Reviewer #1 (Public review):

Summary:

Rossi et al. asked whether gait adaptation is solely a matter of slow perceptual realignment or if it also involves fast/flexible stimulus-response mapping mechanisms. To test this, they conducted a series of split-belt treadmill experiments with ramped perturbations, revealing behavior indicative of a flexible, automatic stimulus-response mapping mechanism.

Strengths:

(1) The study includes a perceptual test of leg speed, which correlates with the perceptual realignment component of motor aftereffects. This indicates that there are motor performances that are not accounted for by perceptual re-alignment.

(2) They study incorporates qualitatively distinct, hypothesis-driven models of adaptation and proposes a new framework that integrates these various mechanisms.

Weaknesses:

(1) The study could benefit from considering other alternative models. As the authors noted in their discussion, while the descriptive models explain some patterns of behaviour/aftereffects, they don't currently account for how these mechanisms influence the initial learning process itself.

a. For example, the pattern of gait asymmetric might differ for perceptual realignment (a smooth, gradual process), structural learning (more erratic, involving hypothesis testing/reasoning to understand the perturbation, see (Tsay et al. 2024) for a recent review on Reasoning), and stimulus-response mapping (possibly through a reinforcement based trial-and-error approach). If not formally doing a model comparison, the manuscript might benefit from clearly laying out the behavioural predictions for how these different processes shape initial learning.

b. Related to the above, the authors noted that the absence of difference during initial learning suggests that the differences in Experiment 2 in the ramp-up phase are driven by two distinct processes: structural learning and memory-based processes. If the assumptions about initial learning are not clear, this logic of this conclusion is hard to follow.

c. The authors could also test a variant of the dual-rate state-space model with two perceptual realignment processes where the constraints on retention and learning rate are relaxed. This model would be a stronger test for two perceptual re-alignment processes: one that is flexible and another that is rigid, without mandating that one be fast learning and fast forgetting, and the other be slow learning and slow forgetting.

(2) The authors claim that stimulus-response mapping operates outside of explicit/deliberate control. While this could be true, the survey questions may have limitations that could be more clearly acknowledged.

a. Specifically, asking participants at the end of the experiments to recall their strategies may suffer from memory biases (e.g., participants may be biased by recent events, and forget about the explicit strategies early in the experiment), be susceptible to the framing of the questions (e.g., participants not being sure what the experimenter is asking and how to verbalize their own strategy), and moreover, not clear what is the category of explicit strategies one might enact here which dictates what might be considered "relevant" and "accurate".

b. The concept of perceptual realignment also suggests that participants are somewhat aware of the treadmill's changing conditions; therefore, as a thought experiment, if the authors have asked participants throughout/during the experiment whether they are trying different strategies, would they predict that some behaviour is under deliberate control?

(3) The distinction between structural and memory-based differences in the two subgroups was based on the notion that memory-based strategies increase asymmetry. However, an alternative explanation could be that unfamiliar perturbations, due to the ramping up, trigger a surprise signal that leads to greater asymmetry due to reactive corrections to prevent one's fall - not because participants are generalizing from previously learned representations (e.g., (Iturralde & Torres-Oviedo, 2019)).

Further contextualization:

Recognizing the differences in dependent variables (reaching position vs. leg speed/symmetry in walking), could the Proprioceptive/Perceptual Re-alignment model also apply to gait adaptation (Tsay et al., 2022; Zhang et al., 2024)? Recent reaching studies show a similar link between perception and action during motor adaptation (Tsay et al., 2021) and have proposed a model aligning with the authors' correlations between perception and action. The core signal driving implicit adaptation is the discrepancy between perceived and desired limb position, integrating forward model predictions with proprioceptive/visual feedback.

References

Iturralde, P. A., & Torres-Oviedo, G. (2019). Corrective Muscle Activity Reveals Subject-Specific Sensorimotor Recalibration. eNeuro, 6(2). https://doi.org/10.1523/ENEURO.0358-18.2019

Tsay, Jonathan S., Hyosub E. Kim, Samuel D. McDougle, Jordan A. Taylor, Adrian Haith, Guy Avraham, John W. Krakauer, Anne G. E. Collins, and Richard B. Ivry. 2024. "Fundamental Processes in Sensorimotor Learning: Reasoning, Refinement, and Retrieval." ELife 13 (August). https://doi.org/10.7554/eLife.91839.

Tsay, Jonathan S., Hyosub E. Kim, Darius E. Parvin, Alissa R. Stover, and Richard B. Ivry. 2021. "Individual Differences in Proprioception Predict the Extent of Implicit Sensorimotor Adaptation." Journal of Neurophysiology, March. https://doi.org/10.1152/jn.00585.2020.

Tsay, Jonathan S., Hyosub Kim, Adrian M. Haith, and Richard B. Ivry. 2022. "Understanding Implicit Sensorimotor Adaptation as a Process of Proprioceptive Re-Alignment." ELife 11 (August). https://doi.org/10.7554/eLife.76639.

Zhang, Zhaoran, Huijun Wang, Tianyang Zhang, Zixuan Nie, and Kunlin Wei. 2024. "Perceptual Error Based on Bayesian Cue Combination Drives Implicit Motor Adaptation." ELife. https://doi.org/10.7554/elife.94608.1.

Reviewer #1 (Public Review):

In this work, the authors aimed to understand how titins derived from different nuclei within the syncytium are organized and integrated after cell fusion during skeletal muscle development and remodeling. The authors developed mCherry titin knock-in mice with the fluorophore mCherry inserted into titin's Z-disk region to track the titin during cell fusion. The results suggested that titin exhibited homogenous distribution after cell fusion. The authors also probed on how titin behaves during muscle injury by implantation of titin-eGFP myoblasts into adult mCherry-titin mice. Interestingly, titin is retained at the proximal nucleus and does not diffuse across the whole syncytium in this system. The findings of the study are novel and interesting. The experimental approaches are appropriate. The results are described well. However, the manuscript needs revisions to enhance its clarity.

(1) In this work, the authors have not described the statistical analysis appropriately. In most of the figures, significance levels are not described. The information on the biological and technical replicates is missing in almost all the figures. This information is critical for understanding the strength of the experimentation.<br /> (2) The in vivo experiments are underpowered. The authors have used only 3 animals in the cardiotoxin injury experiment and eliminated another 3 animals from the analysis. How did they determine insufficient myoblast integration?<br /> (3) Similarly, the in vitro imaging experiments, especially the in vitro titin mobility assays used only 3 cells (Fig 2b) or 6-9 cells (Fig 2c-2e). The number of cells imaged is insufficient to derive a valid conclusion. What is the variability in the results between cells? Whether all the cells behave similarly in titin mobility assays?<br /> (4) Figure 1c-e, Figure 2a, Figure 3, Figure 4, Figure 5, Figure 6- please describe the replicates and also if possible, quantify the data and present them as separate figures.<br /> (5) Figure 2- the authors excluded samples with an obvious decrease in cell quality during imaging from the analysis. How do the authors assess the cell quality? Simply by visual examination? Or were the samples that did not show fluorescence recovery eliminated? I am wondering what percentage of cells showed poor cell quality. How do they avoid the bias? I recommend that the authors include these cells also for the analysis of data presented in Figures 2b, 2c, and 2f.<br /> (6) It is unclear how the authors identified the different stages of cell fusion in the microscopy images i.e. early fusion, distribution, and complete distribution.

Reviewer #1 (Public Review):

Lactobacillus plantarum is a beneficial bacterium renowned for its positive physiological effects and probiotic functions. Fu et al. conducted an investigation into the involvement of this bacterium in host purine metabolism. Initially, they employed microbiomics to analyze changes in L. plantarum within a hyperuricemia model, followed by isolation of the bacterium from this model. The gene map associated with purine nucleoside metabolism was determined through whole-genome analysis. Metabolic shifts in L. plantarum under nucleoside-enriched conditions were assessed using HPLC and metabolomics, while underlying mechanisms were explored through gene knockout experiments. Finally, the efficacy of L. plantarum was validated in hyperuricemia models involving goslings and mice. The authors presented their findings coherently and logically, addressing key questions using appropriate methodologies and yielding significant and innovative results. The authors demonstrated that host-derived Lactobacillus plantarum alleviates host hyperuricemia by influencing purine metabolism. However, their study primarily focused on this bacterium without delving deeper into the mechanisms underlying hyperuricemia beyond verification through two models. Nevertheless, these findings are sufficient to support their conclusion effectively. Additionally, further research is warranted to investigate the metabolites of Lactobacillus plantarum.

Reviewer #1 (Public review):

Theoretical principles of viscous fluid mechanics are used here to assess likely mechanisms of transport in the ER. A set of candidate mechanisms are evaluated, making good use of imaging to represent ER network geometries. Evidence is provided that contraction of peripheral sheets provides a much more credible mechanism than contraction of individual tubules, junctions or perinuclear sheets.

The work has been conducted carefully and comprehensively, making good use of underlying physical principles. There is good discussion of the role of slip; sensible approximations (low volume fraction, small particle size, slender geometries, pragmatic treatment of boundary conditions) allow tractable and transparent calculations; clear physical arguments, including an analysis of energy budgets, provide useful bounds; stochastic and deterministic features of the problem are well integrated.

Reviewer #1 (Public review):

Summary:

This paper by Watanabe et al described an expression system that can express the paired heavy and light chains of IgG antibodies from single cell B cells. In addition, they used FACS sorting for specific antigen to screen/select the specific populations for more targeted cloning of mAb genes. By staining with multiple antigens, they were able to zoom in to cross-reactive antibodies.

Strengths:

A highly efficient process which combines selection/screening with dua expression of both antibody chains. It is particularly suitable for isolation of cross-reactive antibodies against conserved epitopes of different antigens, such as surface proteins of related viruses.

Weaknesses:

(1) The overall writing is very difficult to follow and the authors need to work on significant re-writing<br /> (2) The paper in its current form really lacks detail and it is not possible for readers to repeat or follow their methods. For example: a) It is not clear whether the authors checked the serum to see if the mice were producing antibodies before they sacrificed them to harvest spleen/blood i.e. using ELISA? b) How long after administration of the second dose were the mice sacrificed? c) What cell types are taken for single B cell sorting? Splenocytes or PBMC? These are just some of the questions which need to be addressed.<br /> (3) According to the authors, 77 clones were sorted from the PR8+ and H2+ double positive quadrant. It is surprising that after transfection and re-analysing of bulk antibody presenting EXPI cells on FACS from, only 13 clones (or 8 clones? - unclear) seemed to be truly cross reactive. If that is the case, the approach is not as efficient as the authors claimed.

The authors have adequately addressed the issues raised

Reviewer #1 (Public review):

Summary:

This work sought to demonstrate that gut microbiota dysbiosis may promote the colonization of mycobacteria, and they tried to prove that Nos2 down-regulation was a key mediator of such gut-lung pathogenesis transition.

Strengths:

They did large-scale analysis of RNAs in lungs to analyze the gene expression of mice upon gut dysbiosis in MS-infected mice. This might help provide overview of gene pathways and critical genes for lung pathology in gut dysbiosis. This data is somewhat useful and important for the TB field.

Weaknesses:

(1) They did not use wide-type Mtb strain (e.g. H37Rv) to develop mouse TB infection models, and this may lead to the failure for establishment of TB granuloma and other TB pathology icons.<br /> (2) The usage of in vitro assays based on A542 to examine the regulation function of Nos2 expression on NO and ROS may not be enough. A542 is not the primary Mtb infection target in the lungs.<br /> (3) They did not examine the lung pathology upon gut dysbiosis to examine the true significance of increased colonization of Mtb.<br /> (4) Most of the studies are based on MS-infected mouse models with lack of clinical significance.

Reviewer #2 (Public Review):

Olszyński et al. claim that they identified a "new-type" ultrasonic vocalization around 44 kHz that occurs in response to prolonged fear conditioning (using foot-shocks of relatively high intensity, i.e. 1 mA) in rats. Typically, negative 22-kHz calls and positive 50-kHz calls are distinguished in rats, commonly by using a frequency threshold of 30 or 32 kHz. Olszyński et al. now observed so-called "44-kHz" calls in a substantial number of subjects exposed to 10 tone-shock pairings, yet call emission rate was low (according to Fig. 1G around 15%, according to the result text around 7.5%). They also performed playback experiments and concluded that "the responses to 44-kHz aversive calls presented from the speaker were either similar to 22-kHz vocalizations or in-between responses to 22-kHz and 50-kHz playbacks".

Strengths: Detailed spectrographic analysis of a substantial data set of ultrasonic vocalizations recorded during prolonged fear conditioning, combined with playback experiments.

Reviewer #1 (Public review):

Devakinandan et al. present a revised version of their manuscript. Their scRNA-seq data is a valuable resource to the community, and they further validate their findings via in situ hybridizations and electron microscopy. Overall, they have addressed my major concerns. I only have two minor comments.

(1) The authors note in Figure 4I, and K that because the number of C2 V2Rs or H2-Mv receptors increased while the normalized expression of Gnao1 remained constant (and likewise for V1Rs and Gnai2 in Figure 4-S4C) that their results are unlikely to be capturing doublets. I'm not sure that this is the case. If the authors added together two V2R cells the total count of every gene might double, but the normalized expression of Gnao1 would remain the same. To address this concern, the authors should also show the raw counts for Gnao1 as well as the total number of UMIs for these cells.

(2) As requested, the authors have now added a colorbar to the pseudocolored images in Figures 7. However, this colorbar still doesn't have any units. Can the authors add some units, or clarify in the methods how the raw data relates to the colors (e.g. is it mapped linearly, at a logscale, with gamma or other adjustments, etc.)? Moreover, it's also unclear what the dots in the backgrounds of plots like Figure 7E mean. Are they pixels? Showing the individual lines, the average for each animal, or omitting them entirely, might make more sense.

Reviewer #1 (Public review):

Summary:

In this study, Liu, Jiang, Diao et.al. investigated the role of GSDMD in psoriasis-like skin inflammation in mice. The authors have used full-body GSDMD knock-out mice and Gsdm floxed mice crossed with the S100A8- Cre. In both mice, the deficiency of GSDMD ameliorated the skin phenotype induced by the imiquimod. The authors also analyzed RNA sequencing data from the psoriatic patients to show an elevated expression of GSDMD in the psoriatic skin.

Overall, this is a potentially interesting study, however, the manuscript in its current format is not completely a novel study.

Strengths:

It has the potential to unravel the new role of neutrophils.

Weaknesses:

The main claims are only partially supported and have scope to improve

Disease: Von-willebrand Disorder, type 1

Patient 1 Variant: VWF NM_000552.5: c.4135C>T p.(Arg1379Cys) Exon 28

Family: History not mentioned

Patient 1 phenotype: near normal VWF:Ag and WVF:RCo levels RIPA within normal range Platelet VWF levels were normal normal pattern for multimeric analysis of plasma Slightly reduced VWF levels

In silico predictions available:

I-Mutant 3.0 value = -1.36 PYMOL prediction = substitution of ARG 1379 with a cysteine results in the loss of hydrogen bonds with Lys1407 and Lys1408, predicted change in secondary structure of A1 domain

Joint Public Reviews:

De Waele et al. framed the mass-spectrum-based prediction of antimicrobial resistance (AMR) prediction as a drug recommendation task. Neural networks were trained on the recently available DRIAMS database of MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry data and their associated antibiotic susceptibility profiles (Weis et al. 2022). Weis et al. (2022) also introduced the benchmark models which take as the input a single species and are trained to predict resistance to a single drug. Instead here, a pair of drugs and spectrum are fed to two neural network models to predict a resistance probability. In this manner, knowledge from different drugs and species can be shared through the model parameters. Questions asked: What is the best way to encode the drugs? Does the dual neural network outperform the single spectrum-drug network?

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.

Strengths:

• A single antimicrobial resistance 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 /> • The 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:

• Authors do not offer information about the model features associated with resistance. While reviewers understand that it is difficult to map 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 (Hrabak et al. 2013, Torres-Sangiao et al. 2022).

References:

Hrabak et al. (2013). Clin Microbiol Rev 26. doi: 10.1128/CMR.00058-12.<br /> Torres-Sangiao et al. (2022). Front Med 9. doi: 10.3389/fmed.2022.850374.<br /> Weis et al. (2022). Nat Med 28. doi: 10.1038/s41591-021-01619-9.

Reviewer #2 (Public review):

Most neurodegenerative diseases are characterized by the self-templated misfolding of a particular protein in a manner that enables progressive spread throughout the central nervous system. In diseases including Parkinson's disease (PD) and multiple system atrophy (MSA), the protein a-synuclein misfolds into unique strains, which use this self-replicating mechanism to encode disease-specific information. Previous research suggests that a major contributor to the lack of successful clinical trials across neurodegenerative diseases is the lack of disease-relevant strains used in preclinical testing. While MSA patient samples are known to replicate efficiently in cell and mouse models of disease, Lewy body disease (LBD) patient samples do not. To overcome this obstacle, the seeding amplification assay (SAA) uses recombinant a-synuclein to amplify the misfolded protein structure present in a human patient sample. The resulting fibrils are then widely used by many laboratories as a model of PD. In this manuscript, Lee et al., set out to compare the strain properties of a-synuclein fibrils isolated from LBD and MSA patient samples with the resulting amplified fibrils following SAA. Using orthogonal biochemical and structural approaches to strengthen their analyses, the authors report that the SAA-amplified fibrils do not recapitulate the disease-relevant strains present in the patient samples. Moreover, their data suggest that regardless of which strain is used to seed the SAA reaction, the same strain is generated. These results clearly demonstrate that the SAA-amplified material is likely not disease-relevant. SAA fibrils are broadly used throughout academic and pharmaceutical laboratories. They are used in ongoing drug discovery efforts and recombinant fibrils broadly inform much of what is known about a-synuclein strain biology in LBD patients. The implications of the reported work are, therefore, expansive. These findings add to the growing ledger of reasons that the use of SAA fibrils in research should be halted until improved methods for amplification with high fidelity are developed.

Reviewer #1 (Public review):

Summary:

Assessment of cardiac LEC transcriptomes post-MI may yield new targets to improve lymphatic function. scRNAseq is a valid approach as cardiac LECs are rare compared to blood vessel endothelial cells.

Strengths:

Extensive bioinformatics approaches employed by the group

Weaknesses:

Too few cells included in scRNAseq data set and the spatial transcriptomics data that was exploited has little relevance, or rather specificity, for cardiac lymphatics. This study seems more a collection of preliminary transcriptomic data than a true scientific report to help advance the field.

Reviewer #1 (Public Review):

Tsai and Seymen et al. investigate associations between RTE expression and methylation and age and inflammation, using multiple public datasets. The text of the manuscript has been polished and the phrasing of several findings has been made clearer and more precise. The authors also provided ample discussion to the prior reviewer comments in their rebuttal, including new analyses.

Reviewer #2 (Public Review):

Summary:

The authors aimed to identify which regions of DVL2 contribute to its endogenous/basal clustering, as well as the relevance of such domains to condensate/phase separation and WNT activation.

Strengths:

A strength of the study is the focus on endogenous DVL2 to set up the research questions, as well as the incorporation of various techniques to tackle it. I found also quite interesting that DVL2-CFR addition to DVL1 increased its MW in density gradients.

Weaknesses:

The authors have addressed important drawbacks regarding the overexpression experiments, most notably by including DVL tKO cells in collaboration with Vita. I think that this part has clearly improved.

Unfortunately, I still stand with my key concern: at this stage in the field, with many papers on DVL over expression, there is a clear need to address how endogenous DVL behaves. While the attempts to o/e low levels of DVL mutants in tKO cells are useful for validation experiments, the manuscript does not -in my opinion - address the requirements of DVL2 condensation for WNT signalling. Of note, several of the described effects are partial, including in tKO cells, often indicating that the targeted domains contribute, but are not required, for these processes. I understand that generating endogenous tagged lines or targeting specific endogenous domains is not trivial. But, as indicated in both initial reviews, I think that monitoring endogenous proteins is required to fully address the proposed research question.

In my opinion, the current manuscript A) shows that endogenous DVL2 forms large complexes in a higher proportion as DVL1/3, B) identifies and describes a couple of motifs that contribute to clustering and signalling in overexpressed DVL, including in tKO cells* C) shows that one of those motifs (CFR) rewires o/e DVL1 into behaving similarly as DVL2.

*On a minor note, I am not sure how DVL tKO cells partially react to Wnt3a in Figure 7G

Reviewer #1 (Public review):

Summary:

In the manuscript the authors describe a new pipeline to measure changes in vasculature diameter upon opt-genetic stimulation of neurons.<br /> The work is useful to better understand the hemodynamic response on a network /graph level.

Strengths:

The manuscript provides a pipeline that allows to detect changes in the vessel diameter as well as simultaneously allows to locate the neurons driven by stimulation.<br /> The resulting data could provide interesting insights into the graph level mechanisms of regulating activity dependent blood flow.

Weaknesses:

(1) The manuscript contains (new) wrong statements and (still) wrong mathematical formulas.<br /> (2) The manuscript does not compare results to existing pipelines for vasculature segmentation (opensource or commercial).<br /> Comparing performance of the pipeline to a random forest classifier (illastik) on images that are not preprocessed (i.e. corrected for background etc.) seems not a particularly useful comparison.<br /> (3) The manuscript does not clearly visualize performance of the segmentation pipeline (e.g. via 2d sections, highlighting also errors etc.). Thus, it is unclear how good the pipeline is, under what conditions it fails or what kind of errors to expect.<br /> (4) The pipline is not fully open-source due to use of matlab. Also, the pipeline code was not made available during review contrary to the authors claims (the provided link did not lead to a repository). Thus, the utility of the pipeline was difficult to judge.

Detailed remarks to the revision and new manuscript:

- Generalizability: The authors addressed the point of generalizability by applying the pipeline to other data sets. This demonstrates that their pipeline can be applied to other data sets and makes it more useful.<br /> However, from the visualizations it's unclear to see the performance of the pipeline, where the pipelines fails etc. The 3d visualizations are not particularly helpful in this respect .<br /> In addition, the dice measure seems quite low, indicating roughly 20-40% of voxels do not overlap between inferred and ground truth. I did not notice this high discrepancy earlier. A through discussion of the errors appearing in the segmentation pipeline would be necessary in my view to better asses the quality of the pipeline.

Reviewer #1 (Public review):

Summary:

This is a detailed description of the role of PKCδ in Drosophila learning and memory. The work is based on a previous study (Placais et al. 2017) that has already shown that for the establishment of long-term memory, the repetitive activity of MP1 dopaminergic neurons via the dopamine receptor DAMB is essential to increase mitochondrial energy flux in the mushroom body. In this paper, the role of PKCδ is now introduced. PKCδ is a molecular link between the dopaminergic system and the mitochondrial pyruvate metabolism of mushroom body Kenyon cells. For this purpose, the authors establish a genetically encoded FRET-based fluorescent reporter of PKCδ-specific activity, δCKAR.

Strengths:

This is a thorough study on the long-term memory of Drosophila. The work is based on the extensive, high-quality experience of the senior authors. This is particularly evident in the convincing use of behavioral assays and imaging techniques to differentiate and explore various memory phases in Drosophila. The study also establishes a new reporter to measure the activity of PKCδ - the focus of this study - in behaving animals. The authors also elucidate how recurrent spaced training sessions initiate a molecular gating mechanism, linking a dopaminergic punishment signal with the regulation of mitochondrial pyruvate metabolism. This advancement will enable a more precise molecular distinction of various memory phases and a deeper comprehension of their formation in the future.

Weaknesses:

The study offers novel insights into the molecular mechanisms underlying long-term memory formation and presents no apparent weaknesses in either content or methodology.

Reviewer #1 (Public review):

Summary:

The authors use the teleost medaka as an animal model to study the effect of seasonal changes in day-length on feeding behaviour and oocyte production. They report a careful analysis of how day-length affects female medakas and a thorough molecular genetic analysis of genes potentially involved in this process. They show a detailed analysis of two genes and include a mutant analysis of one gene to support their conclusions

Strengths:

The authors pick their animal model well and exploit the possibilities to examine in this laboratory model the effect of a key environmental influence, namely the seasonal changes of day-length. The phenotypic changes are carefully analysed and well-controlled. The mutational analysis of the agrp1 by a ko-mutant provides important evidence to support the conclusions. Thus this report exceeds previous findings on the function of agrp1 and npyb as regulators of food-intake and shows how in medaka these genes are involved in regulating the organismal response to an environmental change. It thus furthers our understanding of how animals react to key exogenous stimuli for adaptation.

Weaknesses:

The authors are too modest when it comes to underscoring the importance of their findings. Previous animal models used to study the effect of these neuropeptides on feeding behaviour have either lost or were most likely never sensitive to seasonal changes of day length. Considering the key importance of this parameter on many aspects of plant and animal life it could be better emphasised that a suitable animal model is at hand that permits this.<br /> The molecular characterization of the agrp1 ko-mutant that the authors have generated lacks some details that would help to appreciate the validity of the mutant phenotype. Additional data would help in this respect.

Reviewer #1 (Public review):

Summary:

In this manuscript, Shibata describes a method to assess rapidly fluctuating CpG sites (fCpGs) from single-cell methylation sequencing (sc-MeSeq) data. Assuming that fCpGs are largely consistent over time with changes induced by inheritable events during replication, the author infers lineage relationships in available brain-derived sc-MeSeq. Supplementing current lineage tracing through genomic and mitochondrial mosaic variants is an interesting concept that could supplement current work or allow additional lineage analysis in existing data.

However, the author failed to convincingly show the power of fCpG analysis to determine lineages in the human brain. While the correlation with cellular division and distinction of cell types appears plausible and strong, the application to detect specific lineages is less convincing. Aspects of this might be due to a lack of clarity in presentation and erroneous use of developmental concepts. However, without addressing these problems it is challenging for a reader to come to the same conclusions as the author.

On the flip side, this novel application of fCpGs will allow the re-use of existing sc-MeSeq to infer additional features that were previously unavailable, once the biological relevance has been further elucidated.

Strengths:

(1) Novel re-analysis application of methylation data to infer the status of fCpGs and the use as a lineage marker.

(2) Application of this method to an innovative existing data set to benchmark this framework against existing developmental knowledge.

Weaknesses:

(1) Insufficient clarity when presenting results (this includes an incredible shortness of the methods section making an informed assessment very difficult). This makes it hard to fully grasp and evaluate the presented results.

(2) Inconsistent or erroneous use of neurodevelopmental concepts which hinders appropriate interpretation of the results.

(3) Lack of consideration for alternative explanations for the observed data (i.e., considering fCpGs as a cellular division clock that diverges over 'time').

Reviewer #1 (Public review):

Summary:

This theoretical paper addresses how to optimize reward-rate-maximizing decisions in certain foraging-style environments. It presents a series of equations and graphical illustrations for quantities such as reward rates and time-related costs that a decision maker could estimate as a basis for such decisions. One of the main takeaways is that if the hypothetical agent underweights the time spent outside a focal reward pursuit relative to the time spent within it, this can predict a broadly realistic pattern of impatience in two alternative intertemporal choices paired with well-calibrated take-it-or-leave-it decisions. Another takeaway is that if the optimally estimated subjective value of a reward pursuit is plotted as a function of a range of temporal durations, the result resembles a hyperbolic discounting function and is affected in empirically realistic ways by the magnitude and sign of the reward. Thus, the rate-maximization framework might lead to a hypothesis about the basis for the magnitude and sign effects in discounting.

Strengths:

The paper makes a useful contribution by broadening the application of reward-rate maximization to time-related decision scenarios. The paper's breadth of scope includes applying the same framework to accept/reject decisions and multi-alternative discounting decisions. The figures take a creative approach to illustrating the internal quantities in the model. It's particularly useful that the paper gives consideration to internal distortions that could give rise to documented anomalies in decision behavior.

Weaknesses:

(1) Although there are many citations acknowledging relevant previous work, there often isn't a very granular attribution of individual previous findings to their sources. In the results section, it's sometimes ambiguous when the paper is recapping established background and when it is breaking new ground. For example, around equation 8 in the results (sv = r - rho*t), it would be good to refer to previous places where versions of this equation have been presented. Offhand, McNamara 1982 (Theoretical Population Biology) is one early instance and Fawcett et al. 2012 (Behavioural Processes) is a later one. Line 922 of the discussion seems to imply this formulation is novel here.

(2) The choice environments that are considered in detail in the paper are very simple. The simplicity facilitates concrete examples and visualizations, but it would be worth further consideration of whether and how the conclusions generalize to more complex environments. The paper considers "forgo" scenario in which the agent can choose between sequences of pursuits like A-B-A-B (engaging with option B at all opportunities, which are interleaved with a default pursuit A) and A-A-A-A (forgoing option B). It considers "choice" scenarios where the agent can choose between sequences like A-B-A-B and A-C-A-C (where B and C are larger-later and smaller-sooner rewards, either of which can be interleaved with the default pursuit). Several forms of additional complexity would be valuable to consider. One would be a greater number of unique pursuits, not repeated identically in a predictable sequence, akin to a prey-selection paradigm. It seems to me this would cause t_out and r_out (the time and reward outside of the focal prospect) to be policy-dependent, making the 'apportionment cost' more challenging to ascertain. Another relevant form of complexity would be if there were variance or uncertainty in reward magnitudes or temporal durations or if the agent had the ability to discontinue a pursuit such as in patch-departure scenarios.

(3) I had a hard time arriving at a solid conceptual understanding of the 'apportionment cost' around Figure 5. I understand the arithmetic, but it would help if it were possible to formulate a more succinct verbal description of what makes the apportionment cost a useful and meaningful quality to focus on. I think Figure 6C relates to this, but I had difficulty relating the axis labels to the points, lines, and patterned regions in the plot. I also was a bit confused by how the mathematical formulation was presented. As I understood it, the apportionment cost essentially involves scaling the rest of the SV expression by t_out/(t_in + t_out). The way this scaling factor is written in Figure 5C, as 1/(1 + (1/t_out)t_in), seems less clear than it could be. Also, the apportionment cost is described in the text as being subtracted from SV rather than as a multiplicative scaling factor. It could be written as a subtraction, by subtracting a second copy of the rest of the SV expression scaled by t_in/(t_in + t_out). But that shows the apportionment cost to depend on the opportunity cost, which is odd because the original motivation on line 404 was to resolve the lack of independence between terms in the SV expression.

(4) In the analysis of discounting functions (line 664 and beyond), the paper doesn't say much about the fact that many discounting studies take specific measures to distinguish true time preferences from opportunity costs and reward-rate maximization. In many of the human studies, delay time doesn't preclude other activities. In animal studies, rate maximization can serve as a baseline against which to measure additional effects of temporal discounting. This is an important caveat to claims about discounting anomalies being rational under rate maximization (e.g., line 1024).

(5) The paper doesn't feature any very concrete engagement with empirical data sets. This is ok for a theoretical paper, but some of the characterizations of empirical results that the model aims to match seem oversimplified. An example is the contention that real decision-makers are optimal in accept/reject decisions (line 816 and elsewhere). This isn't always true; sometimes there is evidence of overharvesting, for example.

(6) Related to the point above, it would be helpful to discuss more concretely how some of this paper's theoretical proposals could be empirically evaluated in the future. Regarding the magnitude and sign effects of discounting, there is not a very thorough overview of the several other explanations that have been proposed in the literature. It would be helpful to engage more deeply with previous proposals and consider how the present hypothesis might make unique predictions and could be evaluated against them. A similar point applies to the 'malapportionment hypothesis' although in this case there is a very helpful section on comparisons to prior models (line 1163). The idea being proposed here seems to have a lot in common conceptually with Blanchard et al. 2013, so it would be worth saying more about how data could be used to test or reconcile these proposals.

Reviewer #1 (Public review):

Summary:

This manuscript by Shan, Guo, Zhang, Chen et al., shows a raft of interesting data including the first cryo-EM structures of human PIEZO1. Clearly, the molecular basis of PIEZO channel inactivation is of great interest and as such this manuscript provides some valuable extra information that may help to ultimately build a molecular picture of PIEZO channel inactivation. However, the current manuscript though does not provide any compelling evidence for a detailed mechanism of PIEZO inactivation.

Strengths:

This manuscript documents the first cryo-EM structures of human PIEZO1 and the gain of function mutants associated with hereditary anaemia. It is also the first evidence showing that PIEZO1 gain of function mutants are also regulated by the auxiliary subunit MDFIC.

Weaknesses:

While the structures are interesting and clear differences can be seen in the presence of the auxiliary subunit MDFIC the major conclusions and central tenets of the paper, especially a role for pore lipids in inactivation, lack data to support them. The post-translational modification of PIEZOs auxiliary subunit MDFIC is not modelled as a covalent interaction.

Reviewer #1 (Public review):

Summary:

The concept that trained immunity, as defined, can be beneficial to subsequent immune challenges is important in the broad context of health and disease. The significance of this manuscript is the finding that trained immunity is actually a two-edged sword, herein, detrimental in the context of LPS-induced Acute Lung Injury that is mediated by AMs.

Strengths:

Several lines of evidence in different mouse models support this conclusion. The postulation that differences in immune responses in individuals are linked to differences in the mycobiome and consequent B-glucan makeup is provocative.

Weaknesses:

The findings that the authors state are relevant to sepsis, are actually confined to a specific lung injury model and not classically-defined sepsis. In addition, the ontogeny of the reprogrammed AMs is uncertain. Links in the proposed signaling pathways need to be strengthened.

Reviewer #1 (Public Review):

Summary:

This study investigates the hypoxia rescue mechanisms of neurons by non-neuronal cells in the brain from the perspective of exosomal communication between brain cells. Through multi-omics combined analysis, the authors revealed this phenomenon and logically validated this intercellular rescue mechanism under hypoxic conditions through experiments. The study proposed a novel finding that hemoglobin maintains mitochondrial function, expanding the conventional understanding of hemoglobin. This research is highly innovative, providing new insights for the treatment of hypoxic encephalopathy.

Overall, the manuscript is well organized and written, however, there are some minor/major points that need to be revised before this manuscript is accepted.

Major points:

(1) Hypoxia can induce endothelial cells to release exosomes carrying hemoglobin, however, how neurons are able to actively take up these exosomes? It is possible for other cells to take up these exosomes also? This point needs to be clarified in this study.

(2) The expression of hemoglobin in neurons is important for mitochondrial homeostasis, but its relationship with mitochondrial homeostasis needs to be further elucidated in the study.

Reviewer #1 (Public review):

The authors examine how probabilistic reversal learning is affected by dopamine by studying the effects of methamphetamine (MA) administration. Based on prior evidence that the effects of pharmacological manipulation depend on baseline neurotransmitter levels, they hypothesized that MA would improve learning in people with low baseline performance. They found this effect, and specifically found that MA administration improved learning in noisy blocks, by reducing learning from misleading performance, in participants with lower baseline performance. The authors then fit participants' behavior to a computational learning model and found that an eta parameter, responsible for scaling learning rate based on previously surprising outcomes, differed in participants with low baseline performance on and off MA.

Questions:

(1) It would be helpful to confirm that the observed effect of MA on the eta parameter is responsible for better performance in low baseline performers. If performance on the task is simulated for parameters estimated for high and low baseline performers on and off MA, does the simulated behavior capture the main behavioral differences shown in Figure 3?

(2) In Figure 4C, it appears that the main parameter difference between low and high baseline performance is inverse temperature, not eta. If MA is effective in people with lower baseline DA, why is the effect of MA on eta and not IT?

Also, this parameter is noted as temperature but appears to be inverse temperature as higher values are related to better performance. The exact model for the choice function is not described in the methods.

Reviewer #1 (Public review):

Summary:

The current work explored the link between the pulvinar intrinsic organisation and its functional and structural connectivity patterns of the cortex using different dimensional reduction techniques. Overall they find relationships between pulvinar-cortical organization and cortico-cortical organization, and little evidence for clustered organization. Moreover, they investigate PET maps to understand how neurotransmitter/receptor distributions vary within the pulvinar and along its structural and functional connectivity axes.

Strengths:

There is a replication dataset and different modalities are compared against each other to understand the structural and functional organisation of the pulvinar complex.

Weaknesses:

(1) What is the motivation of the study and how does this work extend previous assessments of the organization of the complete thalamus within the gradient framework?

(2) Why is the current atlas chosen for the delineation of the pulvinar and individualised maps not considered? Given the size of the pulvinar, more validation of the correctness of the atlas may be helpful.

(3) Overall the study feels a little incremental and a repetition of what others have done already in the thalamus. It would be good to know how focussing only on the pulvinar changes interpretation, for example by comparing thalamic and pulvinar gradients?

(4) Could it be that the gradient patterns stem from lacking anatomical and functional resolutions (or low SNR) therefore generating no sharp boundaries?

Reviewer #1 (Public review):

Summary:

The overall analysis and discovery of the common motif is important and exciting. Very few human/primate ribozymes have been published and this manuscript presents a detailed analysis of two of them. The minimized domains appear to be some of the smallest known self-cleaving ribozymes.

Strengths:

The manuscript is rooted in deep mutational analysis of the human OR4K15 and LINE1 ribozymes and subsequently in modeling of their active site based on the closely-related core of the TS ribozyme. The experiments support the HTS findings and provide convincing evidence that the ribozymes are structurally related to the core of the TS ribozyme, which has not been found in primates prior to this work.

Reviewer #1 (Public review):

Summary:

The aim of the present work is to evaluate the role of BMP9 and BMP10 in liver by depleting Bmp9 and Bmp10 from the main liver cell types (endothelial cells (EC), hepatic stellate cells (HSC), Kupffer cells (KC) and hepatocytes (H)) using cell-specific cre recombinases. They show that HSCs are the main source of BMP9 and BMP10 in the liver. Using transgenic ALK1 reporter mice, they show that ALK1, the high affinity type 1 receptor for BMP9 and BMP10, is expressed on KC and EC. They have also performed bulk RNAseq analyses on whole liver, and cell-sorted EC and KC, and showed that loss of Bmp9 and Bmp10 decreased KC signature and that KC are replaced by monocyte-derived macrophages. EC derived from these Bmp9fl/flBmp10fl/flLratCre mice also lost their identity and transdifferentiated into continuous ECs. Liver iron metabolism and metabolic zonation were also affected in these mice. In conclusion, this work supports that BMP9 and BMP10 produced by HSC play a central role in mediating liver cell-cell crosstalk and liver homeostasis.

Strengths:

This work further supports the role of BMP9 and BMP10 in liver homeostasis. Using a specific HSC-Cre recombinase, the authors show for the first time that it is the BMP9 and BMP10 produced by HSC that play a central role in mediating liver cell-cell crosstalk to maintain a healthy liver. Although the overall message of the key role of BMP9 in liver homeostasis has been described by several groups, the role of hepatic BMP10 has not been studied before. Thus, one of the novelties of this work is to have used liver cell specific Cre recombinase to delete hepatic Bmp9 and Bmp10. The second novelty is the demonstration of the role of BMP9 and BMP10 in KC Differentiation/homeostasis which has already been slightly addressed by this group by knocking out ALK1, the high affinity receptor of BMP9 and BMP10 (Zhao et al. JCI, 2022).

Weaknesses:

This work remains rather descriptive and the molecular mechanisms are barely touched upon and could have been more explored.

Reviewer #1 (Public review):

The study by Korona and colleagues presents a rigorous experimental strategy for generating and maintaining a nearly complete set of monosomic yeast lines, thereby establishing a new standard for studying monosomes. Their careful approach in generating and handling monosome yeast lines, coupled with their use of high-throughput DNA sequencing and RNA sequencing, addresses concerns related to genomic instability and is commendable. However, I would like to express my concerns regarding the second part of the study, particularly the calculation of epistasis and the conclusion that vast positive epistatic effects have been observed. I believe that the conclusion of positive epistasis for fitness might be premature due to potential errors in estimating the expected fitness.

The method used to calculate fitness expectation (1 + sum(di), where di = rDRi - 1) may be inappropriate. The logarithm transformation mentioned by the authors is designed to transform the exponential growth curve into a linear relation for estimating doubling rate, and thus the fitness expectation should be calculated as the product of rDRi values. As an illustration, if gene A exhibits a 20% reduction in fitness when halved (A/-) and gene B exhibits a 30% reduction (B/-), the expected fitness of A/- B/- should be 56%, rather than the 50% estimated in the study. In other words, the formula used by the authors could underestimate the fitness expectation.

This issue is evident in Figure 2b, where negative values were obtained due to the use of an incorrect formula for estimating fitness expectations. It is worth noting that Figure 2a shows rDR values around one, indicating that no further logarithmic transformation was applied.

While widespread positive epistasis in yeast has been reported by other studies (e.g., doi: 10.1038/ng.524, but not to the extent reported in this study), the conclusion of the current study might not be sufficiently supported. I recommend that the authors revisit their calculation methods to provide a more convincing conclusion on the presence of positive epistasis for fitness in their dataset. Overall, I appreciate the authors' efforts in this study but believe that addressing these concerns is essential for strengthening the validity of their findings.

Comments on revised version:

The authors have adequately addressed all my previous concerns during revision.

Reviewer #2 (Public review):

Summary:

The authors set out to study whether the cooling agent binding site in TRPM8, which is located between the S1-S4 and the TRP domain, is conserved within the TRPM family of ion channels. They specifically chose the TRPM4 channel as the model system, which is directly activated by intracellular Ca2+. Using electrophysiology, the authors characterized and compared the Ca2+ sensitivity and the voltage-dependence of TRPM4 channels in the absence and presence of synthetic cooling agonist icilin. They also analyzed the mutational effects of residues (A867G and R901H; equivalent mutations in TRPM8 were shown involved in icilin sensitivity) on Ca2+ sensitivity and voltage-dependence of TRPM4 in the absence and presence of Ca2+. Based on the results as well as structure/sequence alignment, the authors concluded that icilin likely binds to the same pocket in TRPM4 and suggested that this cooling agonist binding pocket is conserved in TRPM channels.

Strengths:

The authors gave a very thorough introduction of the TRPM channels. They have nicely characterized the Ca2+ sensitivity and the voltage-dependence of TRPM4 channels and demonstrated icilin potentiates the Ca2+ sensitivity and diminishes the outward rectification of TRPM4. These results indicate icilin modulates TRPM4 activation by Ca2+.

The authors have incorporated additional data analysis and control experiments in the revised manuscript to strengthen their findings. They have well addressed the concerns raised by reviewers in the responses.

Weaknesses:

The study is conducted based on an assumption that TRPM4 activation is controlled by Ca2+ binding to a single site in the S1-S4 pocket in each subunit, and the second Ca2+ site in the cytoplasmic MHRs is simplified.

Despite the technical reasons presented by the authors in the rebuttal, the conclusion of this study can be strengthened if more cooling compounds- the most well-studied natural cooling agonist menthol, and/or other cooling agonists such as WS-12 and/or C3-are tested for their effects on TRPM4 and several other TRPM channels.

Reviewer #1 (Public review):

In this manuscript, Yang et al report a novel regulatory role of SIRT4 in the progression of kidney fibrosis. The authors showed that in the fibrotic kidney, SIRT4 exhibited an increased nuclear localization. Deletion of Sirt4 in renal tubule epithelium attenuated the extent of kidney fibrosis following injury, while overexpression of SIRT4 aggravates kidney fibrosis. Employing a battery of in vitro and in vivo experiments, the authors demonstrated that SIRT4 interacts with U2AF2 in the nucleus upon TGF-β1 stimulation or kidney injury and deacetylates U2AF2 at K413, resulting in elevated CCN2 expression through alternative splicing of Ccn2 gene to promote kidney fibrosis. The authors further showed that the translocation of SIRT4 is through the BAX/BAK pore complex and is dependent on the ERK1/2-mediated phosphorylation of SIRT4 at S36, and consequently the binding of SIRT4 to importin α1. This fundamental work substantially advances our understanding of the progression of kidney fibrosis and uncovers a novel SIRT4-U2AF2-CCN2 axis as a potential therapeutic target for kidney fibrosis.

Comment on revised version:

In the new version of the manuscript, the authors have addressed most of my concerns . Overall, the authors have done an extensive, well-performed study. The results are convincing, and the conclusions are mostly well supported by the data. The message is interesting to a wider community working on kidney fibrosis, protein acetylation and SIRT4 biology. This work substantially advances our understanding of the mechanism of kidney fibrosis and uncovers a novel SIRT4-U2AF2-CCN2 axis as a potential therapeutic target for kidney fibrosis.

Reviewer #1 (Public review):

Summary:

The authors want to determine the role of the sperm hook of the house mouse sperm in movement through the uterus. They use transgenic lines with fluorescent labels to sperm proteins, and they cross these males to C57BL/6 females in pathogen-free conditions. They use 2-photon microscopy on ex vivo uteri within 3 hours of mating and the appearance of a copulation plug. There are a total of 10 post-mating uteri that were imaged with 3 different males. They provide 10 supplementary movies that form that basis for some of the quantitative analysis in the main body figures. Their data suggest that the role of the sperm hook is to facilitate movement along the uterine wall.

Strengths:

Ex vivo live imaging of fluorescently labeled sperm with 2-photon microscopy is a powerful tool for studying the behavior of sperm.

Weaknesses:

The paper is descriptive and the data are correlations.

The authors cannot directly test their proposed function of the sperm hook in sliding and preventing backward slipping.

Reviewer #1 (Public Review):

Summary:

This is a well-written and detailed manuscript showing important results on the molecular profile of 4 different cohorts of female patients with lung cancer.

Strengths:

The authors used several different methods to identify potential novel targets for therapeutic interventions.

Weaknesses:

Statistical test results need to be provided in comparisons between cohorts. This was addressed by the authors in the revisions.

Reviewer #1 (Public review):

Summary:

Arman Angaji and his team delved into the intricate world of tumor growth and evolution, utilizing a blend of computer simulations and real patient data from liver cancer.

Strengths:

Their analysis of how mutations and clones are distributed within tumors revealed an interesting finding: tumors don't just spread from their edges as previously believed. Instead, they expand both from within and the edges simultaneously, suggesting a unique growth mode. This mode naturally indicates that external forces may play a role in cancer cells dispersion within the tumor. Moreover, their research hints at an intriguing phenomenon - the high death rate of progenitor cells and extremely slow pace in growth in the initial phase of tumor expansion. Understanding this dynamic could significantly impact our comprehension of cancer development.

Weaknesses:

It's important to note, however, that this study relies on specific computer models, metrics derived from inferred clones, and a limited number of patient data. While the insights gained are promising, further investigation is essential to validate these findings. Nonetheless, this work opens up exciting avenues for comprehending the evolution of cancers.

Comments on revised submission:

The authors have effectively addressed my concerns. This revision is excellent.

Reviewer #1 (Public Review):

Summary:

This manuscript provides a solid advance to the scavenger receptor field by reporting the crystal structures of the domains of SCARF1 that bind modified LDL such as oxidized LDL and acylated LDL. The crystal packing reveals a new interface for homodimerization of SCARF1. The authors characterize SCARF1 binding to modified LDL using flow cytometry, ELISA, and fluorescent microscopy. They identify a positively-charged surface on the structure that they predict will bind the LDLs, and they support this hypothesis with several mutant constructs in binding experiments.

Strengths:

The authors have crystallized domains of an understudied scavenger receptor and used the structure to identify a putative binding site for modified LDL particles. An especially strong set of experiments are binding studies with chimeras of SCARF1 and SCARF2, where they show gain-of-function results (binding of modified LDLs) by SCARF2, a related protein that does not normally bind modified LDLs. The paper is a straightforward set of experiments that identify the likely binding site of modified LDL on SCARF1

Weaknesses:

In the current revision, the authors addressed my technical concerns.<br /> Two remaining considerations that may limit the broader impact of this paper are 1) that it does not explain the structural basis for specificity of the binding of SCARF1 to various lipoproteins (i.e. why SCARF1 binds oxLDL and AcLDL but not LDL or HDL) and 2) a lack of a biological assay to interpret the functional consequences of the SCARF1 mutants. These may be addressed in future work.

Reviewer #1 (Public review):

The authors have tried to identify the plausible Na+ entry pathway in an important SLC6 member GAT1, using computational approaches to assess residence times of the ions as they enter the vestibule of GAT1. The authors identify a patch of negative residues in TM6a and implicate them for being important to attract the Na+ ions during their movement towards the binding sites Na1 and Na2. Besides this they also suggest that sodium binding at site 1 is flexible and at times can occupy the primary binding site when the substrate is not available. Na2 site as other literature also suggests is demonstrated to be vital for the stability of the outward-open state.

Studies of ion permeation are challenging given that the states are challenging to trap through structural studies and computational methods are vital for understanding these steps. The authors suggest that two negatively charged residues are vital to attract Na+ ions to the vestibule. Using a combination of simulations and PCA analysis the authors identify the importance of Na+ binding at site 2 that stabilises the outward-open state and the flexibility observed in Na1 site for ion binding which happens alongside substrate in the GABA bound state. The study reconfirms earlier observations in the SLC6 family that Na2 site is critical for conformational transitions and Na1 site is substrate dependent in amino acid transporters.<br /> One of the challenges in such studies is to conclusively establish the presence of additional Na+ sites or regions of ion-binding with experimental structures as they are nearly impossible to trap. Such studies using simulations therefore become the only resort to understand such phenomena.

The work is likely to further provide insights into the transport mechanism of GAT1 and lends credence to some structural studies where the sodium at site1 is displaced but the ion remains proximal to the bound substrate.

Reviewer #1 (Public review):

In their paper, Kang et al. investigate rigidity sensing in amoeboid cells, showing that, despite their lack of proper focal adhesions, amoeboid migration of single cells is impacted by substrate rigidity. In fact, many different amoeboid cell types can durotax, meaning that they preferentially move towards the stiffer side of a rigidity gradient.

The authors observed that NMIIA is required for durotaxis and, buiding on this observation, they generated a model to explain how durotaxis could be achieved in the absence of strong adhesions. According to the model, substrate stiffness alters the diffusion rate of NMAII, with softer substrates allowing for faster diffusion. This allows for NMAII accumulation at the back, which, in turn, results in durotaxis.

The authors responded to all my comments and I have nothing to add. The evidence provided for durotaxis of non adherent (or low-adhering) cells is strong. I am particularly impressed by the fact that amoeboid cells can durotax even when not confined. I wish to congratulate the authors for the excellent work, which will fuel discussion in the field of cell adhesion and migration.

Reviewer #1 (Public review):

Summary:

In the manuscript "FABP4-mediated lipid accumulation and lipolysis in tumor-associated macrophages promote breast cancer metastasis", Yorek, et al. provide a novel mechanism explaining how unsaturated fatty acids induce macrophages to accumulate lipid droplets, which when contained in tumor-associated macrophages (TAMs) are associated with increased metastasis in breast cancer. The authors conclude that unsaturated fatty acids are transported into macrophages by the chaperone FABP4 where they induce C/EBPalpha expression and transcriptional activity resulting in upregulation of enzymes involved in triacylglycerol and lipid droplet biosynthesis. The resulting accumulation of lipid droplets in macrophages creates a store of fatty acids that can subsequently be released through FABP4-dependent lipolysis and thereby stimulate the migration of nearby breast cancer cells. While generally well-written and developed, there are a few concerns about the rigor of experimental evidence that supports some conclusions, including the existence of a FABP4-C/EBPalpha pathway. Overall, the mechanism identified is a valuable contribution to our understanding of how tumor-associated macrophages may influenced by available metabolites to promote the aggressiveness of certain cancers. FABP4 has the potential to be used as a novel biomarker of macrophage-induced cancer aggressiveness and/or a therapeutic target to prevent metastasis.

Strengths:

(1) The study is logically organized and provides extensive evidence in support of the overall model proposed.

(2) Multiple complementary techniques are used to identify and quantify lipid droplets.

(3) Primary macrophages and macrophage cell lines are used and provide consistent data.

(4) Knock-down and knock-out cells are used to assess the contributions of FABP4 and C/EBPalpha to gene expression.

(5) Public gene expression data (GEO, TCGA) is used effectively throughout.

Weaknesses:

(1) After Figure 1, a single saturated (palmitic acid; PA) and a single unsaturated (linoleic acid; LA) fatty acid are used for the remaining studies, bringing into question whether effects are in fact the result of a difference in saturation vs. other potential differences.

(2) While primary macrophages are used in several mechanistic studies, tumor-associated macrophages (TAMs) are not used. Rather, correlative evidence is provided to connect mechanistic studies in macrophage cell lines and primary macrophages to TAMs.

(3) C/EBPalpha and FABP4 clearly regulate LA-induced changes in gene expression. However, whether these two key proteins act in parallel or as a pathway is not resolved by presented data.

(4) It is very interesting that FABP4 regulates both lipid droplet formation and lipolysis, yet is unclear if the regulation of lipolysis is direct or if the accumulation of lipid droplets - likely plus some other signal(s) - induces upregulation of lipolysis genes.

(5) In several places increased expression of genes coding for enzymes with known functions in lipid biology is conflated with an increase in the lipid biology process the enzymes mediate. Additional evidence would be needed to show these processes are in fact increased in a manner dependent on increased enzyme expression.

Reviewer #1 (Public review):

Summary:

The authors show that the Gαs-stimulated activity of human membrane adenylyl cyclases (mAC) can be enhanced or inhibited by certain unsaturated fatty acids (FA) in an isoform-specific fashion. Thus, with IC50s in the 10-20 micromolar range, oleic acid affects 3-fold stimulation of membrane-preparations of mAC isoform 3 (mAC3) but it does not act on mAC5. Enhanced Gαs-stimulated activities of isoforms 2, 7, and 9, while mAC1 was slightly attenuated, but isoforms 4, 5, 6, and 8 were unaffected. Certain other unsaturated octadecanoic FAs act similarly. FA effects were not observed in AC catalytic domain constructs in which TM domains are not present. Oleic acid also enhances the AC activity of isoproterenol-stimulated HEK293 cells stably transfected with mAC3, although with lower efficacy but much higher potency. Gαs-stimulated mAC1 and 4 cyclase activity were significantly attenuated in the 20-40 micromolar by arachidonic acid, with similar effects in transfected HEK cells, again with higher potency but lower efficacy. While activity mAC5 was not affected by unsaturated FAs, neutral anandamide attenuated Gαs-stimulation of mAC5 and 6 by about 50%. In HEK cells, inhibition by anandamide is low in potency and efficacy. To demonstrate isoform specificity, the authors were able to show that membrane preparations of a domain-swapped AC bearing the catalytic domains of mAC3 and the TM regions of mAC5 are unaffected by oleic acid but inhibited by anandamide. To verify in vivo activity, in mouse brain cortical membranes 20 μM oleic acid enhanced Gαs-stimulated cAMP formation 1.5-fold with an EC50 in the low micromolar range.

Strengths:

(1) A convincing demonstration that certain unsaturated FAs are capable of regulating membrane adenylyl cyclases in an isoform-specific manner, and the demonstration that these act at the AC transmembrane domains.

(2) Confirmation of activity in HEK293 cell models and towards endogenous AC activity in mouse cortical membranes.

(3) Opens up a new direction of research to investigate the physiological significance of FA regulation of mACs and investigate their mechanisms as tonic or regulated enhancers or inhibitors of catalytic activity.

(4) Suggests a novel scheme for the classification of mAC isoforms.

Weaknesses:

(1) Important methodological details regarding the treatment of mAC membrane preps with fatty acids are missing.

(2) It is not evident that fatty acid regulators can be considered as "signaling molecules" since it is not clear (at least to this reviewer) how concentrations of free fatty acids in plasma or endocytic membranes are hormonally or otherwise regulated.

Reviewer #1 (Public review):

Summary:

The paper demonstrated through a comprehensive multi-omics study of the oviduct that the transcriptomic and proteomic landscape of the oviduct at 4 different preimplantation periods was dynamic during natural fertilization, pseudopregnancy, and superovulation using three independent cell/tissue isolation and analytical techniques. This work is very important for understanding oviductal biology and physiology. In addition, the authors have made all the results available in a web search format, which will maximize the public's access and foster and accelerate research in the field.

Strengths:

(1) The manuscript addresses an important and interesting question in the field of reproduction: how does the oviduct at different regions adapt to the sperm and embryos for facilitating fertilization and preimplantation embryo development and transport?

(2) Authors used cutting-edge techniques: Integrated multi-modal datasets followed by in vivo confirmation and machine learning prediction.

(3) RNA-seq, scRNA-seq, and proteomic results are immediately available to the scientific community in a web search format.

(4) Substantiated results indicate the source of inflammatory responses was the secretory cell population in the IU region when compared to other cell types; sperm modulate inflammatory responses in the oviduct; the oviduct displays immuno-dynamism.

Weaknesses:

(1) The rationale for using the superovulation model is not clear. The oviductal response to sperm and embryos can be studied by comparing mating with normal and vasectomized mice and comparing pregnancy vs pseudopregnancy (induced by mating with vasectomized males). Superovulation causes supraphysiological hormone levels and other confounding conditions.

(2) This study involves a very complex dataset with three different models at four time points. If possible, it would be very informative to generate a graphic abstract/summary of their major findings in oviductal responses in different models and time points

(3) The resolution of Figures 3A-3C in the submitted file was not high enough to assess the authors' conclusion.

(4) The authors need to double-check influential transcription factors identified by machine learning. Apparently, some of them (such as Anxa2, Ift88, Ccdc40) are not transcription factors at all.

Reviewer #1 (Public review):

Summary:

This work seeks to provide genetic evidence for a role for beta-adrenergic receptors that regulate heart rate and blood flow on cavernous malformation development using a zebrafish model, and to extend information regarding beta-adrenergic drug blockade in cavernous malformation development, with the idea that these drugs may be useful therapeutically.

Strengths:

The work shows that genetic loss of a specific beta-adrenergic receptor in zebrafish, adrb1, prevents embryonic venous malformations and CCM in adult zebrafish brains. Two drugs, propranolol and metoprolol, also blunt CCM in the adult fish brain. These findings are predicted to potentially impact the treatment of human CCM, and they increase understanding of the factors leading to CCM.

Weaknesses:

There are minor weaknesses that detract slightly from enthusiasm, including poor annotation of the Figure panels and lack of a baseline control for the study of Klf2 expression (Figure 4).

Reviewer #1 (Public review):

Summary:

Ma et al. show that melanoma cells induce an EMT-like state in nearby keratinocytes and that when this state is induced experimentally by Twist-overexpression the resulting alteration in keratinocytes is inhibitory for melanoma invasion. These conclusions are based on experiments in vivo with zebrafish and, in vitro, with human cells. The work is carefully done and provides new insights into the interactions between melanoma cells and their environment.

Strengths:

The use of both zebrafish and human cells adds confidence that findings are relevant to human melanomas while also further demonstrating the utility of the zebrafish system for discovering important new features of melanoma biology that could ultimately have clinical impacts. The work also combines a nice suite of approaches including different models for induced melanomagenesis in zebrafish, single-cell RNA-sequencing, and more. Some of the final observations are intriguing as well, especially the possibility of EMT-induced melanocyte-keratinocyte interactions via Jam3 expression; it will be interesting to see if this is indeed a mechanism for restraining melanoma invasion. The paper is clearly written and the inferences are appropriate for the results obtained. Overall the work makes a solid contribution to our understanding of important, but too often neglected, roles of the tumor microenvironment in promoting or inhibiting tumor progression and outcome.

Weaknesses:

No critical weaknesses were noted.

Reviewer #1 (Public review):

In this study, Hama et al. explored the molecular regulatory mechanisms underlying the formation of the ULK1 complex. By employing the AlphaFold structural prediction tool, they showed notable differences in the complex formation mechanisms between ULK1 in mammalian cells and Atg1 in yeast cells. Their findings revealed that in mammalian cells, ULK1, ATG13, and FIP200 form a complex with a stoichiometry of 1:1:2. These predicted interaction regions were validated through both in vivo and in vitro assays, enhancing our understanding of the molecular mechanisms governing ULK1 complex formation in mammalian cells. Importantly, they identified a direct interaction between ULK1 and FIP200, which is crucial for autophagy. However, some aspects of this manuscript require further clarification, validation, and correction by the authors.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteasomal function in an ATP-independent manner. Although there are multiple works that have highlighted the role of this protein in tumours, this study specifically tried to develop a correlation with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.

Strengths:

The observations of the authors hint that beyond PA28y's association with the proteasome, it might also stabilize certain proteins such as C1QBP which influences energy metabolism.

Weaknesses:

The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.<br /> In most of the experiments, the authors have been dependent on the parallel changes in the expression of both the proteins to justify their stabilizing interaction. However, this approach is indirect at best and does not confirm the direct stabilizing effect of this interaction. IP experiments do not indicate direct interaction and have some quality issues. The upregulation of C1QBP might be indirect at best. It is quite possible that PA28y might be degrading some secondary protein/complex that is responsible for C1QBP expression. Since the core idea of the work is PA28y direct interaction with C1QBP stabilizing it, the same should be demonstrated in a more convincing manner.

In all of the assays, C1QBP has been detected as doublet. However, the expression pattern of the two bands varies depending on the experiment. In some cases, the upper band is intensely stained and in some the lower bands. Do C1QBP isoforms exist and are they differentially regulated depending on experiment conditions/tissue types?

Problems with the background of the work: Line 76. This statement is far-fetched. There are presently a number of works of literature that have dealt with the metabolic programming of OSCC including identification of specific metabolites. Moreover, beyond the estimation of OCR, the authors have not conducted any experiments related to metabolism. In the Introduction, the significance of this study and how it will extend our understanding of OSCC needs to be elaborated.

Reviewer #1 (Public review):

Summary:

The authors investigate the role of HSPA2 during mouse preimplantation development. Knocking down HSPA2 in zygotes, the authors describe lower chances of developing into blastocysts, which show a reduced number of inner cell mass cells. They find that HSPA2 mRNA and protein levels show some heterogeneity among blastomeres at the 4-cell stage and propose that HSPA2 could contribute to skewing their relative contribution to embryonic lineages. To test this, the authors try to reduce HSPA2 expression in one of the 2-cell stage blastomere and propose that it biases their contribution to towards extra-embryonic lineages. To explain this, the authors propose that HSPA2 would interact with CARM1, which controls chromatin accessibility around genes regulating differentiation into embryonic lineage.

Strengths:

(1) The study offers simple and straightforward experiments with large sample sizes.

(2) Unlike most studies in the field, this research often relies on both mRNA and protein levels to analyse gene expression and differentiation.

Weaknesses:

(1) Image and statistical analyses are not well described.

(2) The functionality of the overexpression construct is not validated.

(3) Tracking of KD cells in embryos injected at the 2-cell stage with GFP is unclear.

(4) A key rationale of the study relies on measuring small differences in the levels of mRNA and proteins using semi-quantitative methods to compare blastomeres. As such, it is not possible to know whether those subtle differences are biologically meaningful. For example, the lowest HSPA2 level of the embryo with the highest level is much higher than the top cell from the embryo with the lowest level. What does this level mean then? Does this mean that some blastomeres grafted from strong embryos would systematically outcompete all other blastomeres from weaker embryos? That would be very surprising. I think the authors should be more careful and consider the lack of quantitative power of their approach before reaching firm conclusions. Although to be fair, the authors only follow a long trend of studies with the same intrinsic flaw of this approach.

(5) Some of the analyses on immunostaining do not take into account that this technique only allows for semi-quantitative measurements and comparisons.<br /> a) Some of the microscopy images are shown with an incorrect look-up table.<br /> b) Some of the schematics are incorrect and misleading.

Reviewer #1 (Public review):

Summary:

This is a comprehensive study that clearly and deeply investigates the function of GATA6 in human early cardiac development.

Strengths:

This study combines hESC engineering, differentiation, detailed gene expression, genome occupancy, and pathway modulation to elucidate the role of GATA6 in early cardiac differentiation. The work is carefully executed and the results support the conclusions. The use of publicly available data is well integrated throughout the manuscript. The RIME experiments are excellent.

Weaknesses:

Much has been known about GATA6 in mesendoderm development, and this is acknowledged by the authors.

Reviewer #1 (Public Review):

In the current manuscript, the authors use theoretical and analytical tools to examine the possibility of neural projections to engage ensembles of synaptic clusters in active dendrites. The analysis is divided into multiple models that differ in the connectivity parameters, speed of interactions, and identity of the signal (electric vs. second messenger). They first show that random connectivity almost ensures the representation of presynaptic ensembles. As expected, this convergence is much more likely for small group sizes and slow processes, such as calcium dynamics. Conversely, fast signals (spikes and postsynaptic potentials) and large groups are much less likely to recruit spatially clustered inputs. Dendritic nonlinearity in the postsynaptic cells was found to play a highly important role in distinguishing these clustered activation patterns, both when activated simultaneously and in sequence. The authors tackled the difficult issue of noise, showing a beneficiary effect when noise 'happens' to fill in gaps in a sequential pattern but degraded performance at higher background activity levels. Last, the authors simulated selectivity to chemical and electrical signals. While they find that longer sequences are less perturbed by noise, in more realistic activation conditions, the signals are not well resolved in the soma.

While I think the premise of the manuscript is worth exploring, I have a number of reservations regarding the results.

(1) In the analysis, the authors made a simplifying assumption that the chemical and electrical processes are independent. However, this is not the case; excitatory inputs to spines often trigger depolarization combined with pronounced calcium influx; this mixed signaling could have dramatic implications on the analysis, particularly if the dendrites are nonlinear (see below)

(2) Sequence detection in active dendrites is often simplified to investigating activation in a part of or the entirety of individual branches. However, the authors did not do that for most of their analysis. Instead, they treat the entire dendritic tree as one long branch and count how many inputs form clusters. I fail to see why simplification is required and suspect it can lead to wrong results. For example, two inputs that are mapped to different dendrites in the 'original' morphology but then happen to fall next to each other when the branches are staggered to form the long dendrites would be counted as neighbors.

(3) The simulations were poorly executed. Figures 5 and 6 show examples but no summary statistics. The authors emphasize the importance of nonlinear dendritic interactions, but they do not include them in their analysis of the ectopic signals! I find it to be wholly expected that the effects of dendritic ensembles are not pronounced when the dendrites are linear.

To provide a comprehensive analysis of dendritic integration, the authors could simulate more realistic synaptic conductances and voltage-gated channels. They would find much more complicated interactions between inputs on a single site, a sliding temporal and spatial window of nonlinear integration that depends on dendritic morphology, active and passive parameters, and synaptic properties. At different activation levels, the rules of synaptic integration shift to cooperativity between different dendrites and cellular compartments, further complicated by nonlinear interactions between somatic spikes and dendritic events.

While it is tempting to extend back-of-the-napkin calculations of how many inputs can recruit nonlinear integration in active dendrites, the biological implementation is very different from this hypothetical. It is important to consider these questions, but I am not convinced that this manuscript adequately addressed the questions it set out to probe, nor does it provide information that was unknown beforehand.

Not only was I surprised, but the witnesses in the story were as well. This surprise could be related to the truth being revealed or the curiosity about who is responsible.

The passage shows the cruelness of an animal and also illustrates the behavior of animals being skilled at imitation. Though it did it unconsciously, it’s also harmful.

Showing Dupin's prowess in deduction and his unpredictable personality through a random dialogue. Even before the case officially begins, let the reader understand his character

I am not quite sure about the meaning of this sentence, is it means that normal people over interpreting the coincidences as the motivation of murderer?

I think the detective is smart. When he find the sash is difficult to be opened and needed to be fasten inside, he doesn’t quit the possibility that murderer could escape from the window but try to find if the window can fasten by itself.

When it comes to the crime, we will assume that it is committed by human, so the unusual sound could be interpreted as the terrified scream of the women or the shout of the murder; however, it couldn’t be recognized as any kinds of language, implying that the murderer might not be a human.

This may surprise you because it suggests that being clever doesn’t mean someone can analyze things well, which goes against the common belief that cleverness and analytical skill go hand in hand.

Reviewer #1 (Public review):

Summary

In this manuscript, Day et al. present a high-throughput version of expansion microscopy to increase the throughput of this well-established super-resolution imaging technique. Through technical innovations in liquid handling with custom-fabricated tools and modifications to how the expandable hydrogels are polymerized, the authors show robust ~4-fold expansion of cultured cells in 96-well plates. They go on to show that HiExM can be used for applications such as drug screens by testing the effect of doxorubicin on human cardiomyocytes. Interestingly, the effects of this drug on changing DNA organization were only detectable by ExM, demonstrating the utility of HiExM for such studies.

Overall, this is a very well-written manuscript presenting an important technical advance that overcomes a major limitation of ExM - throughput. As a method, HiExM appears extremely useful and the data generally support the conclusions.

Strengths

Hi-ExM overcomes a major limitation of ExM by increasing the throughput and reducing the need for manual handling of gels. The authors do an excellent job of explaining each variation introduced to HiExM to make this work and thoroughly characterize the impressive expansion isotropy. The dox experiments are generally well-controlled and the comparison to an alternative stressor (H2O2) significantly strengthens the conclusions.

Weaknesses

(1) It is still unclear to me whether or not cells that do not expand remain in the well given the response to point 1. The authors say the cells are digested and washed away but then say that there is a remaining signal from the unexpanded DNA in some cases. I believe this is still a concern that potential users of the protocol should be aware of.

(2) Regarding the response to point 9, I think this information should be included in the manuscript, possibly in the methods. It is important for others to have a sense of how long imaging may take if they were to adopt this method.

Joint Public Review:

TRPML1 functions as a lysosomal ion channel whose variants are associated with lysosomal storage disorder mucolipidosis type IV. Understanding the structure and function of sites involved in the allosteric control TRPML1 may provide new molecular moieties to target with prototypic drugs.

Gan et al provide the first high resolution cryo-EM structure of a mutant (Y404W) TRPML1 channel in the open state without any activating ligands. This new structure demonstrates how a mutation at a site some distance away from the pore can influence channel gating. The authors provide compelling electrophysiology evidence which supports the proposed Y404W gain of function effect.

The authors propose an allosteric mechanism whereby the engineered W404 sidechain provides extra van der Waals contacts within a pocket surrounded by helices of the voltage sensor-like domain (VSLD) and causes S4 bending which in turn opens the pore through the S4-S5 linker. Conversely, the authors functionally demonstrate that an alanine mutation at this site causes a loss of function. Although the authors do not provide a structure of the Y404A mutant, they propose that the alanine substitution disrupts the sidechain packing and likely destabilizes the open conformation.

TRPML1 channels are regulated by PIP2 species in the cell. In the lysosomal membrane, PI(3,5)P2 activates the channel, whereas in the plasma membrane PI(4,5)P2 inhibits it. Towards understanding its lipid regulation, the authors solve a cryo-EM structure of TRPML1 bound to PI(4,5)P2 in the closed state and provide functional evidence that PI(4,5)P2 occupancy inhibits TRPML1 currents.

Within this same structure, the authors observe a density which may be attributed to sphingomyelin (or possibly phosphocholine). Using electrophysiology on WT and Y404W channels, the authors report an antagonist effect of sphingomyelin on TRPML1 currents.

Taken together, the study provides convincing evidence for a gating (opening/closing) mechanism of the TRPML1 pore which can be allosterically regulated by altered side-chain packing and by lipid interactions within the VSLD.

Reviewer #1 (Public Review):

Summary:

This manuscript by Meissner and colleagues described a novel take on a classic social cognition paradigm developed for marmosets. The classic pull task is a powerful paradigm that has been used for many years across numerous species, but its analog approach has several key limitations. As such, it has not been feasible to adopt the task for neuroscience experiments. Here the authors capture the spirit of the classic task but provide several fundamental innovations that modernize the paradigm - technically and conceptually. By developing the paradigm for marmosets, the authors leverage the many advantages of this primate model for studies of social brain functions and their particular amenability to freely-moving naturalistic approaches.

Strengths:

The current manuscript describes one of the most exciting paradigms in primate social cognition to be developed in many years. By allowing for freely-moving marmosets to engage in high numbers of trials, while precisely quantifying their visual behavior (e.g. gaze) and recording neural activity this paradigm has the potential to usher in a new wave of research on the cognitive and neural mechanisms underlying primate social cognition and decision-making. This paradigm is an elegant illustration of how naturalistic questions can be adapted to more rigorous experimental paradigms. Overall, I thought the manuscript was well written and provided sufficient details for others to adopt this paradigm.

Reviewer #1 (Public review):

Summary:

Understanding large-scale neural activity remains a formidable challenge in neuroscience. While several methods have been proposed to discover the assemblies from such large-scale recordings, most of previous studies do not explicit modeling the temporal dynamics. This study is an attempt to uncover the temporal dynamics of assemblies using a tool that have been establish in other domains.

The authors previously introduced the compositional Restricted Boltzmann Machine (cRBM) to identify neuron assemblies in zebrafish brain activity. Building upon this, they now employ the Recurrent Temporal Restricted Boltzmann Machine (RTRBM) to elucidate the temporal dynamics within these assemblies. By introducing recurrent connections between hidden units, RTRBM could retrieve neural assemblies and their temporal dynamics from simulated and zebrafish brain data.

Strengths:

The RTRBM has been previously used in other domains. Training the model has been already established. This study is an application of such model to neuroscience. Overall, the paper is well-structured and the methodology is robust, the analysis is solid to support the authors claim.

Weaknesses:

The overall degree of advance is very limited. The performance improvement by RTRBM compared to their cRBM is marginal, and insights into assembly dynamics are limited.

(1) The biological insights from this method are constrained. Though the aim is to unravel neural ensemble dynamics, the paper lacks in-depth discussion on how this method enhances our understanding of zebrafish neural dynamics. For example, the dynamics of assemblies can be analyzed using various tools such as dimensionality reduction methods once we have identified them using cRBM. What information can we gain by knowing the effective recurrent connection between them? It would be more convincing to show this in real data.

(2) Including predicted and measured neural activity traces could aid readers in evaluating model efficacy. The current version only contains comparison of the statistics, such as mean and covariance.

Reviewer #1 (Public Review):

Summary:

This study 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.

Reviewer #1 (Public review):

Summary:

This paper presents a mechanistic study of rDNA origin regulation in yeast by SIR2. Each of the ~180 tandemly repeated rDNA gene copies contains a potential replication origin. Early-efficient initiation of these origins is suppressed by Sir2, reducing competition with origins distributed throughout the genome for rate-limiting initiation factors. Previous studies by these authors showed that SIR2 deletion advances replication timing of rDNA origins by a complex mechanism of transcriptional de-repression of a local PolII promoter causing licensed origin proteins (MCMcomplexes) to re-localize (slide along the DNA) to a different (and altered) chromatin environment. In this study, they identify a chromatin remodeler, FUN30, that suppresses the sir2∆ effect, and remarkably, results in a contraction of the rDNA to about one-quarter it's normal length/number of repeats, implicating replication defects of the rDNA. Through examination of replication timing, MCM occupancy and nucleosome occupancy on the chromatin in sir2, fun30, and double mutants, they propose a model where nucleosome position relative to the licensed origin (MCM complexes) intrinsically determines origin timing/efficiency. While their interpretations of the data are largely reasonable and can be interpreted to support their model, a key weakness is the connection between Mcm ChEC signal disappearance and origin firing. While the cyclical chromatin association-dissociation of MCM proteins with potential origin sequences may be generally interpreted as licensing followed by firing, dissociation may also result from passive replication and as shown here, displacement by transcription and/or chromatin remodeling. Moreover, linking its disappearance from chromatin in the ChEC method with such precise resolution needs to be validated against an independent method to determine the initiation site(s). Differences in rDNA copy number and relative transcription levels also are not directly accounted for, obscuring a clearer interpretation of the results. Nevertheless, this paper makes a valuable advance with the finding of Fun30 involvement, which substantially reduces rDNA repeat number in sir2∆ background. The model they develop is compelling and I am inclined to agree, but I think the evidence on this specific point is purely correlative and a better method is needed to address the initiation site question. The authors deserve credit for their efforts to elucidate our obscure understanding of the intricacies of chromatin regulation. At a minimum, I suggest their conclusions on these points of concern should be softened and caveats discussed. Statistical analysis is lacking for some claims.

Strengths are the identification of FUN30 as suppressor, examination of specific mutants of FUN30 to distinguish likely functional involvement. Use of multiple methods to analyze replication and protein occupancies on chromatin. Development of a coherent model.

Weaknesses are failure to address copy number as a variable; insufficient validation of ChEC method relationship to exact initiation locus; lack of statistical analysis in some cases.

Review of revised version and response letter:

In the response, the authors make some improvements by better quantifying 2D gels, adding some missing statistical analyses, analyzing the effect of fun30 on rDNA replication in strains with reduced rDNA copy number, and using ChIP-seq of MCMs to support the ChEC-seq data. However, these additions do not address the main issue that is at the heart of their model: where initiation precisely occurs and whether the location is altered in the mutant(s). Thus, mechanistic insight is limited.

Under the section "Addressing Alternative Explanations", the authors claim that processes like transcription and passive replication cannot affect the displaced complex specifically. Why? They are not on same DNA (as mentioned in the Fig 1 legend).

The model in Fig 7 implies that initiation sites are different in WT versus the mutants and this determines their timing/efficiency. But they also suggest that the same site might be used with different efficiencies in this response. I agree that both are possibilities and are not resolved.

Supporting their model requires better resolution to determine the actual replication initiation site. While this may be challenging, it should be feasible with methods to map nascent strands like DNAscent, or Okazaki fragment mapping.

The 2D gel analysis of strains with reduced rDNA copy numbers adequately addresses the copy number variable with regard to the replication effect.

Overall, the paper is improved by providing additional data and improved analysis. The paper nicely characterizes the effect of Fun30. The model is reasonable but remains lacking in precise details of mechanism.

Topic: Defining Distance Education Date: 1980 Course/Subject: Distance Education Studies

Notes Main Idea The paper analyzes existing definitions of distance education and identifies six essential components that should be included in a comprehensive definition. The analysis covers different educational philosophies and institutional contexts for distance education.

Key Definitions and Theorists Holmberg's Definition

Distance education involves the separation of teacher and learner. Requires planned, structured learning materials and institutional support. French Law (Loi 71.556 du 12 juillet 1971)

Defines distance education as learning where the teacher is not physically present, except for occasional tasks. Moore's Definition

Highlights separation of teaching and learning behaviors. Emphasizes the use of technical media and the importance of two-way communication. Peters’ Definition

Describes distance education as an industrialized form of education. Incorporates principles like division of labor, mass production, and mechanization in the teaching process. Six Essential Components of Distance Education Separation of Teacher and Student

Physical and temporal separation in the teaching and learning process. Role of Educational Organization

Planning, preparing, and providing learning materials and support. Use of Technical Media

Communication between teacher and learner facilitated by print, radio, television, or computers. Provision of Two-Way Communication

Opportunities for dialogue between teacher and learner. Possibility of Occasional Seminars

Allowing for some face-to-face interaction for didactic or social purposes. Participation in an Industrialized Form of Education

Incorporates aspects of industrial processes in the delivery of education, such as standardized materials and automated communication. Analysis and Discussion Educational Contexts and Models

Different definitions cater to various levels and types of education (e.g., correspondence, open learning, external studies). Theoretical frameworks like Peters' focus on the structural separation and mechanization of distance education. Challenges and Misconceptions

Confusion over terminology, such as correspondence study vs. distance education. Misunderstanding of the relationship between traditional and distance education models. Proposed Solution

The term 'distance education' is suggested as the most suitable to encompass various forms and philosophies of education that involve separation of teacher and learner. Implications The need for clarity in defining distance education for consistent policy development and theoretical analysis. Importance of recognizing the distinct nature of distance education as an industrialized form of learning. Questions/Cues What are the key differences between Holmberg’s and Peters’ definitions of distance education? How does Keegan's six-component framework help in distinguishing distance education from other forms of non-traditional education? Why is Peters’ view on industrialization important in understanding distance education? Summary The paper provides a detailed analysis of distance education by examining prominent definitions and proposing a comprehensive framework. Keegan emphasizes the industrialized nature of distance education and the necessity for clear terminology to distinguish it from other educational forms.

Reference Keegan, D. J. (1980). On defining distance education. Distance Education, 1(1), 13-36.

Reviewer #1 (Public review):

This manuscript describes soluble Uric Acid (sUA) as an endogenous inhibitor of CD38, affecting CD38 activity and NAD+ levels both in vitro and in vivo. Importantly, the inhibition constants calculated supports the claim that sUA inhibits CD38 under physiological conditions. These findings are of extreme importance to understand the regulation of an enzyme that has been shown to be the main NAD+/NMN-degrading enzyme in mammals, which impacts several metabolic processes and has major implications to understanding aging diseases. The manuscript is well written, the figures are self explanatory, and in the experiments presented, the data is very solid. The authors discuss the main limitations of the study, especially in regard to the in vivo results. As a whole, I believe that this is a very interesting manuscript that will be appreciated by the scientific community and that opens a lot of new questions in the field of metabolism and aging.

During the revision process, the authors have performed new experiments to respond to relevant questions raised by the reviewers. In other cases, they have made changes in the text to improve the manuscript.

I believe that this manuscript in its current form is a mature and relevant set of findings that deserve attention and future developments.

Reviewer #1 (Public review):

Summary:

In previous work, the authors described necrosis-induced apoptosis (NiA) as a consequence of induced necrosis. Specifically, experimentally induced necrosis in the distal pouch of larval wing imaginal discs triggers NiA in the lateral pouch. In this manuscript, the authors confirmed this observation and found that while necrosis can kill all areas of the disc, NiA is limited to the pouch and to some extent to the notum, but is excluded from the hinge region. Interestingly and unexpectedly, signaling by the Jak/Stat and Wg pathways inhibits NiA. Further characterization of NiA by the authors reveals that NiA also triggers regenerative proliferation which can last up to 64 hours following necrosis induction. This regenerative response to necrosis is significantly stronger compared to discs ablated by apoptosis. Furthermore, the regenerative proliferation induced by necrosis is dependent on the apoptotic pathway because RNAi targeting the RHG genes is sufficient to block proliferation. However, NiA does not promote proliferation through the previously described apoptosis-induced proliferation (AiP) pathway, although cells at the wound edge undergo AiP. Further examination of the caspase levels in NiA cells allowed the authors to group these cells into two clusters: some cells (NiA) undergo apoptosis and are removed, while others referred to as Necrosis-induced Caspase Positive (NiCP) cells survive despite caspase activity. It is the NiCP cells that repair cellular damage including DNA damage and that promote regenerative proliferation. Caspase sensors demonstrate that both groups of cells have initiator caspase activity, while only the NiA cells contain effector caspase activity. Under certain conditions, the authors were also able to visualize effector caspase activity in NiCP cells, but the level was low, likely below the threshold for apoptosis. Finally, the authors found that loss of the initiator caspase Dronc blocks regenerative proliferation, while inhibiting effector caspases by expression of p35 does not, suggesting that Dronc can induce regenerative proliferation following necrosis in a non-apoptotic manner. This last finding is very interesting as it implies that Dronc can induce proliferation in at least two ways in addition to its requirement in AiP.

Strengths:

This is a very interesting manuscript. The authors demonstrate that epithelial tissue that contains a significant number of necrotic cells is able to regenerate. This regenerative response is dependent on the apoptotic pathway which is induced at a distance from the necrotic cells. Although regenerative proliferation following necrosis requires the initiator caspase Dronc, Dronc does not induce a classical AiP response for this type of regenerative response. In future work, it will be very interesting to dissect this regenerative response pathway genetically.

Weaknesses:<br /> No weaknesses were identified.

Reviewer #1 (Public review):

Summary:

This work develops a simple, rapid, low-cost methodology for assembling combinatorially complete microbial consortia using basic laboratory equipment. The motivation behind this work is to make the study of microbial community interactions more accessible to laboratories that lack specialized equipment such as robotic liquid handlers or microfluidic devices. The method was tested on a library of Pseudomonas aeruginosa strains to demonstrate its practicality and effectiveness. It provided a means to explore the complex functional interactions within microbial communities and identify optimal consortia for specific functions, such as biomass production.

Strengths:

The primary strength of this manuscript lies in its accessibility and practicality. The method proposed by the authors allows any laboratory with standard equipment, such as multichannel pipettes and 96-well plates, to readily construct all possible combinations of microbial consortia from a given set of species. This greatly enhances access to full factorial designs, which were previously limited to labs with advanced technology.

Another strength of the manuscript is the measurement and analysis of the biomass of all possible combinations of 8 strains of P. aeruginosa. This analysis provides a concrete example of how the authors' new methodology can be used to identify the best-performing communities and map pairwise and higher-order functional interactions.

Notably, the authors do exceptionally well in providing a thorough description of the methodology, including detailed protocols and an R script for customizing the method to different experimental needs. This enhances the reproducibility and adaptability of the methodology, making it a valuable resource for researchers wishing to adopt this methodology.

Weaknesses:

While the methodology is robust and well-presented, there are some limitations that should be acknowledged more thoroughly. First, the method's scalability is an important factor. The authors indicate that it should be effective for up to 10-12 species, but there is no discussion of what sets this scale: time, amount of labor, consumables, the likelihood of error, sample volume, etc. Second, this methodology is tailored to construct communities where the abundance of each strain is identical in each combination. Therefore, combinations with a different number of strains also differ in the total initial amount of microbial cells. Second, variations in the initial proportions of the same set of strains cannot be readily explored. Third, the manuscript only discusses how to construct the combinations, and not how to assay them afterward (e.g. for community function, interspecific interactions, etc'). While details on how to achieve these goals are clearly outside the scope of this work, the use of biomass as an example function may obfuscate this caveat, which should be stated more explicitly.

Reviewer #1 (Public review):

Summary:

Odor- and taste-sensing are mediated by two different systems, the olfactory and gustatory systems, and have different behavioral roles. In this study, Wei et al. challenge this dichotomy by showing that odors can activate gustatory receptor neurons (GRNs) in Drosophila to promote feeding responses, including the proboscis extension response (PER) that was previously thought to be driven only by taste. While previous studies suggested that odors can promote PER to appetitive tastants, Wei et al. go further to show that odors alone cause PER, this effect is mediated through sweet-sensing GRNs, and sugar receptors are required. The study also shows that odor detection by bitter-sensing GRNs suppresses PER. The authors' conclusions are supported by behavioral assays, calcium imaging, electrophysiological recordings, and genetic manipulations. The observation that both attractive and aversive odors promote PER leaves an open question as to why this effect is adaptive. Overall, the study sheds new light on chemosensation and multimodal integration by showing that odor and taste detection converge at the level of sensory neurons, a finding that is interesting and surprising while also being supported by another recent study (Dweck & Carlson, Sci Advances 2023).

Strengths:

(1) The main finding that odors alone can promote PER by activating sweet-sensing GRNs is interesting and novel.

(2) The study uses video tracking of the proboscis to quantify PER rather than manual scoring, which is typically used in the field. The tracking method is less subjective and provides a higher-resolution readout of the behavior.

(3) The study uses calcium imaging and electrophysiology to show that odors activate GRNs. These represent complementary techniques that measure activity at different parts of the GRN (axons versus dendrites, respectively) and strengthen the evidence for this conclusion.

(4) Genetic manipulations show that odor-evoked PER is primarily driven by sugar GRNs and sugar receptors rather than olfactory neurons. This is a major finding that distinguishes this work from previous studies of odor effects on PER and feeding (e.g., Reisenman & Scott, 2019; Shiraiwa, 2008) that assumed or demonstrated that odors were acting through olfactory neurons.

Weaknesses/Limitations:

1) The authors may want to discuss why PER to odors alone has not been previously reported, especially as they argue that this is a broad effect evoked by many different odors. Previous studies testing the effect of odors on PER only observed odor enhancement of PER to sugar (Oh et al., 2021; Reisenman & Scott, 2019; Shiraiwa, 2008) and some of these studies explicitly show no effect of odor alone or odor with low sugar concentration; regardless, the authors likely would have noticed if PER to odor alone had occurred. Readers of this paper may also be aware of unpublished studies failing to observe an effect of PER on odor alone (including studies performed by this reviewer and unrelated work by other colleagues in the field), which of course the authors are not expected to directly address but may further motivate the authors to provide possible explanations.

(2) Many of the odor effects on behavior or neuronal responses were only observed at very high concentrations. Most effects seemed to require concentrations of at least 10-2 (0.01 v/v), which is at the high end of the concentration range used in olfactory studies (e.g., Hallem et al., 2004), and most experiments in the paper used a far higher concentration of 0.5 v/v. It is unclear whether these are concentrations that would be naturally encountered by flies.

(3) The calcium imaging data showing that sugar GRNs respond to a broad set of odors contrasts with results from Dweck & Carlson (Sci Adv, 2023) who recorded sugar neurons with electrophysiology and observed responses to organic acids, but not other odors. This discrepancy is not discussed.

(4) Related to point #1, it would be useful to see a quantification of the percent of flies or trials showing PER for the key experiments in the paper, as this is the standard metric used in most studies and would help readers compare PER in this study to other studies. This is especially important for cases where the authors are claiming that odor-evoked PER is modulated in the same way as previously shown for sugar (e.g., the effect of starvation in Figure S4).

(5) Given the novelty of the finding that odors activate sugar GRNs, it would be useful to show more examples of GCaMP traces (or overlaid traces for all flies/trials) in Figure 3. Only one example trace is shown, and the boxplots do not give us a sense of the reliability or time course of the response. A related issue is that the GRNs appear to be persistently activated long after the odor is removed, which does not occur with tastes. Why should that occur? Does the time course of GRN activation align with the time course of PER, and do different odors show differences in the latency of GRN activation that correspond with differences in the latency of PER (Figure S1A)?

(6) Several controls are missing, and in some cases, experimental and control groups are not directly compared. In general, Gal4/UAS experiments should include comparisons to both the Gal4/+ and UAS/+ controls, at least in cases where control responses vary substantially, which appears to be the case for this study. These controls are often missing, e.g. the Gal4/+ controls are not shown in Figure 2C-G and the UAS/+ controls are not shown in Figure 2J-L (also, the legend for the latter panels should be revised to clarify what the "control" flies are). For the experiments in Figure S5, the data are not directly compared to any control group. For several other experiments, the control and experimental groups are plotted in separate graphs (e.g., Figure 2C-G), and they would be easier to visually compare if they were together. In addition, for each experiment, the authors should denote which comparisons are statistically significant rather than just reporting an overall p-value in the legend (e.g., Figure 2H-L).

(7) Additional controls would be useful in supporting the conclusions. For the Kir experiments, how do we know that Kir is effective, especially in cases where odor-evoked PER was not impaired (e.g., Orco/Kir)? The authors could perform controls testing odor aversion, for example. For the Gr5a mutant, few details are provided on the nature of the control line used and whether it is in the same genetic background as the mutant. Regardless, it would be important to verify that the Gr5a mutant retains a normal sense of smell and shows normal levels of PER to stimuli other than sugar, ruling out more general deficits. Finally, as the method of using DeepLabCut tracking to quantify PER was newly developed, it is important to show the accuracy and specificity of detecting PER events compared to manual scoring.

(8) The authors' explanation of why both attractive and aversive odors promote PER (lines 249-259) did not seem convincing. The explanation discusses the different roles of smell and taste but does not address the core question of why it would be adaptive for an aversive odor, which flies naturally avoid, to promote feeding behavior.

Reviewer #1 (Public review):

Summary:

Here the authors address how reinforcement-based sensorimotor adaptation changes throughout development. To address this question, they collected many participants in ages that ranged from small children (3 years old) to adulthood (18+ years old). The authors used four experiments to manipulate whether binary and positive reinforcement was provided probabilistically (e.g., 30 or 50%) versus deterministically (e.g.,100%), and continuous (infinite possible locations) versus discrete (binned possible locations) when the probability of reinforcement varied along the span of a large redundant target. The authors found that both movement variability and the extent of adaptation changed with age.

Strengths:

The major strength of the paper is the number of participants collected (n = 385). The authors also answer their primary question, that reinforcement-based sensorimotor adaptation changes throughout development, which was shown by utilizing established experimental designs and computational modelling.

Weaknesses:

Potential concerns involve inconsistent findings with secondary analyses, current assumptions that impact both interpretation and computational modelling, and a lack of clearly stated hypotheses.

(1) Multiple regression and Mediation Analyses.

The challenge with these secondary analyses is that:<br /> (a) The results are inconsistent between Experiments 1 and 2, and the analysis was not performed for Experiments 3 and 4,<br /> (b) The authors used a two-stage procedure of using multiple regression to determine what variables to use for the mediation analysis, and<br /> (c) The authors already have a trial-by-trial model that is arguably more insightful.

Given this, some suggested changes are to:<br /> (a) Perform the mediation analysis with all the possible variables (i.e., not informed by multiple regression) to see if the results are consistent.<br /> (b) Move the regression/mediation analysis to Supplementary, since it is slightly distracting given current inconsistencies and that the trial-by-trial model is arguably more insightful.

(2) Variability for different phases and model assumptions:

A nice feature of the experimental design is the use of success and failure clamps. These clamped phases, along with baseline, are useful because they can provide insights into the partitioning of motor and exploratory noise. Based on the assumptions of the model, the success clamp would only reflect variability due to motor noise (excludes variability due to exploratory noise and any variability due to updates in reach aim). Thus, it is reasonable to expect that the success clamps would have lower variability than the failure clamps (which it obviously does in Figure 6), and presumably baseline (which provides success and failure feedback, thus would contain motor noise and likely some exploratory noise).

However, in Figure 6, one visually observes greater variability during the success clamp (where it is assumed variability only comes from motor noise) compared to baseline (where variability would come from:<br /> (a) Motor noise.<br /> (b) Likely some exploratory noise since there were some failures.<br /> (c) Updates in reach aim.

Given the comment above, can the authors please:<br /> (a) Statistically compare movement variability between the baseline, success clamp, and failure clamp phases.<br /> (b) The authors have examined how their model predicts variability during success clamps and failure clamps, but can they also please show predictions for baseline (similar to that of Cashaback et al., 2019; Supplementary B, which alternatively used a no feedback baseline)?<br /> (c) Can the authors show whether participants updated their aim towards their last successful reach during the success clamp? This would be a particularly insightful analysis of model assumptions.<br /> (d) Different sources of movement variability have been proposed in the literature, as have different related models. One possibility is that the nervous system has knowledge of 'planned (noise)' movement variability that is always present, irrespective of success (van Beers, R. J. (2009). Motor learning is optimally tuned to the properties of motor noise. Neuron, 63(3), 406-417). The authors have used slightly different variations of their model in the past. Roth et al (2023) directly compared several different plausible models with various combinations of motor, planned, and exploratory noise (Roth A, 2023, "Reinforcement-based processes actively regulate motor exploration along redundant solution manifolds." Proceedings of the Royal Society B 290: 20231475: see Supplemental). Their best-fit model seems similar to the one the authors propose here, but the current paper has the added benefit of the success and failure clamps to tease the different potential models apart. In light of the results of a), b), and c), the authors are encouraged to provide a paragraph on how their model relates to the various sources of movement variability and other models proposed in the literature.<br /> (e) line 155. Why would the success clamp be composed of both motor and exploratory noise? Please clarify in the text

(3) Hypotheses:

The introduction did not have any hypotheses of development and reinforcement, despite the discussion above setting up potential hypotheses. Did the authors have any hypotheses related to why they might expect age to change motor noise, exploratory noise, and learning rates? If so, what would the experimental behaviour look like to confirm these hypotheses? Currently, the manuscript reads more as an exploratory study, which is certainly fine if true, it should just be explicitly stated in the introduction. Note: on line 144, this is a prediction, not a hypothesis. Line 225: this idea could be sharpened. I believe the authors are speaking to the idea of having more explicit knowledge of action-target pairings changing behaviour.

Reviewer #1 (Public review):

Summary:

Sidarta-Oliveira et al. present TopOMetry, a novel dimensionality reduction method based on the eigendecomposition of approximated Laplace-Beltrami Operator. Shortly, TopOMetry is an iterative version of the existing spectral methods (e.g., Laplacian Eigenmap or Diffusion map). It approximates the Laplacian operators twice, once in a "phenotypic space" and then once again in the eigenbases space. By doing this the approximated operator will contain more information of the manifold, which allows for more robust and accurate downstream analyses.

Strengths:

(1) The approach was rigorously tested based on synthetic and real single-cell RNA-seq datasets.

(2) The package is well-made and easily scalable to millions of cells.

(3) The comprehensive documentation helps the end-users to run desired analyses.

Weaknesses:

(1) The method is an extension of the current state-of-art methods, not a fundamentally new one.

(2) Considering the target readers, the paper contains a lot of jargon.

Reviewer #1 (Public review):

Summary:

The signaling pathways regulating the immune response to bacteria and fungi have been well characterized in Drosophila. Using the recently identified anti-parasitoid effector Lectin24A as a read-out, this article describes the signaling pathways regulating the humoral response against parasites.

Strengths:

This study reveals a role of JAK-STAT, Toll, and GATA in the fat body in the regulation of Lectin24A. They also observe an enrichment of binding sites for NF-kB, STAT, and GATA factors upstream of ORFs of genes induced upon encapsulation. Based on this observation, they generalize their findings on the involvement of JAK-STAT, Toll, and Gata in the humoral response to encapsulation. Although roles for the Toll and JAK-STAT pathways in capsule formation have previously been identified, the merit of this article is in analyzing the roles of these pathways in the humoral response using a new gene readout that will be a precious tool in the community.

Weaknesses:

The data are mostly convincing, but not always analyzed with sufficient detail; their conclusions should be reinforced by monitoring Lectin24A gene expression by RT-qPCR, by adding additional time points and by using alternative genetic tools. Using read-outs of the Toll and Imd pathways as comparisons is also important. Thus, this paper is interesting and important in advancing our understanding of Drosophila immunity but not yet enough solid to reach definitive conclusions on the proposed claims.

DOI: 10.1038/s41419-024-06886-1

Resource: RRID:Addgene_81070

Curator: @olekpark

SciCrunch record: RRID:Addgene_81070

What is this?

DOI: 10.1186/s13041-024-01134-1

Resource: RRID:Addgene_92284

Curator: @olekpark

SciCrunch record: RRID:Addgene_92284

What is this?

DOI: 10.1038/s44161-024-00431-1

Resource: RRID:Addgene_73178

Curator: @olekpark

SciCrunch record: RRID:Addgene_73178

What is this?

DOI: 10.1038/s41598-024-70853-1

Resource: RRID:Addgene_26646

Curator: @olekpark

SciCrunch record: RRID:Addgene_26646

What is this?

DOI: 10.1038/s41467-024-51273-1

Resource: Addgene_19506

Curator: @olekpark

SciCrunch record: RRID:Addgene_19506

What is this?

DOI: 10.1038/s41420-024-02158-1

Resource: RRID:Addgene_89308

Curator: @olekpark

SciCrunch record: RRID:Addgene_89308

What is this?

DOI: 10.1038/s44320-024-00058-1

Resource: RRID:Addgene_128263

Curator: @olekpark

SciCrunch record: RRID:Addgene_128263

What is this?

Tiku, N., Schaul, K., & Chen, S. Y. (2023, Nov 12). AI-generated images show a world of stereotypes. The Washington Post Retrieved from https://ezproxy.lib.uwm.edu/login?url=https://www.proquest.com/newspapers/ai-generated-images-show-world-stereotypes/docview/2888705247/se-2

Melissa H. "These new tools let you see for yourself how biased AI image models are," https://www.technologyreview.com/2023/03/22/1070167/these-news-tool-let-you-see-for-yourself-how-biased-ai-image-models-are, found by using Google.

Reviewer #1 (Public review):

Summary:

Tian et al. investigated the effects of emotional signals in biological motion on pupil responses. In this study, subjects were presented with point-light biological motion stimuli with happy, neutral, and sad emotions. Their pupil responses were recorded with an eye tracker. Throughout the study, emotion type (i.e., happy/sad/neutral) and BM stimulus type (intact/inverted/non-BM/local) were systematically manipulated. For intact BM stimuli, happy BM induced a larger pupil diameter than neutral BM, and neutral BM also induced a larger pupil diameter than sad BM. Importantly, the diameter difference between happy and sad BM correlated with the autistic trait of individuals. These effects disappeared for the inverted BM and non-BM stimuli. Interestingly, both happy and sad emotions show superiority in pupil diameter.

Strengths:

(1) The experimental conditions and results are very easy to understand.<br /> (2) The writing and data presentation are clear.<br /> (3) The methods are sound. I have no problems with the experimental design and results.

Reviewer #1 (Public Review):

The authors use a previously established reporter comprising a slow- and a fast-folding fluorescent protein fused to a randomly-generated library of penta-peptides at its amino-terminus and a signal sequence for import into the endoplasmic reticulum (ER). They then determine the stability of these constructs in a high throughput FACS-sorting procedure and identify a set of peptides that route the construct to proteasomal degradation. Increasing the copy number of one of these peptides further decreases the stability of the construct. This polypeptide resembles a "degron" for ER proteins, because it also targets other ER proteins with different topological and folding properties for degradation. It only works when placed at the amino-terminus of a protein and utilizes components of the Hrd1 ubiquitin ligase complex, a well-established quality control ubiquitin ligase in the ER membrane. Importantly, the degron also targets ER-proteins in mammalian cells.

The authors convincingly show that fusion of their newly identified degron to the amino terminus of ER-resident proteins with different topology suffices to target them for proteasomal degradation. The data for this are well-founded and contain appropriate controls. While technically sound, the study does only give superficial information on general properties of the degron and its recognition by cellular factors. Further simple experiments would have addressed a number of important points. The authors only provide data about the composition of the identified amino acid sections from the high-throughput approach and the statistical preference for certain amino acids at individual positions. They do not study degron composition experimentally by substituting individual amino acids with other residues and analyzing protein stability. Increasing the numbers of the initially identified degron pentamer increases substrate turnover, but the basis for this remains unclear. Each copy may be actively involved in better recognition, elongation of the degron may facilitate accessibility by recognition factors or multiplying the short amino acid stretch may generate new signatures at the amino-terminus that are more readily recognized by a quality control machinery. Consequently, this study does not allow conclusions to be drawn about general properties of degron composition and/or structure. The degron also functions with cytoplasmic proteins, suggesting that similar characteristics of a polypeptide attract the attention of quality control systems also in other cellular compartments. However, the authors did not pursue this finding further, e.g. by identifying factors for degron recognition in the cytoplasm. It would have been particularly interesting to test whether the degron would initiate degradation when placed at cytoplasmically-exposed amino termini of membrane-bound ER proteins. Information on degron properties is required to better understand principles of substrate recognition by protein quality control pathways and to design constructs for targeting endogenous proteins via proteolysis targeting chimeras (PROTACs).

Reviewer 1 (Public Review):

Multiple sclerosis (MS) is a debilitating autoimmune disease that causes loss of myelin in neurons of the central nervous system. MS is characterized by the presence of inflammatory immune cells in several brain regions as well as the brain barriers (meninges). This study aims to understand the local immune hallmarks in regions of the brain parenchyma that are adjacent to the leptomeninges in a mouse model of MS. The leptomeninges are known to be a foci of inflammation in MS and perhaps "bleed" inflammatory cells and molecules to adjacent brain parenchyma regions. To do so, they use novel technology called spatial transcriptomics so that the spatial relationships between the two regions remain intact. The study identifies canonical inflammatory genes and gene sets such as complement and B cells enriched in the parenchyma in close proximity to the leptomeninges in the mouse model of MS but not control. The manuscript is very well written and easy to follow. The results will become a useful resource to others working in the field and can be followed by time series experiments where the same technology can be applied to the different stages of the disease.

Reviewer #1 (Public Review):

This is a technically sound paper focused on a useful resource around the DRGP phenotypes which the authors have curated, pooled, and provided a user-friendly website. This is aimed to be a crowd-sourced resource for this in the future. The authors should make sure they coordinate as well as possible with the NC datasets and community and broader fly community.

Reviewer #1 (Public Review):

Kainate receptors play various important roles in synaptic transmission. The receptors can be divided into low affinity kainate receptors (GluK1-3) and high affinity kainate receptos (GluK4-5). The receptors can assemble as homomers (GluK1-3) or low-high affinity heteromers (GluK4-5). The functional diversity is further increased by RNA splicing. Previous studies have investigated C-terminal splice variants of GluK1, but GluK1 N-terminal (exon 9) insertions have not been previously characterized. In this study Dhingra et al investigate the functional implications of a GluK1 splice variant that inserts a 15 amino acid segment into the extracellular N-terminal region of the protein using whole-cell and excised outside-out electrophysiology.

The authors convincingly show that the insertion profoundly impacts the function of GluK1-1a - the channels that have the insertion are slower to desensitize. The data also shows that the insertion changes the modulatory effects of Neto proteins, resulting in altered rates of desensitization and recovery from desensitization. To determine the mechanism by which the insertion exerts these functional effects, the authors perform pull-down assays of Neto proteins, and extensive mutagenesis on the insert.<br /> The electrophysiological part of the study is very rigorous and meticulous.

The biggest weakness of the manuscript is the structural work. Due to issues with preferred orientation (a common problem in cryo-EM), the 3D reconstructions are at a low resolution (in the 5-8 Å range) and cannot offer much mechanistic insight into the effects of the insertion. The authors have opted to keep this data unchanged in the revised manuscript.

Despite this, the study is a valuable contribution to the field because it characterizes a GluK1 variant that has not been studied before and highlights the functional diversity that exists within the kainate receptor family.

Reviewer #1 (Public Review):

This manuscript addresses the regulation of the osmosensing protein kinases, WNK1 and WNK3. Prior work by the authors has shown that these enzymes are activated by PEG400 or ethylene glycol and inhibited by chloride ion, and that activation is associated with a conformational transition from dimer to monomer. In X-ray structures of the WNK1/SA inactive dimer, a water-mediated hydrogen bond network was observed between the catalytic loop (CL) and the activation loop (AL), named CWN1. This led to the proposal that bound water may be part of the osmosensing mechanism.

The current study carries this work further, by applying PEG400 to Xtals of dimeric WNK1/SA. This results in a change in kinase conformation and space group, along with 4-9 fewer waters in CWN1 and the complete disappearance of another water cluster (CWN2) located at the dimer interface. Six conserved residues lining the CWN1 pocket in WNK3 are mutated to determine effects on activity and inhibition by chloride ion (measured by AL autophosphorylation) and monomer-dimer interconversion (light scattering).

The results show that two mutants (E314Q/A in WNK3) at a site central to the water cluster result in increased kinase activity (autophosphorylation), and increased SLS, interpreted as aggregation. Three sites (D279A, Y346F, M301A) inhibit kinase activity with varying effects on oligomerization - Y346A and M301A retain monomer-dimer ratios similar to WT while D279N promotes aggregation. K236A and K307A show activity and monomer:dimer ratios similar to WT. Selected mutants (E314Q, D279N, Y346F) and WT appear to retain osmosensitivity with comparable activation by PEG400.

The study concludes that osmolytes may activate the kinase by removing waters from the CWN1 and CWN2 clusters, suggesting that waters might be considered allosteric ligands that promote the inactive structure of WNKs. The differing effects of mutations may be ascribed to disruption of the water networks as well as inhibitory perturbations at the active site.

Comments on latest version:

The revised manuscript incorporated new experiments that satisfactorily addressed my concerns.

Reviewer #1 (Public review):

The authors construct a pair of E. coli populations that differ by a single gene duplication in a selectable fluorescent protein. They then evolve the two populations under differing selective regimes to assess whether the end result of the selective process is a "better" phenotype when starting with duplicated copies. Importantly, their starting duplicated population is structured to avoid the duplication-amplification process often seen in bacterial artificial evolution experiments. They find that while duplication increases robustness and speed of adaptation, it does not result in more highly adapted final states, in contrast to Ohno's hypothesis.

Comments on revised version:

The authors have addressed my prior concerns, and I have no further comments on the manuscript.

Reviewer #3 (Public review):

Summary:

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

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

Reviewer #1 (Public review):

Summary:

The authors were seeking to define the roles of the Drosophila caspar gene in embryonic development and primordial germ cell (PGC) formation. They demonstrate that PGC number, and the distribution of the germ cell determinant Oskar, change as a result of changes in caspar expression; reduction of caspar reduces PGC number and the domain of Oskar protein expression, while overexpression of caspar does the reverse. They also observe defects in syncytial nuclear divisions in embryos produced from caspar mutant mothers. Previous work from the same group demonstrated that Caspar protein interacts with two partners, TER94 and Vap33. In this paper, they show that maternal knockdown of TER94 results in embryonic lethality and some overlap of phenotypes with reduction of caspar, supporting the idea may work together in their developmental roles. The authors propose models for how Caspar might carry out its developmental functions. The most specific of these is that Caspar and its partners might regulate oskar mRNA stability by recruiting ubiquitin to the translational regulator Smaug.

Strengths:

The work identifies a new factor that is involved in PGC specification and points toward an additional pathway that may be involved in establishing and maintaining an appropriate distribution of Oskar at the posterior pole of the embryo. It also ties together earlier observations about the presence of TER94 in the pole plasm that have not heretofore been linked to a function.

Weaknesses:

(1) A PiggyBac insertion allele casp[c04227] is used throughout the paper and referred to as a loss-of-function allele (casp[lof]). While the authors avoid the terms 'null' or 'amorph' and on one occasion refer to the allele as a 'strong hypomorph', nevertheless terming it a 'loss-of-function' allele is misleading. This is because the phenotype of the allele when homozygous is different from the phenotype produced when heterozygous over a deficiency.

(2) The peptide counts in the mass spectrometry experiment aimed at finding protein partners for Casp are extremely low, except for Casp itself and TER94. Peptide counts of 1-2 seem to me to be of questionable significance.

(3) The pole bud phenotypes from TER94 knockdown and casp mutant shown in Fig 5 appear to be quite different. These differences are unexplained and seem inconsistent with the model proposed that the two proteins work in a common pathway. Whole embryos should also be shown, as the TER94 KD phenotype could result from a more general dysmorphism.

(4) Fig 6 is not quantitative, lacking even a second control staining to check for intensity variation artifacts. Therefore it shows that the distribution of Oskar protein changes in the various genotypes, but not convincingly that the level of Oskar changes as the paper claims.

(5) The error bars are huge in the graphs in Fig 7H, I, and J, and in fact these changes are not statistically significant. Therefore the conclusion that 'Reduction in Casp activity specifically affects Smaug degradation during the MZT' is not supported by the data in this figure.

Reviewer #1 (Public review):

Summary:

The manuscript studies nutrient intake rates for stationary and motile microorganisms to assess the effectiveness of swim vs. stay strategies. This work provides valuable insights on how the different strategies perform in the context of a simplified mathematical model that couples hydrodynamics to nutrient advection and diffusion. The swim and stay strategies are shown to yield similar nutrient flux under a range of conditions.

Strengths:

Strengths of the work include (i) the model prediction in Fig. 3 of nutrient flux applied to a range of microorganisms including an entire clade that are known to use different feeding strategies and (ii) a study of the interaction between cilia and absorption coverage showing the robustness of their predictions provided these regions have sufficient overlap.

Weaknesses:

In the revision, the authors have adequately addressed the weaknesses I raised in the first round of review.

Reviewer #2 (Public review):

Summary:

This computational modeling study addresses the observation that variable observations are interpreted differently depending on how much uncertainty an agent expects from its environment. That is, the same mismatch between a stimulus and an expected stimulus would be less significant, and specifically would represent a smaller prediction error, in an environment with a high degree of variability than in one where observations have historically been similar to each other. The authors show that if two different classes of inhibitory interneurons, the PV and SST cells, (1) encode different aspects of a stimulus distribution and (2) act in different (divisive vs. subtractive) ways, and if (3) synaptic weights evolve in a way that causes the impact of certain inputs to balance the firing rates of the targets of those inputs, then pyramidal neurons in layer 2/3 of canonical cortical circuits can indeed encode uncertainty-modulated prediction errors. To achieve this result, SST neurons learn to represent the mean of a stimulus distribution and PV neurons its variance.

The impact of uncertainty on prediction errors in an understudied topic, and this study provides an intriguing and elegant new framework for how this impact could be achieved and what effects it could produce. The ideas here differ from past proposals about how neuronal firing represents uncertainty. The developed theory is accompanied by several predictions for future experimental testing, including the existence of different forms of coding by different subclasses of PV interneurons, which target different sets of SST interneurons (as well as pyramidal cells). The authors are able to point to some experimental observations that are at least consistent with their computational results. The simulations shown demonstrate that if we accept its assumptions, then the authors' theory works very well: SSTs learn to represent the mean of a stimulus distribution, PVs learn to estimate its variance, firing rates of other model neurons scale as they should, and the level of uncertainty automatically tunes the learning rate, so that variable observations are less impactful in a high uncertainty setting.

Strengths:

The ideas in this work are novel and elegant, and they are instantiated in a progression of simulations that demonstrate the behavior of the circuit. The framework used by the authors is biologically plausible and matches some known biological data. The results attained, as well as the assumptions that go into the theory, provide several predictions for future experimental testing. The authors have taken into account earlier review comments to revise their paper in ways that enhance its clarity.

Weaknesses:

One weakness could be that the proposed theory does rely on a fairly large number of assumptions. However, there is at least some biological support for these. Importantly, the authors do lay out and discuss their key assumptions in the Discussion section, so readers can assess their validity and implications for themselves.

Joint Public Review:

Summary:

This study presents a strategy to efficiently isolate PcrV-specific BCRs from human donors with cystic fibrosis who have/had Pseudomonas aeruginosa (PA) infection. Isolation of mAbs that provide protection against PA may be a key to developing a new strategy to treat PA infection as the PA has intrinsic and acquired resistance to most antibiotic drug classes. Hale et al. developed fluorescently labeled antigen-hook and isolated mAbs with anti-PA activity. Overall, the authors' conclusion is supported by solid data analysis presented in the paper. Four of five recombinantly expressed PcrV-specific mAbs exhibited anti-PA activity in a murine pneumonia challenge model as potent as the V2L2MD mAb (equivalent to gremubamab). However, therapeutic potency for these isolated mAbs is uncertain as the gremubamab has failed in Phase 2 trials. Clarification of this point would greatly benefit this paper.

Strengths:

(1) High efficiency of isolating antigen-specific BCRs using an antigenic hook.

(2) The authors' conclusion is supported by data.

Weaknesses:

Although the authors state that the goal of this study was to generate novel protective mAbs for therapeutic use (P12; Para. 2), it is unclear whether PcrV-specific mAbs isolated in this study have therapeutic potential better than the gremubamab, which has failed in Phase 2 trials. Four of five PcrV-specific mAbs isolated in this study reduced bacterial burdens in mice as potent as, but not superior to, gremubamab-equivalent mAb. Clarification of this concern by revising the text or providing experimental results that show better potential than gremubamab would greatly benefit this paper.

Reviewer #1 (Public review):

Summary:

Animals in natural environments need to identify predator-associated cues and respond with the appropriate behavioral response to survive. In rodents, some chemical cues produced by predators (e.g., cat saliva) are detected by chemosensory neurons in the vomeronasal organ (VNO). The VNO transmits predator-associated information to the accessory olfactory bulb, which in turn projects to the medial amygdala and the bed nucleus of the stria terminalis, two regions implicated in the initiation of antipredator defensive behaviors. A downstream area to these two regions is the ventromedial hypothalamus (VMH), which has been shown to control both active (i.e., flight) and passive (i.e, freezing) antipredator defensive responses via distinct efferent projections to the anterior hypothalamic nucleus or the periaqueductal gray, respectively. However, whether differences in predator-associated sensory information initially processed in the VNO and further conveyed to the VMH can trigger different types of behavioral responses remained unexplored. To address this question, here the authors investigated the behavioral responses of mice exposed to either fresh or old cat saliva, and further compared the underlying neural circuits that are activated by cat saliva with different freshness.

The scientific question of the study is valid, the experiments were well-performed, and the statistical analyses are appropriate. However, there are some concerns that may directly affect the main interpretation of the results.

In this revised version of the manuscript, the authors have made important modifications in the text, inserted new experiments and performed additional data analyses, as recommended. These modifications have significantly improved the quality of the manuscript and addressed all the major concerns detected during the prior submission.

Reviewer #1 (Public review):

This manuscript from Schwintek and coworkers describes a system in which gas flow across a small channel (10^-4-10^-3 m scale) enables the accumulation of reactants and convective flow. The authors go on to show that this can be used to perform PCR as a model of prebiotic replication.

Strengths:

The manuscript nicely extends the authors' prior work in thermophoresis and convection to gas flows. The demonstration of nucleic acid replication is an exciting one, and an enzyme-catalyzed proof-of-concept is a great first step towards a novel geochemical scenario for prebiotic replication reactions and other prebiotic chemistry.

The manuscript nicely combines theory and experiment, which generally agree well with one another, and it convincingly shows that accumulation can be achieved with gas flows and that it can also be utilized in the same system for what one hopes is a precursor to a model prebiotic reaction. This continues efforts from Braun and Mast over the last 10-15 years extending a phenomenon that was appreciated by physicists and perhaps underappreciated in prebiotic chemistry to increasingly chemically relevant systems and, here, a pilot experiment with a simple biochemical system as a prebiotic model.

I think this is exciting work and will be of broad interest to the prebiotic chemistry community.

Weaknesses:

The manuscript states: "The micro scale gas-water evaporation interface consisted of a 1.5 mm wide and 250 µm thick channel that carried an upward pure water flow of 4 nl/s ≈ 10 µm/s perpendicular to an air flow of about 250 ml/min ≈ 10 m/s." This was a bit confusing on first read because Figure 2 appears to show a larger channel - based on the scale bar, it appears to be about 2 mm across on the short axis and 5 mm across on the long axis. From reading the methods, one understands the thickness is associated with the Teflon, but the 1.5 mm dimension is still a bit confusing (and what is the dimension in the long axis?) It is a little hard to tell which portion (perhaps all?) of the image is the channel. This is because discontinuities are present on the left and right sides of the experimental panels (consistent with the image showing material beyond the channel), but not the simulated panels. Based on the authors' description of the apparatus (sapphire/CNC machined Teflon/sapphire) it sounds like the geometry is well-known to them. Clarifying what is going on here (and perhaps supplying the source images for the machined Teflon) would be helpful.

The data shown in Figure 2d nicely shows nonrandom residuals (for experimental values vs. simulated) that are most pronounced at t~12 m and t~40-60m. It seems like this is (1) because some symmetry-breaking occurs that isn't accounted for by the model, and perhaps (2) because of the fact that these data are n=1. I think discussing what's going on with (1) would greatly improve the paper, and performing additional replicates to address (2) would be very informative and enhance the paper. Perhaps the negative and positive residuals would change sign in some, but not all, additional replicates?

The authors will most likely be familiar with the work of Victor Ugaz and colleagues, in which they demonstrated Rayleigh-Bénard-driven PCR in convection cells (10.1126/science.298.5594.793, 10.1002/anie.200700306). Not including some discussion of this work is an unfortunate oversight, and addressing it would significantly improve the manuscript and provide some valuable context to readers. Something of particular interest would be their observation that wide circular cells gave chaotic temperature profiles relative to narrow ones and that these improved PCR amplification (10.1002/anie.201004217). I think contextualizing the results shown here in light of this paper would be helpful. Again, it appears n=1 is shown for Figure 4a-c - the source of the title claim of the paper - and showing some replicates and perhaps discussing them in the context of prior work would enhance the manuscript.

I think some caution is warranted in interpreting the PCR results because a primer-dimer would be of essentially the same length as the product. It appears as though the experiment has worked as described, but it's very difficult to be certain of this given this limitation. Doing the PCR with a significantly longer amplicon would be ideal, or alternately discussing this possible limitation would be helpful to the readers in managing expectations.

DOI: 10.1038/s41598-018-20394-1

Resource: RRID:BDSC_32219

Curator: @maulamb

SciCrunch record: RRID:BDSC_32219

What is this?

Reviewer #1 (Public review):

Summary:

Lejeune et al. demonstrated sex-dependent differences in the susceptibility to MRSA infection. The authors demonstrated the role of the microbiota and sex hormones as potential determinants of susceptibility. Moreover, the authors showed that Th17 cells and neutrophils contribute to sex hormone-dependent protection in female mice.

Strengths:

The role of microbiota was examined in various models (gnotobiotic, co-housing, microbiota transplantation). The identification of responsible immune cells was achieved using several genetic knockouts and cell-specific depletion models. The involvement of sex hormones was clarified using ovariectomy and the FCG model.

Weaknesses:

The mechanisms by which specific microbiota confer female-specific protection remain unclear.

Reviewer #1 (Public review):

Summary:

The authors measured glutamate transients in the DMS of rats as they performed an action selection task. They identified diverse patterns of behavior and glutamate dynamics depending on the pre-existing behavioral phenotype of the rat (sign tracker or goal tracker). Using pathway-specific DREADDs, they showed that these behavioral phenotypes and their corresponding glutamate transients were differentially dependent on input from the prelimbic cortex to the DMS.

Strengths:

Overall there are some very interesting results that make an important contribution to the field. Notably, the results seem to point to differential recruitment of the PL-DMS pathway in goal-tracking vs sign-tracking behaviors.

Weaknesses:

There is a lot of missing information and data that should be reported/presented to allow a complete understanding of the findings and what was done. The writing of the manuscript was mostly quite clear, however, there are some specific leaps in logic that require more elaboration, and the focus at the start and end on cholinergic neurons and Parkinson's disease are, at the moment, confusing and require more justification.

Reviewer #1 (Public review):

The authors found that the loss of cell-ECM adhesion leads to the formation of giant monocular vacuoles in mammary epithelial cells. This process takes place in a macropinocytosis-like process and involves PI3 kinase. They further identified dynamin and septin as essential machinery for this process. Interestingly, this process is reversible and appears to protect cells from cell death.

Strengths: The data are clean and convincing to support the conclusions. The analysis is comprehensive, using multiple approaches such as SIM and TEM. The discussion on lactation is plausible and interesting.

Weaknesses: As the first paper describing this phenomenon, it is adequate. However, the elucidation of the molecular mechanisms is not as exciting as it does not describe anything new. It is hoped that novel mechanisms will be elucidated in the future. Especially the molecules involved in the reversing process could be quite interesting.

Reviewer #2 (Public review):

Summary:

This very interesting study originated from a serendipitous observation that the deletion of the disordered N-terminal tail of human SUMO1 enhances its binding to its interaction partners. This suggested that the N terminus of SUMO1 might be an intrinsic competitive inhibitor of SUMO-interacting motif (SIM) binding to SUMO1. Subsequent experiments support this mechanism, showing that in humans it is specific to SUMO1 and does not extend to SUMO2 or SUMO3 (except, perhaps, when the N terminus of SUMO2 becomes phosphorylated, as the authors intriguingly suggest - and partially demonstrate). The auto-inhibition of SUMO1 via its N-terminal tail apparently explains lower binding of SUMO1 compared to SUMO2 to some SIMs and lower SIM-dependent SUMOylation of some substrates with SUMO1 compared to SUMO2, thus adding an important element to the puzzle of SUMO paralogue preference. In line with this explanation, N-terminally truncated SUMO1 was equally efficient to SUMO2 in the studied cases. The inhibitory role of SUMO1's N terminus appears conserved in other species including S. cerevisiae and C. elegans, both of which contain only one SUMO. The study also elucidates the molecular mechanism by which the disordered N-terminal region of SUMO1 can exert this auto-inhibitory effect. This appears to depend on the transient, very highly dynamic physical interaction between the N terminus and the surroundings of the SIM-binding groove based mostly on electrostatic interactions between acidic residues in the N terminus and basic residues around the groove.

Strengths:

A key strength of this study is the interplay of different techniques, including biochemical experiments, NMR, molecular dynamics simulations, and, at the end, in vivo experiments. The experiments performed with these different techniques inform each other in a productive way and strengthen each others' conclusions. A further strength is the detailed and clear text, which patiently introduces, describes, and discusses the study. Finally, in terms of the message, the study has a clear, mechanistic message of fundamental importance for various aspects of the SUMO field, and also more generally for protein biochemists interested in the functional importance of intrinsically disordered regions. In revision, the authors have further improved the text.

Weaknesses:

In the future, further experimental validation will be required, particularly with regards to the biological importance of the uncovered mechanism. These limitations are satisfactorily pointed out by the authors themselves in the revised manuscript.

Reviewer #1 (Public review):

Summary:

In this study, Abidi and colleagues used Notch pathway neutralizing antibodies to inhibit sebaceous glands in the skin. The authors find that blocking either the Notch1 receptor or the Jag2 ligand caused loss of the glands and increased retention of sebaceous progenitor cells. Moreover, these glands began to reappear 14 days after treatment.

Strengths:

Overall, this study definitively identifies the Notch receptor/ligand combination that maintains these glands in the adult. The manuscript is clearly written and the figures are beautifully made.

In this resubmitted manuscript, the authors have adequately addressed all the previous critiques.

Reviewer #2 (Public review):

Summary:

The preprint by Fawaz et al. presents the findings of a study that aimed to assess the relationship between somatic mutations associated with clonal hematopoiesis (CHIP) and the prevalence of myocardial infarction (MI). The authors conducted targeted DNA sequencing analyses on samples from 149 MI patients and 297 non-MI controls from a separate cohort. Additionally, they investigated the impact of the loss of the Y chromosome (LOY), another somatic mutation frequently observed in clonally expanded blood cells. The results of the study primarily demonstrate no significant associations, as neither CHIP nor LOY were found to be correlated with an increased prevalence of MI. The null findings regarding CHIP are partly in conflict with several larger studies in the literature. However, it must be noted that the authors did find trends to an association between CHIP and a higher incidence of MI during follow-up among those without a history of MI at baseline, which is more consistent with previous research work. The association with incident MI reached statistical significance in men, particularly in those not showing LOY, suggesting potential interactions between different clonally-expanded somatic mutations.

Strengths:

Overall, this is a useful research work on an emerging risk factor for cardiovascular disease (CVD). The use of a targeted sequencing approach is a strength, as it offers higher sensitivity than the whole exome sequencing approaches used in many previous studies. Reporting null findings is definitely relevant in an emerging field such as the role of somatic mutations in cardiovascular disease.

Weaknesses:

The study suffers from important limitations, which cast some doubts onto the authors' conclusions, as detailed below:

(1) The small sample size of the study population is a critical limitation, particularly when reporting null findings that conflict (partly) with positive findings in much larger studies, totaling hundreds of thousands of individuals (e.g. Zekavat et al, Nature CVR 2023, Vlasschaert et al, Circulation 2023; Zhao et al, JAMA Cardio 2024). The authors claim that they have 90% power to detect an effect size of CHIP on MI comparable to that in previous reports (a hazard ratio of 1.7, mainly based on the findings by Jaiswal et al, NEJM 2014,2017). However, this analysis is simply based on the predicted prevalence of CHIP in MI(+) and MI(-) patients, and it does not consider the complex relationship between age CHIP and atherosclerotic disease. More advanced approaches to calculate statistical power may have provided a more accurate estimation. It must also be noted that recent work in much larger populations suggest that the overall effect of CHIP on atherosclerotic CVD is smaller than 1.7, most likely due to the heterogeneity of effects of different mutated genes (e.g. Zekavat et al, Nature CVR 2023, Vlasschaert et al, Circulation 2023; Zhao et al, JAMA Cardio 2024). In addition, several analyses in the current manuscript are conducted separately in MI(+) (n= 149) and MI(-) (N=297) individuals, further limiting statistical power. Power is even lower in the investigation of the effects of LOY and its interaction with CHIP, as only men are included in these analyses. Overall, I believe the study is underpowered from a statistical point of view, so the authors' findings need to be interpreted with caution.

(2) Related to the above, it is widely accepted that the effects of CHIP on CVD are highly heterogeneous, as some mutated genes appear to have a strong impact on atherosclerosis, whereas the effect of others is negligible (e.g. Zekavat et al, Nature CVR 2023, Vlasschaert et al, Circulation 2023, among others). TET2 mutations are frequently considered a "positive control", given the multiple lines of evidence suggesting that these mutations confer a higher risk of atherosclerotic disease. However, no association with MI or related variables was found for TET2 mutations in the current work, which likely reflects the limited statistical power of the study to assess accurately the effects of CHIP mutations on atherosclerotic disease.

(3) One of the most essential features of CHIP is the tight correlation with age. In this study, the effect of age on CHIP (e.g. Supp. Tables S5, S6) is statistically significant, but substantially milder than in previous studies. Given the relatively modest effect size of age on CHIP here, it is not surprising that no association with MI or atherosclerotic disease was found, considering that this association would have a much smaller effect size. It must be considered, however, that the advanced age of the population may have confounded the analysis of these relationships, as acknowledged by the authors.

(4) CHIP represents just one type of clonal hematopoiesis (e.g. see https://doi.org/10.1182/blood.2023022222). In this context, it must be noted that the mutated genes included in the definition of "CHIP" here are markedly different than in most previous studies, particularly when considering specifically the studies that demonstrated an association between CHIP and atherosclerotic CVD. For instance, the definition of CHIP in this manuscript includes genes such as ANKRD26, CALR, CCND2, DDX41... that are not prototypical CHIP genes. This is unlikely to have major impact on the main results, as the vast majority of mutations detected are indeed in bona fide CHIP genes, but it needs to be considered when interpreting the authors' findings. Furthermore, the strategy used here for CHIP variant calling and curation is substantially different than that used in previous studies. This is important, because such differences in the definition of CHIP and the curation of variants are at the basis of most conflicting findings in the literature regarding the effects of this condition. The authors estimate that the effect of these discrepancies on the definition of CHIP is limited, but small differences can have substantial impact in a study with limited sample size.

(5) A major limitation of the current study is the cross-sectional design of most of the analyses. For instance, it is not surprising that no association is found between CHIP and prevalent atherosclerosis burden by ultrasound imaging, considering that many individuals may have developed atherosclerosis years or decades before the expansion of the mutant clones, limiting the possible effect of CHIP on atherosclerosis burden. Similarly, the analysis of the relationship between CHIP and a history of MI may be confounded by the potential effects of MI on the expansion of mutant clones. In this context, it is noteworthy that the only positive results here are found in the analysis of the relationship between CHIP at baseline and incident MI development over follow-up. A larger sample size in these longitudinal analyses would provide deeper insights into the relationship between CHIP and MI.

Reviewer #1 (Public review):

Summary:

Bacteria exhibit species-specific numbers and localization patterns of flagella. How specificity in number and pattern is achieved is poorly understood but often depends on a soluble GTPase called FlhF. Here the authors take an unbiased protein-pulldown approach to identify a protein FipA in V. parahaemolyticus that interacts with FlhF. They show that FipA co-occurs with FlhF in the genomes of bacteria with polarly-localized flagella and study the role of FipA in three different bacteria: V. parahaemolyticus, S. purtefaciens, and P. putida. In each case, they show that FipA contributes to FlhF polar localization, flagellar assembly, flagellar patterning, and motility to different species-specific extents.

Strengths:

The authors perform a comprehensive analysis of FipA, including phenotyping of mutants, protein localization, localization dependence, and domains of FipA necessary for each. Moreover, they perform a time-series analysis indicating that FipA localizes to the cell pole likely prior to, or at least coincident with, flagellar assembly. They also show that the role of FipA appears to differ between organisms in detail but the overarching idea that it is a flagellar assembly/localization factor remains convincing.

Weaknesses:

For me the comparative analysis in the different organism was on balance, a weakness. By mixing the data for each of the organisms together, I found it difficult to read, and take away key points from the results. In its current form, the individual details seem to crowd out the model.

Reviewer #1 (Public Review):

Summary:

Zeng et al. have investigated the impact of inhibiting lactate dehydrogenase (LDH) on glycolysis and the tricarboxylic acid cycle. LDH is the terminal enzyme of aerobic glycolysis or fermentation that converts pyruvate and NADH to lactate and NAD+ and is essential for the fermentation pathway as it recycles NAD+ needed by upstream glyceraldehyde-3-phosphate dehydrogenase. As the authors point out in the introduction, multiple published reports have shown that inhibition of LDH in cancer cells typically leads to a switch from fermentative ATP production to respiratory ATP production (i.e., glucose uptake and lactate secretion are decreased, and oxygen consumption is increased). The presumed logic of this metabolic rearrangement is that when glycolytic ATP production is inhibited due to LDH inhibition, the cell switches to producing more ATP using respiration. This observation is similar to the well-established Crabtree and Pasteur effects, where cells switch between fermentation and respiration due to the availability of glucose and oxygen. Unexpectedly, the authors observed that inhibition of LDH led to inhibition of respiration and not activation as previously observed. The authors perform rigorous measurements of glycolysis and TCA cycle activity, demonstrating that under their experimental conditions, respiration is indeed inhibited. Given the large body of work reporting the opposite result, it is difficult to reconcile the reasons for the discrepancy. In this reviewer's opinion, a reason for the discrepancy may be that the authors performed their measurements 6 hours after inhibiting LDH. Six hours is a very long time for assessing the direct impact of a perturbation on metabolic pathway activity, which is regulated on a timescale of seconds to minutes. The observed effects are likely the result of a combination of many downstream responses that happen within 6 hours of inhibiting LDH that causes a large decrease in ATP production, inhibition of cell proliferation, and likely a range of stress responses, including gene expression changes.

Strengths:

The regulation of metabolic pathways is incompletely understood, and more research is needed, such as the one conducted here. The authors performed an impressive set of measurements of metabolite levels in response to inhibition of LDH using a combination of rigorous approaches.

Weaknesses:

Glycolysis, TCA cycle, and respiration are regulated on a timescale of seconds to minutes. The main weakness of this study is the long drug treatment time of 6 hours, which was chosen for all the experiments. In this reviewer's opinion, if the goal was to investigate the direct impact of LDH inhibition on glycolysis and the TCA cycle, most of the experiments should have been performed immediately after or within minutes of LDH inhibition. After 6 hours of inhibiting LDH and ATP production, cells undergo a whole range of responses, and most of the observed effects are likely indirect due to the many downstream effects of LDH and ATP production inhibition, such as decreased cell proliferation, decreased energy demand, activation of stress response pathways, etc.

Reviewer #1 (Public review):

Summary:

Lodhiya et al. demonstrate that antibiotics with distinct mechanisms of action, norfloxacin, and streptomycin, cause similar metabolic dysfunction in the model organism Mycobacterium smegmatis. This includes enhanced flux through the TCA cycle and respiration as well as a build-up of reactive oxygen species (ROS) and ATP. Genetic and/or pharmacologic depression of ROS or ATP levels protect M. smegmatis from norfloxacin and streptomycin killing. Because ATP depression is protective, but in some cases does not depress ROS, the authors surmise that excessive ATP is the primary mechanism by which norfloxacin and streptomycin kill M. smegmatis. In general, the experiments are carefully executed; alternative hypotheses are discussed and considered; the data are contextualized within the existing literature. Clarification of the effect of 1) ROS depression on ATP levels and 2) ADP vs. ATP on divalent metal chelation would strengthen the paper, as would discussion of points of difference with the existing literature. The authors might also consider removing Figures 9 and 10A-B as they distract from the main point of the paper and appear to be the beginning of a new story rather than the end of the current one. Finally, statistics need some attention.

Strengths:

The authors tackle a problem that is both biologically interesting and medically impactful, namely, the mechanism of antibiotic-induced cell death.

Experiments are carefully executed, for example, numerous dose- and time-dependency studies; multiple, orthogonal readouts for ROS; and several methods for pharmacological and genetic depletion of ATP.

There has been a lot of excitement and controversy in the field, and the authors do a nice job of situating their work in this larger context.

Inherent limitations to some of their approaches are acknowledged and discussed e.g., normalizing ATP levels to viable counts of bacteria.

Weaknesses:

The authors have shown that treatments that depress ATP do not necessarily repress ROS, and therefore conclude that ATP is the primary cause of norfloxacin and streptomycin lethality for M. smegmatis. Indeed, this is the most impactful claim of the paper. However, GSH and dipyridyl beautifully rescue viability. Do these and other ROS-repressing treatments impact ATP levels? If not, the authors should consider a more nuanced model and revise the title, abstract, and text accordingly.

Does ADP chelate divalent metal ions to the same extent as ATP? If so, it is difficult to understand how conversion of ADP to ATP by ATP synthase would alter metal sequestration without concomitant burst in ADP levels.

Some of the results in the paper diverge from what has been previously reported by some of the referenced literature. These discrepancies should be clarified.

Joint Public Review:

Summary:

This work provides a new general tool for predicting post-ERCP pancreatitis before the procedure depending on pancreatic calcification, female sex, intraductal papillary mucinous neoplasm, a native papilla of Vater, or the use of pancreatic duct procedures. Even though it is difficult for the endoscopist to predict before the procedure which case might have post-ERCP pancreatitis, this new model score can help with the maneuver and when the patient is at high risk of pancreatitis, sometimes can be deadly), so experienced endoscopists can do the procedure from the start. This paper provides a model for stratifying patients before the ERCP procedure into low, moderate, and high risk for pancreatitis. To be validated, this score should be done in many countries and on large numbers of patients. Risk factors can also be identified and added to the score to increase rank.

Strengths:

(1) One of the severe complications of endoscopic retrograde cholangiopancreatography procedure is pancreatitis, so investigators try all the time to find a score that can predict which patients will probably have pancreatitis after the procedure. Most scores depend on the intraprocedural maneuver. Some studies discuss the preprocedural score that can predict pancreatitis before the procure. This study discusses a new preprocedural score for post-ERCP pancreatitis.

(2) Depending on this score that identifies low, moderate, and high-risk patients for post-pancreatitis, so from the start, experienced and well-trained endoscopists can do the procedure or can refer patients to tertiary hospitals or use interventional radiology or endoscopic retrograde cholangiopancreatography.

(3) The number of patients in this study is sufficient to analyze data correctly.

Weaknesses:

(1) It is a single-country, retrospective study.

(2) Many cases were excluded, so the score cannot be applied to those patients.

(3) Many other studies, e.g., https://link.springer.com/article/10.1007/s00464-021-08491-1, https://pubmed.ncbi.nlm.nih.gov/36344369/, that have been published before discussing the same issue, so what is the new with this score?

(4) The discussion section needs reformulation to express the study's aim and results.

(5) Why did the authors select these items in their scoring system and did not add more variables?

Reviewer #1 (Public review):

Summary:

In this manuscript, Muramoto and colleagues have examined a mechanism by which the executioner caspase Drice is activated in a non-lethal context in Drosophila. The authors have comprehensively examined this in the Drosophila olfactory receptor neurons using sophisticated techniques. In particular, they had to engineer a new reporter by which non-lethal caspase activation could be detected. The authors conducted a proximity labeling experiment and identified Fasciclin 3 as a key protein in this context. While the removal of Fascilin 3 did not block non-lethal caspase activation (likely because of redundant mechanisms), its overexpression was sufficient to activate non-lethal caspase activation.

Strengths:

While non-lethal functions of caspases have been reported in several contexts, far less is known about the mechanisms by which caspases are activated in these non-lethal contexts. So, the topic is very timely. The overall detail of this work is impressive and the results for the most part are well-controlled and justified.

Weaknesses:

The behavioral results shown in Figure 6 need more explanation and clarification (more details below). As currently shown, the results of Figure 6 seem uninterpretable. Also, overall presentation of the Figures and description in legends can be improved.

Reviewer #1 (Public review):

Summary:

The authors of this valuable study use linearly polarized UV light rotating at different angular velocities to stimulate photoreceptors in bumblebees and study the response of TL3 neurons to polarized light. Previous work has typically used a single constant rotation velocity of the polarized light, while the authors of this study explore a range of constant rotational velocities spanning from 30deg/s to 1920deg/s. The authors also use linearly polarized UV light rotating at continuously varying velocities following the angular velocity of the head of a flying bumblebee. 

Strengths:

The authors investigate the neuronal responses of TL3 neurons to a variety of rotational velocities. This approach has the potential to reveal the neuronal response to dynamically changing stimuli experienced by the animal as it moves around its environment.

The authors make good use of physiology and modeling to validate their hypotheses and findings.  If done right, this line of investigation has the potential to provide a very useful methodology for utilizing more complex stimuli in studies of the visual pathway and central complex than traditionally. 

Weaknesses: 

The attempt of the authors to use more naturalistic stimuli than previous studies is very important, but the stimulus they use, i.e. linearly polarized UV light projected on the whole dorsal rim of the animal's eyes, is very different from the circular pattern of UV light polarization coming through the sky. In particular, as a bumblebee turns under the sky, the light projected on each ommatidium of the dorsal rim area will not smoothly change like the rotating linearly polarized light used in the experiments. The authors need to discuss this and other limitations of their study. 

The authors should also commend the light intensity confound common in polarized light setups as discussed by Reinhard Wolf et al, J. Comp. Physiol. 1980 and in the thesis of Peter Weir, California Institute of Technology, 2013. It is unclear whether the authors performed measurements to quantify the intensity pattern and if they took measures to compensate and make the polarized light intensity uniform. 

The authors show that the neuronal responses of TL3 neurons depend on the recent history of the polarized light stimulus. They use as evidence, the different neuronal firing rates measured when arriving at the same polarization stimulus by following two different preceding stimulus sequences. It would have been worthwhile to investigate to what extent the difference in neuronal response is due to the history alone and to what extent it is due to spike timing stochasticity inherent in the neurons. According to the raster plots in Figure 2F, there is substantial stochasticity in the timing of the action potential firing events.

The authors appear to base their delay calculations and analysis on the response of one single neuron (Figures 2 and 3) even though they have recorded the responses of several TL3 neurons. There is no reason for the authors not to use all neuron recordings in their calculations and analysis.

Another concern is that while the authors make good use of modeling, like any model, the presented models only partially explain the observed phenomena. However, a discussion about the limitations of their model needs to be provided.  Actually, observing the discrepancies between the model's output and the intracellular recordings reveals what the model is missing. That is, careful consideration of the discrepancies would have led the authors to try adding some noise in their model, which would partially resolve the differences observed at the lower rotational speeds (see stars deviating from the fitted line in Figure 2A) and to consider that introducing an asymmetry between the post-stimulus inhibition and excitation time constants could result in a model not deviating as much at the higher rotation velocities during counter-clockwise rotation of the polarized light (see stars deviating from the fitted line in Figure 2A). 

In the end, the authors use the observation that during saccades, the average activity in their model-with-history increases to claim that when the animal does not turn, it uses less neuronal activity and energy. This is not a convincing line of reasoning. To make a claim about energy efficiency, the authors must instead compare their model with alternatives and show that the neuronal activity of their model during straight flight is indeed lower than those alternative models. Note that such a comparison would be meaningful only if the alternative models compared against capture physiology equally well in all other respects. However, the evident deviations of the presented model from the physiology measurements and the short duration of the test stimulus used would make any such claims difficult to substantiate. 

Finally, for most experiments, the models are stimulated with a single short yaw sequence lasting a few seconds to measure responses. Given the dependence of the model on history, using such a small sample, we cannot see how generalizable the observations are. The authors need to show that the same effect is produced using multiple different trajectories.

DOI: 10.1038/s41598-017-18989-1

Resource: RRID:BDSC_10403

Curator: @maulamb

SciCrunch record: RRID:BDSC_10403

What is this?

Reviewer #1 (Public review):

Summary:

The authors use microscopy experiments to track the gliding motion of filaments of the cyanobacteria Fluctiforma draycotensis. They find that filament motion consists of back-and-forth trajectories along a "track", interspersed with reversals of movement direction, with no clear dependence between filament speed and length. It is also observed that longer filaments can buckle and form plectonemes. A computational model is used to rationalize these findings.

Strengths:

Much work in this field focuses on molecular mechanisms of motility; by tracking filament dynamics this work helps to connect molecular mechanisms to environmentally and industrially relevant ecological behavior such as aggregate formation.

The observation that filaments move on tracks is interesting and potentially ecologically significant.

The observation of rotating membrane-bound protein complexes and tubular arrangement of slime around the filament provides important clues to the mechanism of motion.

The observation that long filaments buckle has the potential to shed light on the nature of mechanical forces in the filaments, e.g. through the study of the length dependence of buckling.

Weaknesses:

The manuscript makes the interesting statement that the distribution of speed vs filament length is uniform, which would constrain the possibilities for mechanical coupling between the filaments. However, Figure 1C does not show a uniform distribution but rather an apparent lack of correlation between speed and filament length, while Figure S3 shows a dependence that is clearly increasing with filament length. Also, although it is claimed that the computational model reproduces the key features of the experiments, no data is shown for the dependence of speed on filament length in the computational model. The statement that is made about the model "all or most cells contribute to propulsive force generation, as seen from a uniform distribution of mean speed across different filament lengths", seems to be contradictory, since if each cell contributes to the force one might expect that speed would increase with filament length.

The computational model misses perhaps the most interesting aspect of the experimental results which is the coupling between rotation, slime generation, and motion. While the dependence of synchronization and reversal efficiency on internal model parameters are explored (Figure 2D), these model parameters cannot be connected with biological reality. The model predictions seem somewhat simplistic: that less coupling leads to more erratic reversal and that the number of reversals matches the expected number (which appears to be simply consistent with a filament moving backwards and forwards on a track at constant speed).

Filament buckling is not analysed in quantitative detail, which seems to be a missed opportunity to connect with the computational model, eg by predicting the length dependence of buckling.

Reviewer #1 (Public review):

This is a clever and well-done paper. The authors sought to craft a method, applicable to biobank-scale data but without necessarily using genotyping or sequencing, to detect the presence of de novo mutations and rare variants that stand out from the polygenic background of a given trait. Their method depends essentially on sibling pairs where one sibling is in an extreme tail of the phenotypic distribution and whether the other sibling's regression to the mean shows a systematic deviation from what is expected under a simple polygenic architecture.

Their method is successful in that it builds on a compelling intuition, rests on a rigorous derivation, and seems to show reasonable statistical power in the UK Biobank. (More biobanks of this size will probably become available in the near future.) It is somewhat unsuccessful in that rejection of the null hypothesis does not necessarily point to the favored hypothesis of de novo or rare variants. The authors discuss the alternative possibility of rare environmental events of large effect.

Comments on current version:

The authors have addressed the concerns of the reviewers. I have no further comments.

Reviewer #1 (Public Review):

The authors demonstrate that it is possible to carry out eQTL experiments for the model eukaryote S. cerevisiae, in "one pot" preparations, by using single-cell sequencing technologies to simultaneously genotype and measure expression. This is a very appealing approach for investigators studying genetic variation in single-celled and other microbial systems, and will likely inspire similar approaches in non-microbial systems where comparable cell mixtures of genetically heterogeneous individuals could be achieved.

While eQTL experiments have been done for nearly two decades (the corresponding author's lab are pioneers in this field), this single-cell approach creates the possibility for new insights about cell biology that would be extremely challenging to infer using bulk sequencing approaches. The major motivating application shown here is to discover cell occupancy QTL, i.e. loci where genetic variation contributes to differences in the relative occupancy of different cell cycle stages. The authors dissect and validate one such cell cycle occupancy QTL, involving the gene GPA1, a G-protein subunit that plays a role in regulating the mating response MAPK pathway. They show that variation at GPA1 is associated with proportional differences in the fraction of cells in the G1 stage of the cell cycle. Furthermore, they show that this bias is associated with differences in mating efficiency.

Reviewer #1 (Public review):

Summary:

This important study investigates the neurobiological mechanisms underlying the stable operation and maintenance of functionally appropriate rhythmic motor patterns during changing environmental conditions - temperature in this study in the crab Cancer borealis stomatogastric neural pattern generating network producing the pyloric motor rhythm, which is naturally subjected to temperature perturbations over a substantial range. This study is relevant to the general problem that some rhythmic motor systems adjust to changing environmental conditions and state changes by increasing the cycle frequency in a smooth monotonic fashion while maintaining the relative timing of different network activity pattern phases that determine proper motor coordination. How this is achieved mechanistically in complex dynamic motor networks is not understood, particularly how the frequency and phase adjustments are achieved as conditions change while avoiding operational instabilities on different time scales. The authors specifically studied the contributions of the hyperpolarization-activated inward current (Ih), which is involved in rhythm control, to the adjustments of frequency and phases in the pyloric rhythmic pattern as the temperature was altered from 11 degrees C to 21 degrees C. They present compelling evidence that this current is a critical biophysical feature in the ability of this system to adjust transiently and persistently to temperature perturbations appropriately. After blocking Ih in the pyloric network with cesium, the network was unable to reliably produce its characteristic rapid and smooth increase in the frequency of the triphasic rhythmic motor pattern in response to increasing temperature or its typical steady-state increase in frequency over this Q10 temperature range.

Strengths:

(1) The authors addressed this problem by technically rigorous experiments in the crab Cancer borealis stomatogastric ganglion (STG) in vitro, which readily allows for neuronal activity recording in a behaviorally and architecturally defined rhythmic neural circuit in conjunction with the application of blockers of Ih and synaptic receptors to disrupt circuit interactions. This approach is an effective way to experimentally investigate how complex rhythmic networks, at least in poikilotherms, mechanistically adjust to environmental perturbations such as temperature.

(2) While previous work demonstrated that Ih increases in pyloric neurons as temperature increases, the authors here establish that this increase is necessary for normal responses of STG neural activity to temperature, which consist of a smooth monotonic increase in the frequency of rhythmic activity with increasing temperature.

(3) The data shows that blocking Ih with cesium causes the frequency to transiently decrease ("jags") when the temperature increases and then increases after the temperature stabilizes at a steady state, revealing a non-monotonic frequency response to temperature perturbations.

(4) The authors dissect some of the underlying neuronal and circuit dynamics, presenting evidence that after blocking Ih, the non-monotonic jags in the frequency response are mediated by intrinsic properties of pacemaker neurons, while in the steady state, Ih determined the overall frequency change (i.e., temperature sensitivity) through network interactions.

(5) The authors' results highlight more complex dynamic responses to increasing temperature for the first time, suggesting a longer timescale process than previously recognized that may result from interactions between multiple channels and/or ion channel kinetics.

Weaknesses:

(1) The involvement of Ih in achieving the frequency and phase adjustments as conditions change and allowing smooth transitions to avoid operational instabilities in other complex rhythmic motor networks, for example, in homeotherms, is not established, so the present results may have limited general extrapolations.

Reviewer #1 (Public review):

Summary:

Recruitment of neutrophils to the lungs is known to drive susceptibility to infection with M. tuberculosis. In this study, the authors present data in support of the hypothesis that neutrophil production of the cytokine IL-17 underlies the detrimental effect of neutrophils on disease. They claim that neutrophils harbor a large fraction of Mtb during infection, and are a major source of IL-17. To explore the effects of blocking IL-17 signaling during primary infection, they use IL-17 blocking antibodies, SR221 (an inverse agonist of TH17 differentiation), and celecoxib, which they claim blocks Th17 differentiation, and observe modest improvements in bacterial burdens in both WT and IFN-γ deficient mice using the combination of IL-17 blockade with celecoxib during primary infection. Celecoxib enhances control of infection after BCG vaccination.

Strengths:

The most novel finding in the paper is that treatment with celecoxib significantly enhances control of infection in BCG-vaccinated mice that have been challenged with Mtb. It was already known that NSAID treatments can improve primary infection with Mtb.

Weaknesses:

The major claim of the manuscript - that neutrophils produce IL-17 that is detrimental to the host - is not strongly supported by the data. Data demonstrating neutrophil production of IL-17 lacks rigor. The experiments examining the effects of inhibitors of IL-17 on the outcome of infection are very difficult to interpret. First, treatment with IL-17 inhibitors alone has no impact on bacterial burdens in the lung, either in WT or IFN-γ KO mice. This suggests that IL-17 does not play a detrimental role during infection. Modest effects are observed using the combination of IL-17 blocking drugs and celecoxib, however, the interpretation of these results mechanistically is complicated. Celecoxib is not a specific inhibitor of Th17. Indeed, it affects levels of PGE2, which is known to have numerous impacts on Mtb infection separate from any effect on IL-17 production, as well as other eicosanoids. Finally, the human data simply demonstrates that neutrophils and IL-17 both are higher in patients who experience relapse after treatment for TB, which is expected and does not support their specific hypothesis. The use of genetic ablation of IL-17 production specifically in neutrophils and/or IL-17R in mice would greatly enhance the rigor of this study. The authors do not address the fact that numerous studies have shown that IL-17 has a protective effect in the mouse model of TB in the context of vaccination. Finally, whether and how many times each animal experiment was repeated is unclear.

Reviewer #1 (Public review):

Summary:

In this paper Homan et al used mouse models of Metabolic Dysfunction-Associated Steatotic Liver Disease and different specific target deletions in cells to rule out the role of Complement 3a Receptor 1 in the pathogenesis of disease. They provided limited evidence and only descriptive results that despite C3aR being relevant in different contexts of inflammation, however, these tenets did not hold true.

Weaknesses:

(1) The results are based on readouts showing that C3aR is not involved in the pathogenesis of liver metabolic disease.

(2) The description of the mouse models they used to validate their findings is not clear. Lysm-cre mice - which are claimed to delete C3aR in (?) macrophages are not specific for these cells, and the genetic strategy to delete C3aR in Kupffer cells is not clear.

(3) Taking this into account, it is very challenging to determine the validity of these data, also considering that they are merely descriptive and correlative.