchers aim to uncover the mechanisms by which dact1/2 modulates Wnt signaling during embryonic development and patterning. They propose distinct spatiotemporal roles for Dact1 and Dact2 proteins in zebrafish embryonic development, particularly their involvement in modulating noncanonical Wnt signaling during convergent extension events. The findings demonstrate that dact1 and dact2 have unique spatiotemporal expression domains during development and that mutations in dact1/2 lead to convergent extension defects. Furthermore, the study attempts to link these defects to craniofacial abnormalities resulting from dact1/2 mutations. Compound mutants were used to investigate the connection between dact1 and dact2, as single mutants did not exhibit craniofacial phenotypes. The research also includes comprehensive transcriptomics and pathway analyses of differentially expressed genes in dact1/2 mutants, revealing the overexpression of calpain 8, a calcium-dependent cysteine protease. The study suggests that the upregulation of calpain 8 is linked to the observed craniofacial dysmorphology in dact1/2 mutants, implying a potential connection between calpain 8 expression and craniofacial abnormalities.

      Strengths:

      • The study effectively recapitulates previous findings on the role of dact1/2 in modulating convergent extension during zebrafish embryogenesis.<br /> • A combination of multiple approaches, including in vivo time-lapse imaging, is used to elucidate the etiology of the rod-like neurocranial phenotype in dact1/2 double mutants.<br /> • The study utilizes both traditional and newly created mutant lines, analyzing them through single-cell transcriptomics.

      Weaknesses:

      (1) The authors successfully addressed reviewers' suggestions with revised experiments and explanations. However, the overall narrative struggles to build a more coherent storyline.<br /> (2) The potential activity of truncated and upregulated dact mRNAs (Fig S2) and partially functional dact proteins needs further clarification.<br /> (3) Data-rich figures, specifically Figs 6, 7, and 8D, could be simplified for better clarity.

    1. Reviewer #1 (Public Review):

      In this manuscript, Laboy and colleagues investigated upstream regulators of MML-1/Mondo, a key transcription factor that regulates aging and metabolism, using the nematode C. elegans and cultured mammalian cells. By performing a targeted RNAi screen for genes encoding enzymes in glucose metabolism, the authors found that two hexokinases, HXK-1 and HXK-2, regulate nuclear localization of MML-1 in C. elegans. The authors showed that knockdown of hxk-1 and hxk-2 suppressed longevity caused by germline-deficient glp-1 mutations. The authors demonstrated that genetic or pharmacological inhibition of hexokinases decreased nuclear localization of MML-1, via promoting mitochondrial β-oxidation of fatty acids. They found that genetic inhibition of hxk-2 changed the localization of MML-1 from the nucleus to mitochondria and lipid droplets by activating pentose phosphate pathway (PPP). The authors further showed that the inhibition of PPP increased the nuclear localization of mammalian MondoA in cultured human cells under starvation conditions, suggesting the underlying mechanism is evolutionarily conserved. This paper provides compelling evidence for the mechanisms by which novel upstream metabolic pathways regulate MML-1/Mondo, a key transcription factor for longevity and glucose homeostasis, through altering organelle communications, using two different experimental systems, C. elegans and mammalian cells. This paper will be of interest to a broad range of biologists who work on aging, metabolism, and transcriptional regulation.

    1. Reviewer #1 (Public Review):

      Summary:

      By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to the identification of a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.

      Strengths:

      In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.

      Weaknesses:

      The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as, for example, species-specific gamete recognition. Additionally, the use of a TPM >1 threshold for protein-coding transcripts - which, as the authors pointed out, was a necessary compromise due to the high transcriptional noise of the system under study - may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis. Moreover, as mentioned in the Discussion, future variations of the pipeline described in the manuscript may allow us to extend the reach of the present analysis.

    1. Reviewer #2 (Public Review):

      Summary:

      The authors used state-of-the-art microscopy to analyze the structural changes that occur in sperm tails after the acrosome reaction. They found that midpiece contraction and actin reorganization occurred, which is associated with the cessation of flagellar motility during sperm-egg fusion. The mechanism by which flagellar motility is arrested during sperm-oocyte fusion is unknown, and this study proposes its novel mechanism and provides important insights for cell and reproductive biologists.

      In the revised manuscript, the authors addressed most of my concerns.

      Strength:

      Various microscopy techniques including super-resolution microscopy and scanning electron microscopy were used to analyze structural organization of the midpiece in detail.

    1. Reviewer #1 (Public Review):

      Summary

      This study was designed to investigate changes in gene expression and associated chromatin accessibility patterns in spermatogonia in mice at different postnatal stages from pups to adults. The objective was to describe dynamic changes in these patterns that potentially correlate with functional changes in spermatogonia as a function of development and reproductive maturation. The potential utility of this information is to serve as a reference against which similar data from animals subjected to various disruptive environmental influences can be compared.

      Major Strengths and Weaknesses of the Methods and Results

      A strength of the study is that it reviews previously published datasets describing gene expression and chromatin accessibility patterns in mouse spermatogonia. A weakness of the study is that it is not clear what new information is provided by the data provided that was not already known from previously published studies (see below). Specific weaknesses include the following...

      - Terminology - In the Abstract and first part of the Introduction the authors use the generic term "spermatogonial cells" in a manner that seems to be referring primarily to spermatogonial stem cells (SSCs) but initially ignores the well-known heterogeneity among spermatogonia - particularly the fact that only a small proportion of developing spermatogonia become SSCs - and ONLY those SSCs and NOT other developing spermatogonia - support steady-state spermatogenesis by retaining the capacity to either self-renew or contribute to the differentiating spermatogenic lineage throughout the male reproductive lifespan. The authors eventually mention other types of developing male germ cells, but their description of prospermatogonial stages that precede spermatogonial stages is deficient in that M-prospermatogonia - which occur after PGCs but before T1-prospermatogonia - are not mentioned. This description also seems to imply that all T2-prospermatogonia give rise to SSCs which is far from the case. It is the case that prospermatogonia give rise to spermatogonia, but only a very small proportion of undifferentiated spermatogonia form the foundational SSCs and ONLY SSCs possess the capacity to either self-renew or give rise to sequential waves of spermatogenesis.

      - Introduction - Statements regarding distinguishing transcriptional signatures in spermatogonia at different postnatal stages appear to refer to ALL subtypes of spermatogonia present at each stage collectively, thereby ignoring the well-known fact that there are distinct spermatogonial subtypes present at each postnatal stage and that some of those occur at certain stages but not at others. This brings into question the usefulness of the authors' discussion of what types of genes are expressed and/or what types of changes in chromatin accessibility are detected in spermatogonia at each stage.

      - Methodology - The authors based recovery (enrichment) of spermatogonia from male pups on FACS sorting for THY1 and RMV-1. While sorting total testis cells for THY1+ cells does enrich for spermaogonia, this approach is now known to not be highly specific for spermatogonia (somatic cells are also recovered) and definitely not for SSCs. There are more effective means for isolating SSCs from total testis cells that have been validated by transplantation experiments (e.g. use of the Id4/eGFP transgene marker).

      The authors then used "deconvolution" of bulk RNA-seq data in an attempt to discern spermatogonial subtype-specific transcriptomes. It is not clear why this is necessary or how it is beneficial given the availability of multiple single-cell RNA-seq datasets already published that accomplish this objective quite nicely - as the authors essentially acknowledge. Beyond this concern, a potential flaw with the deconvolution of bulk RNA-seq data is that this is a derivative approach that requires assumptions/computational manipulations of apparent mRNA abundance estimates that may confound interpretation of the relative abundance of different cellular subtypes within the hetergeneous cell population from which the bulk RNA-seq data is derived. Bottom line, it is not clear that this approach affords any experimental advantage over use of the publicly available scRNA-seq datasets and it is possible that attempts to employ this approach may be flawed yielding misleading data.

      - Results & Discussion - In general, much of the information reported in this study is not novel. The authors' discussion of the makeup of various spermatogonial subtypes in the testis at various ages does not really add anything to what has been known for many years on the basis of classic morphological studies. Further, as noted above, the gene expression data provided by the authors on the basis of their deconvolution of bulk RNA-seq data does not add any novel information to what has been shown in recent years by multiple elegant scRNA-seq studies - and, in fact, as also noted above - represents an approach fraught with potential for misleading results. The potential value of the authors' report of "other cell types" not corresponding to major somatic cell types identified in earlier published studies seems quite limited given that they provide no follow-up data that might indicate the nature of these alternative cell types. Beyond this, much of the gene expression and chromatin accessibility data reported by the authors - by their own admission given the references they cite - is largely confirmatory of previously published results. Similarly, results of the authors' analyses of putative factor binding sites within regions of differentially accessible chromatin also appear to confirm previously reported results. Ultimately, it is not at all novel to note that changes in gene expression patterns are accompanied by changes in patterns of chromatin accessibility in either related promoters or enhancers. The discussion of these observations provided by the authors takes on more of a review nature than that of any sort of truly novel results. As a result, it is difficult to discern how the data reported in this manuscript advance the field in any sort of novel or useful way beyond providing a review of previously published studies on these topics.

      Likely impact - The likely impact of this work is relatively low because, other than the value it provides as a review of previously published datasets, the new datasets provided are not novel and so do not advance the field in any significant manner.

    1. Reviewer #1 (Public Review):

      Summary:<br /> The manuscript provides a novel method for the automated detection of scent marks from urine and feces in rodents. Given the importance of scent communication in these animals and their role as model organisms, this is a welcome tool.

      Strengths:<br /> The method uses a single video stream (thermal video) to allow for the distinction between urine and feces. It is automated.

      Weaknesses:<br /> The accuracy level shown is lower than may be practically useful for many studies. The accuracy of urine is 80%. This is understandable given the variability of urine in its deposition, but makes it challenging to know if the data is accurate. If the same kinds of mistakes are maintained across many conditions it may be reasonable to use the software (i.e., if everyone is under/over counted to the same extent). Differences in deposition on the scale of 20% would be challenging to be confident in with the current method, though differences of the magnitude may be of biological interest. Understanding how well the data maintain the same relative ranking of individuals across various timing and spatial deposition metrics may help provide further evidence for the utility of the method.

    1. Reviewer #1 (Public Review):

      Summary:

      De Waele et al. reported a dual-branch neural network model for predicting antibiotic resistance profiles using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry data. Neural networks were trained on the recently available DRIAMS database of MALDI-TOF mass spectrometry data and their associated antibiotic susceptibility profiles. The authors used dual branch neural network to simultaneously represent information about mass spectra and antibiotics for a wide range of species and antibiotic combinations. The authors showed consistent performance of their strategy to predict antibiotic susceptibility for different spectrum and antibiotic representations (i.e., embedders). Remarkably, the authors showed how small datasets collected at one location can improve the performance of a model trained with limited data collected at a second location. The authors also showed that species-specific models (trained in multiple antibiotic resistance profiles) outperformed both the single recommender model and the individual species-antibiotic combination models. Despite the promising results, the authors should explain in more detail some of the analyses reported in the manuscript (see weaknesses).

      Strengths:

      • A single AMR recommender system could potentially facilitate the adoption of MALDI-TOF based antibiotic susceptibility profiling into clinical practices by reducing the number of models to be considered, and the efforts that may be required to periodically update them.<br /> • Authors tested multiple combinations of embedders for the mass spectra and antibiotics while using different metrics to evaluate the performance of the resulting models. Models trained using different spectrum embedder-antibiotic embedder combinations had remarkably good performance for all tested metrics. The average ROC AUC scores for global and species-specific evaluations were above 0.8.<br /> • Authors developed species-specific recommenders as an intermediate layer between the single recommender system and single species-antibiotic models. This intermediate approach achieved maximum performance (with one type of the species-specific recommender achieving a 0.9 ROC AUC), outlining the potential of this type of recommenders for frequent pathogens.<br /> • Authors showed that data collected in one location can be leveraged to improve the performance of models generated using a smaller number of samples collected at a different location. This result may encourage researchers to optimize data integration to reduce the burden of data generation for institutions interested in testing this method.

      Weaknesses:

      • Section 4.3 ("expert baseline model"): the authors need to explain how the probabilities defined as baselines were exactly used to predict individual patient susceptible profiles.<br /> • Authors do not offer information about the model features associated with resistance. Although I understand the difficulty of mapping mass spectra to specific pathways or metabolites, mechanistic insights are much more important in the context of AMR than in the context of bacterial identification. For example, this information may offer additional antimicrobial targets. Thus, authors should at least identify mass spectra peaks highly associated with resistance profiles. Are those peaks consistent across species? This would be a key step towards a proteomic survey of mechanisms of AMR. See previous work on this topic: PMIDs: 35586072 and 23297261.

    1. Joint Public Review:

      The study offers a compelling molecular model for the organization of rootlets, a critical organelle that links cilia to the basal body. Striations have been observed in rootlets, but their assembly, composition, and function remain unknown. While previous research has explored rootlet structure and organization, this study delivers an unprecedented level of resolution, valuable to the centrosome and cilia field. The authors isolated rootlets from mice's eyes. They apply EM to partially purified rootlets (first negative stain, then cryoET). From these micrographs, they observed striations along the membranes along the rootlet but no regular spacing was observed.

      The thickness of the sample and membranes prevented good contrast in the tomograms. Thus they further purified the rootlets using detergent, which allowed them to obtain cryoET micrographs of the rootlets with greater details. The tomograms were segmented and further processed to improve the features of the rootlet structures. They proposed that a number of proteins, including rootletin, form parallel coiled coils that run along the rootlet longitudinally. They described how the cross-striations form 3 types of periodic structures -D1/D2/A bands- connected perpendicularly to filaments along the length of the rootlets and to membranes. Overall their data provide a detailed model for the molecular organization of the rootlet.

      The major strength is that this high-quality study uses state-of-the-art cryo-electron tomography, sub-tomogram averaging, and image analysis to provide a model of the molecular organization of rootlets. The micrographs are exceptional, with excellent contrast and details, which also implies the sample preparation was well optimized to provide excellent samples for cryo-ET. The manuscript is also clear and accessible.

      This research marks a significant step forward in our understanding of rootlets' molecular organization.

    1. Reviewer #1 (Public review):

      Summary:

      Ctnnb1 encodes β-catenin, an essential component of the canonical Wnt signaling pathway. In this study, the authors identify an upstream enhancer of Ctnnb1 responsible for the specific expression level of β-catenin in the gastrointestinal track. Deletion of this promoter in mice and analyses of its association with human colorectal tumors support that it controls the dosage of Wnt signaling critical to the homeostasis in intestinal epithelia and colorectal cancers.

      Strengths:

      This study has provided convincing evidence to demonstrate the functions of a gastrointestinal enhancer of Ctnnb1 using combined approaches of bioinformatics, genomics, in vitro cell culture models, mouse genetics, and human genetics. The results support the idea that the dosage of Wnt/β-catenin signaling plays an important role in pathophysiological functions of intestinal epithelia. The experimental designs are solid and the data presented are of high quality. This study significantly contributes to the research fields of Wnt signaling, tissue-specific enhancers, and intestinal homeostasis.

      Weaknesses:

      Insufficient discussion on some findings was a major weakness in the previous submission, which has been addressed in the revised submission.

    1. Reviewer #1 (Public Review):

      The authors developed a rigorous methodology for identifying all Cancer Driving Nucleotides (CDNs) by leveraging the concept of massively repeated evolution in cancer. By focusing on mutations that recur frequently in pan-cancer, they aimed to differentiate between true driver mutations and neutral mutations, ultimately enhancing the understanding of the mutational landscape that drives tumorigenesis. Their goal was to call a comprehensive catalogue of CDNs to inform more effective targeted therapies and address issues such as drug resistance.

      Strengths

      (1) The authors introduced a concept of using massively repeated evolution to identify CDNs. This approach recognizes that advantageous mutations recur frequently (at least 3 times) across cancer patients, providing a lens to identify true cancer drivers.

      (2) The theory showed the feasibility of identifying almost all CDNs if the number of sequenced patients increases to 100,000 for each cancer type.

      Weaknesses

      (1) The methodology remains theoretical and no novel true driver mutations were identified in this study.

      (2) Different cancer types have unique mutational landscapes. The methodology, while robust, might face challenges in uniformly identifying CDNs across various cancers with distinct genetic and epigenetic contexts.

      (3) L223, the statement "In other words, the sequences surrounding the high-recurrence sites appear rather random.". Since it was a pan-cancer analysis, the unique patterns of each cancer type could be strongly diluted in the pan-cancer data.

      (4) To solidify the findings, the results need to be replicated in an independent dataset.

      (5) The key scripts and the list of key results (i.e., CDN sites with i{greater than or equal to}3) need to be shared to enable replication, validation, and further research. So far, only CDN sites with i{greater than or equal to}20 have been shared.

      (6) The versions of data used in this study are not clearly detailed, such as the specific version of gnomAD and the version and date of TCGA data downloaded from the GDC Data Portal.

    1. Reviewer #1 (Public Review):

      Summary:

      Cai et al have investigated the role of msiCAT-tailed mitochondrial proteins that frequently exist in glioblastoma stem cells. Overexpression of msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore formation/opening. These changes in mitochondrial properties provide resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells.

      Strengths:

      The CAT-tailing concept has not been explored in cancer settings. Therefore, the present provides new insights for widening the therapeutic avenue.

      Weaknesses:

      Although the paper does have strengths in principle, the weaknesses of the paper are that these strengths are not directly demonstrated. The conclusions of this paper are mostly well-supported by data, but some aspects of image acquisition and data analysis need to be clarified and extended.

    1. Reviewer #1 (Public Review):

      Summary:

      This work contributes several important and interesting observations regarding the heterotolerance of non-growing Escherichia coli and Pseudomonas aeruginosa to the antimicrobial peptide tachyplesin. The primary mechanism of action of tachyplesin is thought to be disruption of the bacterial cell envelope, leading to leakage of cellular contents after a threshold level of accumulation. Although the MIC for tachyplesin in exponentially growing E. coli is just 1 ug/ml, the authors observe that a substantial fraction of a stationary phase population of bacteria survive much higher concentrations, up to 64 ug/ml. By using a fluorescently-labelled analogue of tachyplesin, the authors show that the amount of per-cell intracellular accumulation of tachyplesin displays a bimodal distribution and that the fraction of "low accumulators" correlates with the fraction of survivors.

      Using a microfluidic device, they show that low accumulators exclude propidium iodide, suggesting that their cell envelopes remain largely intact, while high accumulators of tachyplesin also stain with propidium iodide. They show that this phenomenon holds for several clinical isolates of E. coli with different genetic determinants of antibiotic resistance, and for a strain of Pseudomonas aeruginosa. However, the bimodal distribution does not occur in these organisms for several other antimicrobial peptides, or for tachyplesin in Klebsiella pneumoniae or Staphylococcus aureus, indicating some degree of specificity in the interaction between AMP and bacterial cell envelope. They next explore the dynamics of the fluorescent tachyplesin accumulation and show interestingly that a high degree of accumulation is initially seen in all cells, but that the "low accumulator" subpopulation manages to decrease the amount of intracellular fluorescence over time, while the "high accumulator" subpopulation continues to increase its intracellular fluorescence. Focusing on increased efflux as a hypothesised mechanism for the "low accumulator" phenotype, based on transcriptomic analysis of the two subpopulations, the authors screen putative efflux inhibitors to see if they can block the formation of the low accumulator subpopulation. They find that both the protonophore CCCP and the SSRI sertraline can block the formation of this subpopulation and that a combination of sertraline plus tachyplesin kills a greater fraction of the stationary phase cells than either agent alone, similar to the killing observed when growing cells are treated with tachyplesin.

      Strengths:

      This study provides new insight into the heterogeneous behaviours of non-growing bacteria when exposed to an antimicrobial peptide, and into the dynamics of their response. The single-cell analysis by FACS and microscopy is compelling. The results provide a much-needed single-cell perspective on the phenomenon of tolerance to AMPs and a good starting point for further exploration.

      Weaknesses:

      My main concerns surround the conclusions drawn about the physiological underpinnings of these behaviours, based in part on transcriptomic analysis and also on the observation of the dynamics. I think deeper consideration of the relative contributions of influx and efflux to the observed accumulation dynamics, and the slow/non-growing context of the observations would be helpful. In particular, these issues seem important:

      (1) The initial high accumulation by all cells followed by the emergence of a sub-population that has reduced its intracellular levels of tachyplesin is a key observation and I agree with the authors' conclusion that this suggests an induced response to the AMP is important in facilitating the bimodal distribution. However, I think the conclusion that upregulated efflux is driving the reduction in signal in the "low accumulator" subpopulation is not fully supported. Steady-state amounts of intracellular fluorescent AMP are determined by the relative rates of influx and efflux and a decrease could be caused by decreasing influx (while efflux remained unchanged), increasing efflux (while influx remained unchanged), or both decreasing influx and increasing efflux. Given the transcriptomic data suggest possible changes in the expression of enzymes that could affect outer membrane permeability and outer membrane vesicle formation as well as efflux, it seems very possible that changes to both influx and efflux are important. The "efflux inhibitors" shown to block the formation of the low accumulator subpopulation have highly pleiotropic or incompletely characterised mechanisms of action so they also do not exclusively support a hypothesis of increased efflux.

      (2) A conclusion of the transcriptomic analysis is that the lower accumulating subpopulation was exhibiting "a less translationally and metabolically active state" based on less upregulation of a cluster of genes including those involved in transcription and translation. This conclusion seems to borrow from well-described relationships referred to as bacterial growth laws in which the expression of genes involved in ribosome production and translation is directly related to the bacterial growth (and metabolic) rate. However, the assumptions that allow the formulation of the bacterial growth laws (balanced, steady state, exponential growth) do not hold in growth arrest. A non-growing cell could express no genes at all or could express ribosomal genes at a very low level, or efflux pumps at a high level. The distribution of transcripts among the functional classes of genes does not reveal anything about metabolic rates within the context of growth arrest - it only allows insight into metabolic rates when the constraint of exponential growth can be assumed. Efflux pumps can be highly metabolically costly; for example, Tn-Seq experiments have repeatedly shown that mutants for efflux pump gene transcriptional repressors have strong fitness disadvantages in energy-limited conditions. There are no data presented here to disprove a hypothesis that the low accumulators have high metabolic rates but allocate all of their metabolic resources to fortifying their outer membranes and upregulating efflux. This could be an important distinction for understanding the vulnerabilities of this subpopulation. Metabolic rates can be more directly estimated for single cells using respiratory dyes or pulsed metabolic labelling, for example, and these data could allow deeper insight into the metabolic rates of the two subpopulations.

      The observation that adding nutrients to the stationary phase cultures pushes most of the cells to the "high accumulator" state is presented as support of the hypothesis that the high accumulator state is a higher metabolism/higher translational activity state. However, it is important to note that adding nutrients will cause most or all of the cells in the population to start to grow, thus re-entering the familiar regime in which bacterial growth laws apply. This is evident in the slightly larger cell sizes seen in the nutrient-amended condition. In contrast to stationary phase cells, growing cells largely do not exhibit the bimodal distribution, and they are much more sensitive to tachyplesin, as demonstrated clearly in the supplement. Growing cells are not necessarily the same as the high-accumulating subpopulation of non-growing cells.

      It might also be worth adding some additional context around the potential to employ efflux inhibitors as therapeutics. It is very clear that obtaining sufficient antimicrobial drug accumulation within Gram-negative bacteria is a substantial barrier to effective treatments, and large concerted efforts to find and develop therapeutic efflux pump inhibitors have been undertaken repeatedly over the last 25 years. Sufficiently selective inhibitors of bacterial efflux pumps with appropriate drug-like properties have been challenging to find and none have entered clinical trials. Multiple psychoactive drugs have been shown to impact efflux in bacteria but usually using concentrations in the 10-100 uM range (as here). Meanwhile, the Ki values for their human targets are usually in the sub- to low-nanomolar range. The authors rightly note that the concentration of sertraline they have used is higher than that achieved in patients, but this is by many orders of magnitude, and it might be worth expanding a bit on the substantial challenge of finding efflux inhibitors that would be specific and non-toxic enough to be used therapeutically. Many advances in structural biology, molecular dynamics, and medicinal chemistry may make the quest for therapeutic efflux inhibitors more fruitful than it has been in the past but it is likely to remain a substantial challenge.

    1. Reviewer #1 (Public Review):

      Summary:

      The study addresses the growing threat of multi-drug-resistant (MDR) pathogens, focusing on the efficacy of colistin (COL), a last-resort antibiotic, and its enhanced activity when combined with artesunate (AS) and ethylenediaminetetraacetic acid (EDTA) against colistin-resistant Salmonella strains. The researchers aim to explore whether these combinations can restore the effectiveness of colistin and understand the underlying mechanisms. The study used a combination of microbiological and molecular techniques to evaluate the antibacterial activity and mechanisms of action of COL, AS, and EDTA.

      Key methods include:

      (1) Antimicrobial Susceptibility Testing: Determining minimum inhibitory concentrations (MICs) of COL, AS, and EDTA, both alone and in combination, against various Salmonella strains;

      (2) Time-Kill Assays: Measuring bacterial growth inhibition over time with different drug combinations;

      (3) Fluorescent Probe-Permeability Assays: Assessing cell membrane integrity using fluorescent dyes;

      (4) Proton Motive Force Assay: Evaluating the impact on the electrochemical proton gradient (PMF);

      (5) Reactive Oxygen Species (ROS) Measurement: Quantifying intracellular ROS levels; (vi) Scanning Electron Microscopy (SEM): Observing morphological changes in bacterial cells; and

      (6) Omics Analysis: Transcriptome and metabolome profiling to identify differentially expressed genes (DEGs) and significant differential metabolites (SDMs).

      The combination of COL, AS, and EDTA (AEC) showed significant antibacterial activity against colistin-resistant Salmonella strains, reducing the MICs and enhancing bacterial killing compared to individual treatments. The AEC treatment caused extensive damage to both the outer and inner bacterial membranes, as evidenced by increased fluorescence of membrane-impermeant dyes and SEM images showing deformed cell membranes. AEC treatment selectively collapsed the Δψ component of PMF, indicating disruption of vital cellular processes. The combination therapy increased intracellular ROS levels, contributing to bacterial killing. Transcriptome data revealed changes in genes related to two-component systems, flagellar assembly, and ABC transporters. Metabolome analysis highlighted disruptions in pathways such as arachidonic acid metabolism. The findings suggest that AS and EDTA can potentiate the antibacterial effects of colistin by disrupting bacterial membranes, collapsing PMF, and increasing ROS levels. This combination therapy could serve as a promising approach to combat colistin-resistant Salmonella infections.

      Strengths:

      (1) The study employs a wide range of techniques to thoroughly investigate the antibacterial mechanisms and efficacy of the drug combinations.

      (2) The results are consistent across multiple assays and supported by both in vitro and in vivo data.

      (3) Combining AS and EDTA with COL represents a novel strategy to tackle antibiotic resistance.

      Weaknesses:

      (1) The study focuses on a limited number of Salmonella strains, and broader testing on various MDR pathogens would strengthen the findings.

      (2) While the study elucidates several mechanisms, further molecular details could provide deeper insights into the interactions between these drugs and bacterial targets.

      (3) The time-kill experiment was conducted over 12 hours instead of the recommended 24 hours. To demonstrate a synergistic effect among the drugs, a reduction of at least 2 log10 in colony count should be shown in a 24-hour experiment. Additionally, clarifying the criteria for selecting drug concentrations is important to improve the interpretation of the results.

      (4) While the combination of EDTA, artesunate, and colistin shows promising in vitro results against Salmonella strains, the clinical application of this combination warrants careful consideration due to potential toxicity issues associated with these compounds.

    1. Reviewer #1 (Public Review):

      Summary of the Study:

      The manuscript delves into the COVID-19 virus membrane protein M1-subtype and its IgM responses in COVID-19 cohorts. The authors conducted an extensive epitope screening and prediction through Differential Antibody Screening Assay (DASA) and validated their findings across multiple cohorts in Europe. The study aims to provide novel insights into the immune responses to COVID-19 and explore potential clinical implications for long COVID prognostics.

      Strengths:

      (1) Innovative Approach:<br /> The use of DASA for epitope screening is innovative and allows for detailed mapping of immune responses.

      (2) Validation Across Cohorts:<br /> The study's validation of findings across multiple European cohorts adds robustness and generalizability to the results.

      (3) Comprehensive Analysis:<br /> The manuscript presents a thorough analysis of IgM responses, contributing valuable data to the understanding of immune responses in COVID-19.

      Weaknesses:

      (1) Lack of Clarity on T-Independent B Cell Reactions:<br /> The rationale and results regarding T-independent B cell reactions are not well-explained, requiring additional bridging sentences or data for better comprehension.

      (2) Limited Sample Size for B Cell Stimulation:<br /> The in vitro B cell stimulation experiments involve a very small number of individuals (2 reacted vs 1 unreacted), which weakens the strength of the conclusions drawn from these experiments.

      (3) Insufficient Exploration of Comorbidities:<br /> The manuscript could benefit from exploring correlations with other clinical data on comorbidities or sub-grouping the long COVID cohort by specific outcomes.

      Appraisal of the Study's Aims and Conclusions :

      The authors have partially achieved their aims by providing novel insights into COVID-19 immune responses and highlighting the potential for using IgM responses in long COVID prognostics. However, the conclusions would be more convincing with additional data and clarity on certain aspects, such as the T-independent B cell reactions and the impact of comorbidities.

      Impact on the Field and Utility to the Community:

      This study has the potential to significantly impact the field of COVID-19 research by advancing the understanding of immune responses to the virus. The novel insights into IgM responses and epitope screening could inform future diagnostic and prognostic tools for COVID-19, particularly in the context of long COVID. Additionally, the methods and data presented could be valuable to researchers exploring similar viral immune responses.

      Additional Context:

      For readers and researchers, it is essential to note that while the study offers intriguing results, the manuscript would benefit from more comprehensive data and clearer explanations in certain areas. The inclusion of the DASA equation in the manuscript or a figure would improve readability and contextual comprehension. Further exploration of clinical comorbidities and additional external validation data would enhance the study's robustness and applicability.

    1. Reviewer #1 (Public Review):

      Summary:

      In this study, the authors investigate the effect of mitochondrial transplantation on post-cardiac arrest myocardial dysfunction (PAMD), which is associated with mitochondrial dysfunction. The authors demonstrate that mitochondrial transplantation enhances cardiac function and increases survival rates after the return of spontaneous circulation (ROSC). Mechanistically, they found that myocardial tissues with transplanted mitochondria exhibit increased mitochondrial complex activity, higher ATP levels, reduced cardiomyocyte apoptosis, and lower myocardial oxidative stress post-ROSC.

      Strengths:

      Previous studies have reported that mitochondrial transplantation can improve myocardial recovery after regional ischemia, but its potential for treating myocardial injury following cardiac arrest has not been tested yet. Therefore, the findings are somewhat novel. Remarkably, the increased survival in mitochondria treated group post-ROSC is very promising and highlights its translational potential.

      Weaknesses:

      The organization of the paper, along with the analysis and interpretation of the results, requires significant revision.

    1. Reviewer #1 (Public Review):

      Drp1 supports mitochondrial fission (doi: 10.1038/s41586-019-1296-y). Viral sensing triggers mitochondrial fusion, leading to MAVS aggregation and improved type-1 IFN response. It was suggested that impairment of Drp1 upon phosphorylation by Tbk1 enhances mitochondrial fusion in virus-infected cells (doi.org/10.1016/j.molcel.2020.10.018). In this manuscript, Fang et al. describe an unexpected role of caspases activated upon Rift Valley fever virus (RVFV) infection in inactivating Drp1. They show that Drp1 is targeted by multiple caspases, including caspase-3, -6, -7 and -8. Indeed, cleavage of Drp1 leads to mitochondrial elongation, boosting the type-1 IFN response of infected cells. Finally, the authors establish the generalisability of the proposed mechanism in the context of cellular infections with H1N1, SeV, and HSV-1. Caspase-dependent and independent cell death processes provide important host defence mechanisms against obligatorily intracellular viral pathogens. This work suggests that caspases reinforce antiviral response involving also the mitochondria-type 1 IFN axis. As such, the manuscript is well written, and the proposal pertaining to caspase-mediated targeting of Drp1 may have implications beyond host-virus interaction studies. However, several loose ends remain, and these concerns need to be addressed to substantiate the mechanistic model.

  3. Aug 2024
    1. Reviewer #1 (Public Review):

      Summary:

      The current manuscript uses electron spin resonance spectroscopy to understand how the dynamic behavior and conformational heterogeneity of the LPS transport system change during substrate transport and in response to the membrane, bound nucleotide (or transition state analog) and accessory subunits. The study builds on prior structural studies to expand our molecular understanding of this highly significant bacterial transport system.

      Strengths

      This series of well-designed and well-executed experiments provide new mechanistic insights into the dynamic behavior of the LPS transport system. Notable new insights provided by this study include its indication of the spatial organization of the LptC domain, which was poorly resolved in structures, and how the LptC domain modulates the dynamic behavior of the gate through which lipids access the binding site. In addition, a mass spectrometry approach designed to examine LPS binding at different stages in the nucleotide-dependent conformational cycle provides insight into the order of operations of LPS binding and transport.