- Dec 2023
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The authors set up a pipeline for automated high-throughput single-molecule fluorescence imaging (htSMT) in living cells and analysis of molecular dynamics.
Strengths:<br /> htSMT reveals information on the diffusion and bound fraction of molecules, dose-response curves, relative estimates of binding rates, and temporal changes of parameters. It enables the screening of thousands of compounds in a reasonable time and proves to be more sensitive and faster than classical cell-growth assays. If the function of a compound is coupled to the mobility of the protein of interest, or affects an interaction partner, which modulates the mobility of the protein of interest, htSMT allows identifying the modulator and getting the first indication of the mechanism of action or interaction networks, which can be a starting point for more in-depth analysis.
Weaknesses:<br /> While elegantly showcasing the power of high-throughput measurements, the authors disclose little information on their microscope setup and analysis procedures. Thus, reproduction by other scientists is limited. Moreover, a critical discussion about the limits of the approach in determining dynamic parameters, the mechanism of action of compounds, and network reconstruction for the protein of interest is missing. In addition, automated imaging and analysis procedures require implementing sensitive measures to assure data and analysis quality, but a description of such measures is missing.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors performed single nucleus RNA-seq for perirenal adipose tissue (PRAT) at different ages. They concluded a distinct subpopulation of adipocytes arises through brown-to-white conversion and can convert to a thermogenic phenotype upon cold exposure.
Strengths:
PRAT adipose tissue has been reported as an adipose tissue that undergoes browning. This study confirms that brown-to-white and white-to-beige conversions also exist in PRAT, as previously reported in the subcutaneous adipose tissue.
Weaknesses:
1. There is overall a disconnection between single nucleus RNA-seq data and the lineage chasing data. No specific markers of this population have been validated by staining.<br /> 2. It would be nice to provide more evidence to support the conclusion shown in lines 243 to 245 "These results indicated that new BAs induced by cold exposure were mainly derived from UCP1- adipocytes rather than de novo ASPC differentiation in puPRAT". Pdgfra-negative progenitor cells may also contribute to these new beige adipocytes.<br /> 3. The UCP1Cre-ERT2; Ai14 system should be validated by showing Tomato and UCP1 co-staining right after the Tamoxifen treatment.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The authors have presented data showing that there is a greater amount of spontaneous differentiation in human pluripotent cells cultured in suspension vs static and have used PKCβ and Wnt signaling pathway inhibitors to decrease the amount of differentiation in suspension culture.
Strengths:<br /> This is a very comprehensive study that uses a number of different rector designs and scales in addition to a number of unbiased outcomes to determine how suspension impacts the behaviour of the cells and in turn how the addition of inhibitors counteracts this effect. Furthermore, the authors were also able to derive new hiPSC lines in suspension with this adapted protocol.
Weaknesses:<br /> The main weakness of this study is the lack of optimization with each bioreactor change. It has been shown multiple times in the literature that the expansion and behaviour of pluripotent cells can be dramatically impacted by impeller shape, RPM, reactor design, and multiple other factors. It remains unclear to me how much of the results the authors observed (e.g. increased spontaneous differentiation) was due to not having an optimized bioreactor protocol in place (per bioreactor vessel type). For instance - was the starting seeding density, RPM, impeller shape, feeding schedule, and/or any other aspect optimized for any of the reactors used in the study, and if not, how were the values used in the study determined?
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www.researchsquare.com www.researchsquare.com
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Reviewer #1 (Public Review):
Summary:<br /> The authors develop a method to fluorescently tagged peptides loaded onto dendritic cells using a three step method. These include a pre blocking step to block endogenous cysteine motifs on the DC surface, loading a tetracystein motif modified peptide on surface MHC and a labelling step done on the surface of live DC using a dye with high affinity for the added motif. The results are convincing in demonstrating in vitro and in vivo T cell activation and efficient label transfer to specific T cells in vivo. The label transfer technique will be useful to identify T cell that have recognised a DC presenting a specific peptide antigen to allow the isolation of the T cell and cloning of its TCR subunits, for example. It may also be useful as a general assay for in vitro or in vivo T-DC communication that can allow detection of genetic or chemical modulators.
Strengths:<br /> The study include both in vitro and in vivo analysis including flow cytometry and two photon laser scanning microscopy. The results are convincing and the level of T cell labelling with the fluorescent pMHC is surprisingly robust and suggests that the approach is potentially revealing something about fundamental mechanisms beyond the state of the art. They also provide practical information about the challenges of the method and discuss limitations.
Weaknesses:<br /> The method is demonstrated only at high pMHC density and it would need to be re-optimised to determine if it can be used at lower densities that may often be encountered physiologically.
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Reviewer #1 (Public Review):
In this manuscript, Shimonty and colleagues study the effects of FNDC5/irisin deletion on osteocytes in a sex-specific manner using models of lactation-induced bone loss and bone loss due to low calcium diet (LCD). Consistent with the previous findings of Kim et al. (2018), the authors report 'protective' effects of irisin deficiency in lactating female FNDC5-null mice due to reduced osteocytic osteolysis. Interestingly, FNDC5 null mice show distinct changes when placed on LCD, with mutant females showing some protection from hyperparathyroidism-induced bone loss, while mutant males (which have more cortical bone at baseline) show increased LCD-induced bone loss. Furthermore, new insights into irisin's role in osteocytes regarding cellular energetic metabolism were provided by sex and gene-dependent transcriptomic datasets. Strengths of the well-written manuscript include a clear description of sex-dependent effects, strong transcriptomic datasets, and a focus on cortical bone changes using microCT, histomorphometry, BSEM, and serum analysis. Despite these strengths, important weaknesses are noted (below) which could be addressed to improve the impact of the work for a broad audience.
Major comments:
1. Overall, the magnitude of the effect size due to FNDC5 deficiency in both male and female mice is rather modest. Looking at the data from a qualitative perspective, it is clear that knockout females still lose bone during lactation and on the low calcium diet (LCD). It is difficult to assess the physiologic consequence of the modest quantitative 'protection' seen in FNDC5 mutants since the mutants still show clear and robust effects of lactation and LCD on all parameters measured. Similarly, the magnitude of the 'increased' cortical bone loss in FNDC5 mutant males is also modest and perhaps could be related to the fact that these mice are starting with slightly more cortical bone. Since the authors do not provide a convincing molecular explanation for why FNDC5 deficiency causes these somewhat subtle changes, I would like to offer a suggestion for the authors to consider (below, point #2) which might de-emphasize the focus of the manuscript on FNDC5. If the authors chose not to follow this suggestion, the manuscript could be strengthened by addressing the consequences of the modest changes observed in WT versus FNDC5 KO mice.
2. The bone RNA-seq findings reported in Figures 4-6 are quite interesting. Although Youlten et al previously reported that the osteocyte transcriptome is sex-dependent, the work here certainly advances that notion to a considerable degree and likely will be of high interest to investigators studying skeletal biology and sexual dimorphism in general. To this end, one direction for the authors to consider might be to refocus their manuscript toward sexually-dimorphic gene expression patterns in osteocytes and the different effects of LCD on male versus female mice. This would allow the authors to better emphasize these major findings, and to then use FNDC5 deficiency as an illustrative example of how sexually-dimorphic osteocytic gene expression patterns might be affected by deletion of an osteocyte-acting endocrine factor. Ideally, the authors would confirm RNA-seq data comparing male versus female mice in osteocytes using in situ hybridization or immunostaining.
3. Along the lines of point #2 (above), the presentation of the RNA-seq studies in Figures 4-6 is somewhat confusing in that the volcano plot titles seem to be reversed. For example, Figure 4A is titled "WT M: WT F", but the genes in the upper right quadrant appear to be up-regulated in female cortical bone RNA samples. Should this plot instead be titled "WT F: WT M"? If so, then all other volcano plots should be re-titled as well.
4. Have the authors compared male versus female transcriptomes of LCD mice?
5. It would be appreciated if the authors could provide additional serum parameters (if possible) to clarify incomplete data in both lactation and low-calcium diet models: RANKL/OPG ratio, Ctx, PTHrP, and 1,25-dihydroxyvitamin D levels.
6. Lastly, the data that overexpressing irisin improved bone properties in Fig 2G was somewhat confusing. Based on Kim et al.'s (2018) work, irisin injection increased sclerostin gene expression and serum levels, thus reducing bone formation. Were sclerostin levels affected by irisin overexpression in this study? Was irisin's role in modulating sclerostin levels attenuated with additional calcium deficiency?
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Review #1 (Public Review)
Watanuki et al used metabolomic tracing strategies of U-13C6-labeled glucose and 13C-MFA to quantitatively identify the metabolic programs of HSCs during steady-state, cell-cycling, and OXPHOS inhibition. They found that 5-FU administration in mice increased anaerobic glycolytic flux and decreased ATP concentration in HSCs, suggesting that HSC differentiation and cell cycle progression are closely related to intracellular metabolism and can be monitored by measuring ATP concentration. Using the GO-ATeam2 system to analyze ATP levels in single hematopoietic cells, they found that PFKFB3 can accelerate glycolytic ATP production during HSC cell cycling by activating the rate-limiting enzyme PFK of glycolysis. Additionally, by using Pfkfb3 knockout or overexpressing strategies and conducting experiments with cytokine stimulation or transplantation stress, they found that PFKFB3 governs cell cycle progression and promotes the production of differentiated cells from HSCs in proliferative environments by activating glycolysis. Overall, in their study, Watanuki et al combined metabolomic tracing to quantitatively identify metabolic programs of HSCs and found that PFKFB3 confers glycolytic dependence onto HSCs to help coordinate their response to stress.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
This study used ATAC-Seq to characterize chromatin accessibility during stages of GABAergic neuron development in induced pluripotent stem cells (iPSCs) derived from both Dravet Syndrome (DS) patients and healthy donors. The authors report accelerated GABAergic maturation to a point, followed by further differentiation into a perturbed chromatin profile, in the cells from patients. In a preliminary analysis, valproic acid, an anti-seizure medication commonly used in patients with DS, increased open chromatin in both patient and control iPSCs in a nonspecific manner, and to different degrees in cultures derived from different patients. These findings provide new information about DS-associated changes in chromatin, and provide further evidence for developmental abnormalities in interneurons with DS.
Strengths:
This is a novel study that aims to investigate the epigenetic changes that occur in a sodium channel model of epilepsy; these changes are often ignored but may be an interesting area for future therapeutics. In general, the flow of the paper is good, and the figures are well-designed.
Weaknesses:
The most substantial weakness relates to the observation that DS is often viewed as a monogenic form of epilepsy. It is directly linked to SCN1A gene haploinsufficiency (Yu et al, 2006; Ogiwara et al, 2007). The gene product is Nav1.1, the alpha subunit of voltage-gated sodium channel type I that regulates neuronal excitability. Yet, analysis was conducted at time points of GABAergic interneuron differentiation in which SCN1A is likely not expressed. The paper would be strengthened if SCN1A expression and Nav1.1 protein were examined across the experimental time course. If SCN1A is not yet expressed, this would complicate any explanation of how the observed epigenetic changes might arise. It also seems counterintuitive that the absence of a sodium channel can accelerate differentiation, when, a priori, one might expect the opposite (a 'less neuronal' signal).
Related to this, another important limitation of the study is that the controls are cells derived from healthy individuals and not from isogenic lines. The usage of isogenic lines is extremely relevant for every study in which iPSC-derived somatic cells are used to model a disease, but specifically in diseases like DS, in which the genetic background has an ascertained impact on disease phenotype (Cetica et al, 2017 and others). This serious limitation should be considered. In addition, the authors should provide data on variability across cell lines and differentiations to help convince the reader that the results can be attributed to genetic defects, rather than variability across individuals.
Additionally, the authors acknowledge the variability of the differentiations and cell lines, which is commendable, and they attribute this to "possibly reflecting cell line specific and endogenous differences reported previously", but could also have to do with cell death. This is a large confounding factor for ATAC-seq. Certainly, Sup Fig 1C shows lower FrIP scores, consistent with cell death, and there seems to be a lot of death in the representative images. Moreover, the iGABA neurons are very difficult to keep alive, especially to 65 days, without co-culturing with glia and/or glutamatergic neurons. The authors should comment on how much these factors may have influenced their results.
Finally, changes in gene expression are only inferred, as no RNA levels were measured. If RNA-seq was not possible it would have been good to see at least some of the key genes/findings corroborated with RNA/protein levels vs chromatin accessibility alone, particularly given that these molecular readouts do not always correlate.
Additional Points:
1. Representative images for cell-identity markers for only D65 are shown, and not D0, D19, and D35 though it is stated in the text that this was performed. At a minimum, these representative images should be shown for all lines.<br /> 2. What QC was performed on iPSC lines, i.e. karyotype/CNV analysis and confirmation of genotypes?<br /> 3. Were all experiments performed on a single differentiation? Or multiples? Were the differentiations performed with the same type? If not, was batch considered in the analysis? I also assume that technical replicates were merged, and then all three biological replicates were kept for each analysis and outliers were not removed, e.g. Control_D19_8F seems like an example of an outlier.<br /> 4. In Figure 1C, it is intriguing that the ATACseq signal gets stronger in imN. One might expect it to be strongest in the iPSCs which are undifferentiated and have the highest levels of open chromatin. Is this a function of sequencing depth, or are all the Y-axes normalized across all time points?<br /> 5. In Figure 1F, are these all enriched terms, or were they prioritized somehow?<br /> 6. In Figure 1G (also the same plots in Fig 2/3), are all these images normalized i.e. there is no scale bar for each track, and do they represent and aggregate BAM/bigwig? It would be good to show in supplement the variability across cell lines/diffs - particularly given the variability in the heatmap/PCA - and demonstrate the rigor/reproducibility of these results. This comment applies to all these plots across the 3 figures, particularly as in some instances the samples appear to cluster by individual first and then time point (Sup Fig 3B). How confident are the authors that these effects are driven by genotype and not a single cell line? In the Fig 3D representation of NANOG, it is very difficult to see any difference between patient and control.<br /> 7. For the changes in occupancy annotation (UTR/exon/intron etc), are these differences still significant after correcting for variability from cell line to cell line at each time point? I.e. rather than average across all three samples, what is the range?<br /> 8. The VPA timepoint is not well-justified. Given that VPA would be administered in patients with fully mature inhibitory neurons, it is difficult to determine the biological relevance. I appreciate that this is a limitation of the model, but this should at least be addressed in the manuscript.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The authors provide mechanistic insights into how the loss of function of MBOAT7 promotes alcohol-associated liver disease. They showed that hepatocyte-specific genetic deletion of Mboat7 enhances ethanol-induced hepatic steatosis and increased ALT levels in a murine model of ethanol-induced liver disease. Through lipidomic profiling, they showed that mice with Mboat7 deletion demonstrated augmented ethanol-induced endosomal and lysosomal lipids, together with impaired transcription factor EB (TFEB)-mediated lysosomal biogenesis and accumulation of<br /> autophagosomes.
Strengths:<br /> -Alcohol-induced liver disease (ALD) and metabolic-associated steatotic liver disease (MASLD) are major global health problems, and polymorphism near the gene encoding MBOAT7 has been associated with these conditions. This paper is timely as it is important to gain insights on how loss of MBOAT function contributes to liver disease as this may eventually lead to therapeutic strategies.<br /> -The conclusions of the paper are mostly well supported by data.
Weaknesses:<br /> 1) In regards to circulating levels of MBOAT7 products, a comparison of heavy drinkers with ALD versus heavy drinkers without ALD would be more clinically relevant.<br /> 2) A few typos need to be addressed. For Figure 1 - figure supplement 1, should the second column heading be "Heavy drinkers" instead of "Healthy drinkers"? Also, in the same figure, it is unclear what the "healthy" subcategory under MELD means.<br /> 3) Some of the data in the tables need to be addressed/discussed. For instance, the white blood cell count (WBC) in Figure 1 - figure supplement 1 for "healthy controls" is 34, compared to 13.51 for drinkers. A WBC of 34 is not at all healthy and should be explained. The vast difference between BMI and also between racial distribution within the two cohorts should also be explained. Is it possible that some of these differences contributed to the different levels of circulating MBOAT7 products that were measured?<br /> 4) The representation of the statistical difference between the bars in the results figures by using alphabets is a bit confusing. For instance, in figure 2C, does that mean all the bars labelled A are significantly different from B? The solid black bar seems to be very similar to the open red bar; please double check.
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Reviewer #1 (Public Review):
Summary:<br /> The extra macrochaetae (emc) gene encodes the only Inhibitor of DNA binding protein (Id protein) in Drosophila. Its best-known function is to inhibit proneural genes during development. However, the emc mutants also display non-proneural phenotypes. In this manuscript, the authors examined four non-proneural phenotypes of the emc mutants and reported that they are all caused by inappropriate non-apoptotic caspase activity. These non-neuronal phenotypes are: reduced growth of imaginal discs, increased speed of the morphogenetic furrow, and failure to specify R7 photoreceptor neurons and cone cells during eye development. Double mutants between emc and either H99 (which deletes the three pro-apoptotic genes reaper, grim, and hid) or the initiator caspase dronc suppress these mutant phenotypes of emc suggesting that the cell death pathway and caspase activity are mediating these emc phenotypes. In previous work, the authors have shown that emc mutations elevate the expression of ex which activates the SHW pathway (aka the Hippo pathway). One known function of the SHW pathway is to inhibit Yorkie which controls the transcription of the inhibitor of apoptosis, Diap1. Consistently, in emc clones the levels of Diap1 protein are reduced which might explain why caspase activity is increased in emc clones giving rise to the four non-neural phenotypes of emc mutants. However, this increased caspase activity is not causing ectopic apoptosis, hence the authors propose that this is non-apoptotic caspase activity. In the last part of the manuscript, the authors ruled out that Wg, Dpp, and Hh signaling are the target of caspases, but instead identified Notch signaling as the target of caspases, specifically the Notch ligand Delta. Protein levels of Delta are increased in emc clones in an H99- and dronc-dependent manner. The authors conclude that caspase-dependent non-apoptotic signaling underlies multiple roles of emc that are independent of proneural bHLH proteins.
Strengths:<br /> Overall, this is an interesting manuscript and the findings are intriguing. It adds to the growing number of non-apoptotic functions of apoptotic proteins and caspases in particular. The manuscript is well written and the data are usually convincingly presented.
Weaknesses:<br /> 1. One major concern I have is the observation by the authors in Figure 3C in which protein levels of Diap1 are still reduced in emc H99 double mutant clones. If Diap1 is still reduced in these clones, shouldn't caspases still be de-repressed? Given that emc H99 double mutants rescue all emc phenotypes examined, the observation that Diap1 levels are still reduced in emc H99 clones is inconsistent with the authors' model. The authors need to address this inconsistency.
2. Are Diap1 protein levels reduced in all emc clones, including clones anterior to the furrow? This is difficult to see in Figure 3B. it is also recommended to look in emc mosaic wing discs.
3. The authors speculate that Delta may be a direct target of caspase cleavage (Figure 9B), but then rule it out for a good reason. However, I assume that the increased protein levels of Delta in emc clones (Figure 7) are the results of increased transcription. In that case, shouldn't caspases control the transcriptional machinery leading to Delta expression?
4. How does caspase activity in emc clones cause reduced growth? Is this also mediated through Delta signaling?
5. Figure 1M: Is there a similar result with emc dronc mosaics?
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recyt.fecyt.es recyt.fecyt.es
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La inteligencia artificial (IA) está revolucionando la investigación y el mundo académico, facilitando desde la redacción del manuscrito hasta el análisis de datos. Pero, la IA no “pisa la calle”. La IA sigue confinada al ámbito digital y es incapaz de involucrarse en interacciones humanas para indagar sobre dinámicas interpersonales o comprender el contexto. Ante este panorama, ¿se debería aceptar que la IA revise artículos de investigación enviados a una revista? ¿Cómo debe adaptarse la academia ante el auge de la IA generativa? El presente artículo reflexiona sobre el impacto de la IA en la investigación y el mundo académico, ofreciendo una definición y caracterización de la IA generativa, presentando herramientas de IA que asisten en la investigación, y analizando cuatro escenarios futuros posibles: democratización de la investigación, desaprovechamiento de la IA, aumento de la desigualdad y rezagados digitales. Se concluye sugiriendo estrategias de acción como mayor formación en herramientas de IA, fomento de una educación basada en investigación, y necesidad de abrir un debate sobre el uso de la IA en la gestión de las revistas científicas.
Comentario 21/12/2023 público
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recyt.fecyt.es recyt.fecyt.es
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La inteligencia artificial (IA) está revolucionando la investigación y el mundo académico, facilitando desde la redacción del manuscrito hasta el análisis de datos. Pero, la IA no “pisa la calle”.
Comentario 21/12/2023 público
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Joint Public Review:
Roget et al. build on their previous work developing a simple theoretical model to examine whether ageing can be under natural selection, challenging the mainstream view that ageing is merely a byproduct of other biological and evolutionary processes. The authors propose an agent-based model to evaluate the adaptive dynamics of a haploid asexual population with two independent traits: fertility timespan and mortality onset. Through computational simulations, their model demonstrates that ageing can give populations an evolutionary advantage. Notably, this observation arises from the model without invoking any explicit energy tradeoffs, commonly used to explain this relationship.
Additionally, the theoretical model developed here indicates that mortality onset is generally selected to start before the loss of fertility, irrespective of the initial values in the population. The selected relationship between the fertility timespan and mortality onset depends on the strength of fertility and mortality effects, with larger effects resulting in the loss of fertility and mortality onset being closer together. By allowing for a trans-generational effect on ageing in the model, the authors show that this can be advantageous as well, lowering the risk of collapse in the population despite an apparent fitness disadvantage in individuals. Upon closer examination, the authors reveal that this unexpected outcome is a consequence of the trans-generational effect on ageing increasing the evolvability of the population (i.e., allowing a more effective exploration of the parameter landscape), reaching the optimum state faster.
The simplicity of the proposed theoretical model represents both the major strength and weakness of this work. On one hand, with an original and rigorous methodology, the logic of their conclusions can be easily grasped and generalised, yielding surprising results. Using just a handful of parameters and relying on direct competition simulations, the model qualitatively recapitulates the negative correlation between lifespan and fertility without requiring energy tradeoffs. This alone makes this work an important milestone for the rapidly growing field of adaptive dynamics, opening many new avenues of research, both theoretically and empirically.
On the other hand, the simplicity of the model also makes its relationship with living organisms difficult to gauge, leaving open questions about how much the model represents the reality of actual evolution in a natural context. In particular, a more explicit discussion of how the specifics of the model can impact the results and their interpretation is needed. For example, the lack of mechanistic details on the trans-generational effect on ageing makes the results difficult to interpret. Even if analytical results are obtained, most of the observations appear derived from simulations as they are currently presented. Also, the choice of parameters for the simulations shown in the paper and how they relate to our biological knowledge are not fully addressed by the authors. Finally, the conclusions of evolvability are insufficiently supported, as the authors do not show if the wider genotypic variability in populations with the ageing trans-generational effect is, in fact, selected.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors' earlier deep mutational scanning work observed that allosteric mutations in TetR (the tetracycline repressor) and its homologous transcriptional factors are distributed across the structure instead of along the presumed allosteric pathways as commonly expected. Especially, in addition, the loss of the allosteric communications promoted by those mutations, was rescued by additional distributed mutations. Now the authors develop a two-domain thermodynamic model for TetR that explains these compelling data. The model is consistent with the in vivo phenotypes of the mutants with changes in parameters, which permits quantification. Taken together their work connects intra- and inter-domain allosteric regulation that correlate with structural features. This leads the authors to suggest broader applicability to other multidomain allosteric proteins.
Here the authors follow their first innovative observations with a computational model that captures the structural behavior, aiming to make it broadly applicable to multidomain proteins. Altogether, an innovative and potentially useful contribution.
Weaknesses:
None that I see, except that I hope that in the future, if possible, the authors would follow with additional proteins to further substantiate the model and show its broad applicability. I realize however the extensive work that this would entail.
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Reviewer #1 (Public Review):
Shoemaker and Grilli analyze publicly available sequencing data to quantify how the microbial diversity of ecosystems changes with the taxonomic scale considered (e.g., diversity of genera vs diversity of families). This study builds directly on Grilli's 2020 paper which used this data to show that for many different microbial species, the distribution of abundances of the species across sampling sites belongs to a simple one-parameter family of gamma distributions. In this work, they show that the gamma distribution also describes the distribution of abundances of higher taxonomic levels. The distribution now requires two parameters, but the second parameter can be approximately derived by treating the distributions of lower-level taxonomic units as being independent. The difference between the species-level result and the result at higher taxonomic levels suggests that in some sense microbial species are ecologically meaningful units.
While the higher-level taxon abundance distributions can be well-approximated assuming independence of the constituent species, this approach substantially underestimates variation in community richness and diversity among sampling sites. Much of this extra variability appears to be driven by variability in sample size across sites. It is not clear to me how much this variation in sample size is itself due to variation in sampling effort versus variation in overall microbial densities. This variation in sample size also produces correlations between taxon richness at lower and higher taxonomic levels. For instance, sites with large samples are likely to have both many species within a genus and many genera. The authors also consider taxon diversity (Shannon index, i.e. entropy), which is constructed from frequencies and is therefore less sensitive to sample size. In this case, correlations between diversity across taxonomic scales instead appear to depend on the idiosyncratic correlations among species abundances.
This paper's results are presented in a fairly terse manner, even when they are describing summary statistics that require a lot of thought to interpret. I don't think it would make sense to try to understand it without having first worked through the 2020 paper. But everyone interested in a general understanding of microbial ecology should read the 2020 paper, and once one has done that, this paper is worth reading as well simply for seeing how the major pattern in that paper shifts as one moves up in taxonomic scale.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> This study examined the impact of exogenous microapplication of acetylcholine (Ach) on metrics of novelty detection in the anesthetized rat auditory cortex. The authors found that the majority of units showed some degree of modulation of novelty detection, with roughly similar numbers showing enhanced novelty detection, suppressed novelty detection or no change. Enhanced novelty responses were driven by increases in repetition suppression. Suppressed novelty responses were driven by deviance suppression. There were no compelling differences seen between auditory cortical subfields or layers, though there was heterogeneity in the Ach effects within subfields. Overall, these findings are important because they suggest that fluctations in cortical Ach, which are known to occur during changes in arousal or attentional states, will likely influence the capacity for individual auditory cortical neurons to respond to novel stimuli.
Strengths:<br /> The work addresses an important problem in auditory neuroscience. The main strengths of the study are that the work appears to be systematically done with appropriate controls (cascaded stimuli) and utilizes a classical approach that ensures that drug application is isolated to the micro-environment of the recorded neuron. In addition, the authors do not isolate their study to only primary auditory cortex, but examine the impact of Ach across all known auditory cortical subfields.
Weaknesses:<br /> 1. As acknowledged by the authors, this study explicitly examines a phenomenon of high relevance to active listening, but is done in anesthetized animals, limiting its applicability to the waking state.<br /> 2. The authors do not make any attempt to determine, by spike shape/duration, if their units are excitatory or inhibitory, which may explain some of the variance of the data.<br /> 3. The application of exogenous Ach, potentially in supra-physiological amounts, makes this study hard to extrapolate to a behaving animal. A more compelling design would be to block Ach, particularly at particular receptor types, to determine the effect of endogenous Ach
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The manuscript by Heyndrickx et al describes protein crystal formation and function that bears similarity to Charcot-Leyden crystals made of galectin 10, found in humans under similar conditions. Therefore, the authors set out to investigate CLP crystal formation and their immunological effects in the lung. The authors reveal the crystal structure of both Ym1 and Ym2 and show that Ym1 crystals trigger innate immunity, activated dendritic cells in the lymph node, enhancing antigen uptake and migration to the lung, ultimately leading to induction of type 2 immunity.
Strengths:
We know a lot about expression levels of CLPs in various settings in the mouse, but still know very little about the functions of these proteins, especially in light of their ability to form crystal structures. As such data presented in this paper is a major advance to the field.
Resolving the crystal structure of Ym2 and the comparison between native and recombinant CLP crystals is a strength of this manuscript that will be a very powerful tool for further evaluation and understanding of receptor, binding partner studies including ability to aid mutant protein generation.
The ability to recombinantly generate CLP crystals and study their function in vivo and ex vivo has provided a robust dataset whereby CLPs can activate innate immune responses, aid activation and trafficking of antigen presenting cells from the lymph node to the lung and further enhances type 2 immunity. By demonstrating these effects the authors directly address the aims for the study. A key apoint of this study is the generation of a model in which crystal formation/function an important feature of human eosinophilic diseases, can be studied utilising mouse models. Excitingly, using crystal structures combined with understanding the biochemistry of these proteins will provide a potential avenue whereby inhibitors could be used to dissolve or prevent crystal formation in vivo.
Generation of the crystal structure for Ym2 is a particular strength of the authors work and highlights the similarities between Ym1 and Ym2. Whilst the authors did not specifically examine Ym2 function, they have provided a discussion on this and speculate that Ym2 will function in a similar manner to Ym1.
The data presented flows logically and formulates a well constructed overall picture of exactly what CLP crystals could be doing in an inflammatory setting in vivo. Leaves open a clear and exciting future avenue (currently beyond the scope of this work) for determining whether targeting crystal formation in vivo could limit pathology.
Weaknesses:
It would have been nice for the authors to confirm whether Ym2 has similar functions to Ym1 using the in vivo and in vitro systems. However, they have discussed these points and raised it as a potential for future studies.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This manuscript describes the development of an oral THC consumption model in mice where THC is added to a chocolate flavored gelatin. The authors compared the effects of THC consumed in this highly palatable gelatin (termed E-gel) to THC dissolved in a less palatable gelatin (CTR-gel), and to i.p. injections of multiple doses of THC, on the classic triad of CB1R dependent behaviors (hypolocomotion, antinociception, and body temperature).
The authors found that they could achieve consumption of higher concentrations of THC in the E-gel than the CTR-gel, and that this led to larger total dose exposure and decreases in locomotor activity, antinociception, and body temperature reductions similar to 3-4 mg/kg THC when tested after 2 hour consumption and roughly 10 mg/kg if tested immediately after 1 hour consumption. The majority of THC E-gel consumption was found to occur in the first hour on the first exposure day. THC E-gel consumption was lower than VEH E-gel consumption and this persisted on a subsequent consumption day, suggesting that the animals may form a taste aversion and that THC at the dose consumed likely has aversive properties, consistent with the literature on i.p. dosing. The authors also report the pharmacokinetics in brain and plasma of THC and metabolites after 1 or 2 hour consumption, finding high levels of THC in the brain that begins to dissipate at 2.5 hours is gone 24 hours later. Finally, the authors tested THC effects on the acoustic startle response and found an inverted dose response that was more pronounced in males than females after i.p. dosing and a greater startle response in males after E-gel dosing.
Overall, the authors find that voluntary oral consumption of THC can achieve levels of intake that are consistent with the present and prior reported literature on i.p. dosing.
Strengths:
The strengths of the article include a direct comparison of voluntary oral THC consumption to noncontingent i.p. administration, the use of multiple THC doses and oral THC formulations, the inclusion of multiple assays of cannabinoid agonist effects, and the inclusion of males and females. Additional strengths include monitoring intake over 10 minute intervals and validating that effects are CB1R dependent via antagonist studies.
Weaknesses:
1. The abstract does not discuss the reduction of E-gel consumption that occurs after multiple days of exposure to the THC formulation, but rather implies that a new model for chronic oral self-administration has been developed. Given that only two days of consumption was assessed, it is not clear if the model will be useful to determine THC effects beyond the acute measures presented here. The abstract should clarify that there was evidence of reduced consumption/aversive effects with repeated exposures.<br /> 2. In the results section, the authors sometimes describe effects in terms of the concentration of gel as opposed to the dose consumed in mg/kg, which can make interpretation difficult. For example, the text describing Figure 1i states that significant effects on body temperature were achieved at 4 mg CTR-gel and 5 mg THC-gel, but were essentially equivalent doses consumed? It would be helpful to describe what average dose of THC produced effects given that consumption varied within each group of mice assigned to a particular concentration.<br /> 3. The description of the PK data in Figure 3 did not specify if sex differences were examined. Prior studies have found that males and females can exhibit stark differences in brain and plasma levels of THC and metabolites, even when behavioral effects are similar. However, this does depend on species, route, timing of tissue collection. It would be helpful to describe the PK profile of males and females separately.<br /> 4. In Figure 5, it is unclear how the predicted i.p. THC dose could be 30 mg/kg when 30 mg/kg was not tested by the i.p. route according to the figure, and if it had been it would have likely been almost zero acoustic startle, not the increased startle that was observed in the 2 hr gel group. It seems more likely that it would be equivalent to 3 mg/kg i.p. Could there be an error in the modeling, or was it based on the model used for the triad effects? This should be clarified.
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Reviewer #1 (Public Review):
This study investigates the structuring of long calls in orangutans. The authors demonstrate long calls are structured around full pulses, repeated following a regular tempo (isochronic rhythm). These full pulses are themselves structured around different sub-pulses, themselves repeated following an isochronic rhythm. The authors argue this patterning is evidence for self-embedded, recursive structuring in orangutang long calls.
The analyses conducted are robust and compelling and they support the rhythmicity the authors argue is present in the long calls. Furthermore, the authors went above and beyond and confirmed acoustically the sub-categories identified were accurate.
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Reviewer #1 (Public Review):
Summary:<br /> In this work, the authors study whether the human brain uses long-term priors (acquired during our lifetime) regarding the statistics of auditory stimuli to make predictions respecting auditory stimuli. This is an important open question in the field of predictive processing.
To address this question, the authors cleverly profit from the naturally existing differences between two linguistic groups. While speakers of Spanish use phrases in which function words (short words like articles and prepositions) are followed by content words (longer words like nouns, adjectives, and verbs), speakers of Basque use phrases in the opposite order. Because of this, speakers of Spanish usually hear phrases in which short words are followed by longer words, and speakers of Basque experience the opposite. This difference in the order of short and longer words is hypothesized to result in a long-term duration prior that is used to make predictions regarding the likely durations of incoming sounds, even if they are not linguistic in nature.
To test this, the authors used MEG to measure the mismatch responses (MMN) elicited by the omission of short and long tones that were presented in alternation. The authors report an interaction between the language background of the participants (Spanish, Basque) and the type of omission MMN (short, long), which goes in line with their predictions. They supplement these results with a source-level analysis.
Unfortunately, serious concerns regarding the predictions put forward by the authors, and the interaction effect found, make the interpretation of these results difficult.
Strengths:<br /> This work has many strengths. To test the main question, the authors profit from naturally occurring differences in the everyday auditory experiences of two linguistic groups, which allows them to test the effect of putative auditory priors consolidated over years. This is a direct way of testing the effect of long-term priors.
The fact that the priors in question are linguistic, and that the experiment was conducted using non-linguistic stimuli (i.e. simple tones), allows for testing of whether these long-term priors generalize across auditory domains.
The experimental design is elegant and the analysis pipeline is appropriate. This work is very well written. In particular, the introduction and discussion sections are clear and engaging. The literature review is complete.
Weaknesses:<br /> There are two main issues in this work. The first one pertains to the predictions put forward by the researchers, and the second with the interaction effect reported.
1) With respect to the predictions, the authors propose that the subjects, depending on their linguistic background and the length of the tone in a trial, can put forward one or two predictions. The first is a short-term prediction based on the statistics of the previous stimuli and identical for both groups (i.e. short tones are expected after long tones and vice versa). The second is a long-term prediction based on their linguistic background. According to the authors, after a short tone, Basque speakers will predict the beginning of a new phrasal chunk, and Spanish speakers will predict it after a long tone.
In this way, when a short tone is omitted, Basque speakers would experience the violation of only one prediction (i.e. the short-term prediction), but Spanish speakers will experience the violation of two predictions (i.e. the short-term and long-term predictions), resulting in a higher amplitude MMN. The opposite would occur when a long tone is omitted. So, to recap, the authors propose that subjects will predict the alternation of tone durations (short-term predictions) and the beginning of new phrasal chunks (long-term predictions).
The problem with this is that subjects are also likely to predict the completion of the current phrasal chunk. In speech, phrases are seldom left incomplete. In Spanish is very unlikely to hear a function-word that is not followed by a content-word (and the opposite happens in Basque). On the contrary, after the completion of a phrasal chunk, a speaker might stop talking and a silence might follow, instead of the beginning of a new phrasal chunk.
Considering that the completion of a phrasal chunk is more likely than the beginning of a new one, the prior endowed to the participants by their linguistic background should make us expect a pattern of results actually opposite to the one reported here.
2) The authors report an interaction effect that modulates the amplitude of the omission response, but caveats make the interpretation of this effect somewhat uncertain. The authors report a widespread omission response, which resembles the classical mismatch response (in MEG) with strong activations in sensors over temporal regions. Instead, the interaction found is circumscribed to four sensors that do not overlap with the peaks of activation of the omission response. Furthermore, the boxplot in Figure 2E suggests that part of the interaction effect might be due to the presence of two outliers (if removed, the effect is no longer significant). Overall, it is possible that the reported interaction is driven by a main effect of omission type which the authors report, and find consistently only in the Basque group (showing a higher amplitude omission response for long tones than for short tones).
Because of these points, it is difficult to interpret this interaction as a modulation of the omission response. It should also be noted that in the source analysis, the interaction only showed a trend in the left auditory cortex, but in its current version the manuscript does not report the statistics of such a trend.
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Reviewer #1 (Public Review):
Summary:<br /> The global decline of amphibians is primarily attributed to deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). It is unclear whether and how skin-resident immune cells defend against Bd. Although it is well known that mammalian mast cells are crucial immune sentinels in the skin and play a pivotal role in the immune recognition of pathogens and orchestrating subsequent immune responses, the roles of amphibian mast cells during Bd infections are largely unknown. The current study developed a novel way to enrich X. laevis skin mast cells by injecting the skin with recombinant stem cell factor (SCF), a KIT ligand required for mast cell differentiation and survival. The investigators found an enrichment of skin mast cells provides X. laevis substantial protection against Bd and mitigates the inflammation-related skin damage resulting from Bd infection. Additionally, the augmentation of mast cells leads to increased mucin content within cutaneous mucus glands and shields frogs from the alterations to their skin microbiomes caused by Bd.
Strengths:<br /> This study underscores the significance of amphibian skin-resident immune cells in defenses against Bd and introduces a novel approach to examining interactions between amphibian hosts and fungal pathogens.
Weaknesses:<br /> The main weakness of the study is the lack of functional analysis of X. laevis mast cells. Upon activation, mast cells have the characteristic feature of degranulation to release histamine, serotonin, proteases, cytokines, and chemokines, etc. The study should determine whether X. laevis mast cells can be degranulated by two commonly used mast cell activators IgE and compound 48/80 for IgE-dependent and independent pathways. This can be easily done in vitro. It is also important to assess whether in vivo these mast cells are degranulated upon Bd infection using avidin staining to visualize vesicle releases from mast cells. Figure 3 only showed rSCF injection caused an increase in mast cells in naïve skin. They need to present whether Bd infection can induce mast cell increase and rSCF injection under Bd infection causes a mast cell increase in the skin. In addition, it is unclear how the enrichment of mast cells provides protection against Bd infection and alternations to skin microbiomes after infection. It is important to determine whether skin mast cells release any contents mentioned above.
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Reviewer #1 (Public Review):
Summary: This study brings new information about the function of serotonin-gated ion channels 5-HT3AR, by describing the conformational changes undergoing during ligands binding. These results can be potentially extrapolated to other members of the Cys-loop ligand-gated ion channels. By combining fluorescence microscopy with electrophysiological recordings, the authors investigate structural changes inside and outside the orthosteric site elicited by agonists, partial agonists, and antagonists. The results are convincing and correlate well with the observations from cryo-EM structures. The work will be of important significance and broad interest to scientists working on channel biophysics but also drug development targeting ligand-gated ion channels.
Strengths: The authors present an elegant and well-designed study to investigate the conformational changes on 5-HT3AR where they combine electrophysiological and fluorometry recordings. They determined four positions suitable to act as sensors for the conformational changes of the receptor: two inside and two outside the agonist binding site. They make a strong point showing how antagonists produce conformational changes inside the orthosteric site similarly as agonists do but they failed to spread to the lower part of the ECD, in agreement with previous studies and Cryo-EM structures. They also show how some loss-of-function mutant receptors elicit conformational changes (changes in fluorescence) after partial agonist binding but failed to produce measurable ionic currents, pointing to intermediate states that are stabilized in these conditions. The four fluorescence sensors developed in this study may be good tools for further studies on characterizing drugs targeting the 5-HT3R.
Weaknesses: Although the major conclusions of the manuscript seem well justified, some of the comparison with the structural data may be vague. The claim that monitoring these silent conformational changes can offer insights into the allosteric mechanisms contributing to signal transduction is not unique to this study and has been previously demonstrated by using similar techniques with other ion channels.
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Reviewer #1 (Public Review):
In their revised manuscript, the authors have addressed all the concerns raised earlier (written below for completeness).
Summary:
These types of analyses use many underlying assumptions about the data, which are not easy to verify. Hence, one way to test how the algorithm is performing in a task is to study its performance on synthetic data in which the properties of the variable of interest can be apriori fixed. For example, for burst detection, synthetic data can be generated by injected bursts of known durations, and checking if the algorithm can pick it up. Burst detection is difficult in the spectral domain since direct spectral estimators have high variance (see Subhash Chandran et al., 2018, J Neurophysiol). Therefore, detected burst lengths are typically much lower than injected burst lengths (see their Figure 3). This problem can be solved by doing burst estimation in the time domain itself, for example, using Matching Pursuit (MP). I think the approach presented in this paper would also work since this model is also trained on data in the time domain. Indeed, the synthetic data can be made more "challenging" by injecting multiple oscillatory bursts that are overlapping in time, for which a greedy approach like MP may fail. It would be very interesting to test whether this method can "keep up" as the data is made more challenging. While showing results from brain signals directly (e.g., Figure 7) is nice, it will be even more impactful if it is backed up with results obtained from synthetic data with known properties.
I was wondering about what kind of "synthetic data" could be used for the results shown in Figure 8-12 but could not come up with a good answer. Perhaps data in which different sensory systems are activated (visual versus auditory) or sensory versus movement epochs are compared to see if the activation maps change as expected? We see similarities between states across multiple runs (reproducibility analysis) and across tasks (e.g. Figure 8 vs 9) and even methods (Figure 8 vs 10), which is great. However, we should also expect emergence of new modes specific to sensory activation (say auditory cortex for an auditory task). This will allow us to independently check the performance of this method.
The authors should explain the reproducibility results (variational free energy and best run analysis) in the Results section itself, to better orient the reader on what to look for.
Page 15: the comparison across subjects is interesting, but it is not clear why sensory-motor areas show a difference and the mean lifetime of the visual network decreases. Can you please explain this better? The promised discussion in section 3.5 can be expanded as well.
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Reviewer #1 (Public Review):
Batra, Cabrera, Spence et al. present a model which integrates histone posttranslational modification (PTM) data across cell models to predict gene expression with the goal of using this model to better understand epigenetic editing. This gene expression prediction model approach is useful if a) it predicts gene expression in specific cell lines b) it predicts expression values rather than a rank or bin, c) it helps us to better understand the biology of gene expression, or d) it helps us to understand epigenome editing activity. Problematically for points a) and b) it is easier to directly measure gene expression than to measure multiple PTMs and so the real usefulness of this approach mostly relates to c) and d).
Other approaches have been published that use histone PTM to predict expression (e.g. 27587684, 36588793). Is this model better in some way? No comparisons are made. The paper does not seem to have substantial novel insights into understanding the biology of gene expression. The approach of using this model to predict epigenetic editor activity on transcription is interesting and to my knowledge novel but I doubt given the variability of the predictions (Figures 6 and S7&8) that many people will be interested in using this in a practical sense. As the authors point out, the interpretation of the epigenetic editing data is convoluted by things like sgRNA activity scoring and to fully understand the results likely would require histone PTM profiling and maybe dCas9 ChIP-seq for each sgRNA which would be a substantial amount of work.
Furthermore from the model evaluation of H3K9me3 it seems the model is not performing well for epigenetic or transcriptional editing- e.g. we know for the best studied transcriptional editor which is CRISPRi (dCas9-KRAB) that recruitment to a locus is associated with robust gene repression across the genome and is associated with H3K9me3 deposition by recruitment of KAP1/HP1/SETDB1 (PMID: 35688146, 31980609, 27980086, 26501517). However, it seems from Figures 2&4 that the model wouldn't be able to evaluate or predict this.
The model seems to predict gene expression for endogenous genes quite well although the authors sometimes use expression and sometimes use rank (e.g. Figure 6) - being clearer with how the model predicts expression rather than using rank or fold change would be very useful.
One concern overall with this approach is that dCas9-p300 has been observed to induce sgRNA-independent off-target H3K27Ac (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8349887/ see Figure S5D) which could convolute interpretation of this type of experiment for the model.
Figure 2<br /> It seems this figure presents known rather than novel findings from the authors' description. Please comment on whether there are any new findings in this figure. Please comment on differences in patterns of repressive and activating histone PTMs between cell lines (e.g. H1-Esc H3K27me3 green 25-50% is more enriched than red 0-25%).
Figure 3&4<br /> There are a number of approaches including DeepChrome and TransferChrome that predict endogenous gene expression from histone PTMs. I appreciate that the authors have not used the histone PTM data to predict gene expression levels of an "average cell" but rather that they are predicting expression within specific cell types or for unseen cell types. But from what is presented it isn't clear that the author's model is better or enabling beyond other approaches. The authors should show their model is better than other approaches or make clear why this is a significant advance that will be enabling for the field. For example is it that in this approach they are actually predicting expression levels whereas previous approaches have only predicted expressed or not expressed or a rank order or bin-based ranking?
Figure 5<br /> From the methods, it seems gene activation is measured by qpcr in hek293 transfected with individual sgRNAs and dCas9-p300. The cells aren't selected or sorted before qPCR so how are we sure that some of the variability isn't due to transfection efficiency associated with variable DNA quality or with variable transfection efficiency?
Figure 6<br /> The use of rank in 6D and 6E is confusing. In 6D a higher rank is associated with higher expression while in 6E a higher rank seems to mean a lower fold change e.g. CYP17A1 has a low predicted fold-change rank and qPCR fold-change rank but in Figure 5 a very high qPCR fold change. Labeling this more clearly or explaining it in the text further would be useful.
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Reviewer #1 (Public Review):
Summary:
Glenn et al. present solid evidence that both lab and clinical Enterobacteriaceae strains rapidly migrate towards human serum using an exciting approach that combines microfluidics, structural biology, and genotypic analysis. The authors succeed in bringing to light a novel context for the role of serine as a bacterial chemoattractant as well as documenting what is likely to be a key step in bloodstream entry for some of the main sepsis-associated pathogens during gastrointestinal bleeding. They aim to expand their conclusions from a single lab serovar of Salmonella enterica to a range of clinical serovars and other species within the Enterobacteriaceae family. This is a powerful approach that greatly increases the scope of their findings but I find that some of their conclusions here are not always supported by strong evidence.
I would also like to note that, while I enjoyed the interdisciplinary scope of this study, I am personally not well positioned to review the protein structural aspects of this work.
Strengths:<br /> - The authors first characterise migration towards serum in detail using a well-characterised lab strain but one of the main strengths of this study is that they then expand their scope to observe equivalent behaviours in several clinical serovars. This strongly supports the clinical relevance of the key behaviours that they document.
- The interdisciplinary nature of this study is a real strength and greatly increases the scope of the conclusions presented. Working from a structural understanding of chemoreceptor-ligand binding through to a larger-scale genetic analysis of chemoreceptor phylogeny allows the authors to draw important (although I find not always definitive) conclusions about bacterial migration towards human serum across a wide range of bacterial species (including many important pathogens). This is a very exciting approach and I was particularly interested to see the authors follow this up with observations of migration in C. koseri (a clinical isolate with little known about its chemotactic capabilities).
- The authors use experiments that compete migrating strains against each other and these offer an exciting glimpse into how bacterial movement and navigation could play out in multi-species environments like the human gut.
- The authors successfully identify a single component of human serum (i.e. serine) as one specific attractant driving the bacterial migration response seen here. Teasing apart the response of bacteria to complex stimuli like human serum is an important step here.
Weaknesses:<br /> There are several issues that I would personally like to see addressed in this study:
1) The authors refer to human serum as a chemoattractant numerous times throughout the study (including in the title). As the authors acknowledge, human serum is a complex mixture and different components of it may act as chemoattractants, chemo-repellents (particularly those with bactericidal activities), or may elicit other changes in motility (e.g. chemokinesis). The authors present convincing evidence that cells are attracted to serine within human serum - which is already a well-known bacterial chemoattractant. Indeed, their ability to elucidate specific elements of serum that influence bacterial motility is a real strength of the study. However, human serum itself is not a chemoattractant and this claim should be re-phrased - bacteria migrate towards human serum, driven at least in part by chemotaxis towards serine.
2) Linked to the previous point, several bacterial species (including E. coli - one of the bacterial species investigated here) are capable of osmotaxis (moving up or down gradients in osmolality). Whilst chemotaxis to serine is important here, could movement up the osmotic gradient generated by serum injection play a more general role? It could be interesting to measure the osmolality of the injected serum and test whether other solutions with similar osmolality elicit a similar migratory response. Another important control here would be to treat human serum with serine racemase and observe how this impacts bacterial migration.
3) The inference of the authors' genetic analysis combined with the migratory response of E. coli and C. koseri to human serum shown in Fig. 6 is that Tsr drives movement towards human serum across a range of Enterobacteriaceae species. The evidence for the importance of Tsr here is currently correlative - more causal evidence could be presented by either studying the response of tsr mutants in these two species (certainly these should be readily available for E. coli) or by studying the response of these two species to serine gradients.
4) The migratory response of E. coli looks striking when quantified (Fig. 6C), but is really unclear from looking at Panel B - it would be more convincing if an explanation was offered for why these images look so much less striking than analogous images for other species (E.g. Fig. 6A).
5) It is unclear why the fold-change in bacterial distribution shows an approximately Gaussian shape with a peak at a radial distance of between 50 -100 um from the source (see for example Fig. 2H). Initially, I thought that maybe this was due to the presence of the microcapillary needle at the source, but the CheY distribution looks completely flat (Fig. 3I). Is this an artifact of how the fold-change is being calculated? Certainly, it doesn't seem to support the authors' claim that cells increase in density to a point of saturation at the source. Furthermore, it also seems inappropriate to apply a linear fit to these non-linear distributions (as is done in Fig. 2H and in the many analogous figures throughout the manuscript).
6) The authors present several experiments where strains/ serovars competed against each other in these chemotaxis assays. As mentioned, these are a real strength of the study - however, their utility is not always clear. These experiments are useful for studying the effects of competition between bacteria with different abilities to climb gradients. However, to meaningfully interpret these effects, it is first necessary to understand how the different bacteria climb gradients in monoculture. As such, it would be instructive to provide monoculture data alongside these co-culture competition experiments.
7) Linked to the above point, it would be especially instructive to test a tsr mutant's response in monoculture. Comparing the bottom row of Fig. 3G to Fig. 3I suggests that when in co-culture with a cheY mutant, the tsr mutant shows a higher fold-change in radial distribution than the WT strain. Fig. 4G shows that a tsr mutant can chemotax towards aspartate at a similar, but reduced rate to WT. This could imply that (like the trg mutant), a tsr mutant has a more general motility defect (e.g. a speed defect), which could explain why it loses out when in competition with the WT in gradients of human serum, but actually seems to migrate strongly to human serum when in co-culture with a cheY mutant. This should be resolved by studying the response of a tsr mutant in monoculture.
8) In Fig. 4, the response of the three clinical serovars to serine gradients appears stronger than the lab serovar, whilst in Fig. 1, the response to human serum gradients shows the opposite trend with the lab serovar apparently showing the strongest response. Can the authors offer a possible explanation for these slightly confusing trends?
9) In Fig. S2, it seems important to present quantification of the effect of serine racemase and the reported lack of response to NE and DHMA - the single time-point images shown here are not easy to interpret.
10) Importantly, the authors detail how they controlled for the effects of pH and fluid flow (Line 133-136). Did the authors carry out similar controls for the dual-species experiments where fluorescent imaging could have significantly heated the fluid droplet driving stronger flow forces?
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Reviewer #1 (Public Review):
Strengths:<br /> The authors first perform several important controls to show that the expressed mutant actin is properly folded, and then show that the Arp2/3 complex behaves similarly with WT and mutant actin via a TIRF microscopy assay as well as a bulk pyrene-actin assay. A TIRF assay showed a small but significant reduction in the rate of elongation of the mutant actin suggesting only a mild polymerization defect.
Based on in silico analysis of the close location of the actin point mutation and bound cofilin, cofilin was chosen for further investigation. Faster de novo nucleation by cofilin was observed with mutant actin. In contrast, the mutant actin was more slowly severed. Both effects favor the retention of filamentous mutant actin. In solution, the effect of cofilin concentration and pH was assessed for both WT and mutant actin filaments, with a more limited repertoire of conditions in a TIRF assay that directly showed slower severing of mutant actin.
Lastly, the mutated residue in actin is predicted to interact with the cardiomyopathy loop in myosin and thus a standard in vitro motility assay with immobilized motors was used to show that non-muscle myosin 2A moved mutant actin more slowly, explained in part by a reduced affinity for the filament deduced from transient kinetic assays. By the same motility assay, myosin 5A also showed impaired interaction with the mutant filaments.
The Discussion is interesting and concludes that the mutant actin will co-exist with WT actin in filaments, and will contribute to altered actin dynamics and poor interaction with relevant myosin motors in the cellular context. While not an exhaustive list of possible defects, this is a solid start to understanding how this mutation might trigger a disease phenotype.
Weaknesses:<br /> Potential assembly defects of the mutant actin could be more thoroughly investigated if the same experiment shown in Fig. 2 was repeated as a function of actin concentration, which would allow the rate of disassembly and the critical concentration to also be determined.
The more direct TIRF assay for cofilin severing was only performed at high cofilin concentration (100 nM). Lower concentrations of cofilin would also be informative, as well as directly examining by the TIRF assay the effect of cofilin on filaments composed of a 50:50 mixture of WT:mutant actin, the more relevant case for the cell.
The more appropriate assay to determine the effect of the actin point mutation on class 5 myosin would be the inverted assay where myosin walks along single actin filaments adhered to a coverslip. This would allow an evaluation of class 5 myosin processivity on WT versus mutant actin that more closely reflects how Myo5 acts in cells, instead of the ensemble assay used appropriately for myosin 2.
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Joint Public Review:
The authors are trying to distinguish between four models of the role of glypicans (HSPGs) on the Dpp/BMP gradient in the Drosophila wing, schematized in Fig. 1: (1) "Restricted diffusion" (HSPGs transport Dpp via repetitive interaction of HS chains with Dpp); (2) "Hindered diffusion" (HSPGs hinder Dpp spreading via reversible interaction of HS chains with Dpp); (3) "Stabilization" (HSPGs stabilize Dpp on the cell surface via reversible interaction of HS chains with Dpp that antagonizes Tkv-mediated Dpp internalization); and (4) "Recycling" (HSPGs internalize and recycle Dpp).
To distinguish between these models, the authors generate new alleles for the glypicans Dally and Dally-like protein (Dlp) and for Dpp: a Dally knock-out allele, a Dally YFP-tagged allele, a Dally knock-out allele with 3HA-Dlp, a Dlp knock-out allele, a Dlp allele containing 3-HA tags, and a Dpp lacking the HS-interacting domain. Additionally, they use an OLLAS-tag Dpp (OLLAS being an epitope tag against which extremely high affinity antibodies exist). They examine OLLAS-Dpp or HA-Dpp distribution, phospho-Mad staining, adult wing size.
They find that over-expressed Dally - but not Dlp - expands Dpp distribution in the larval wing disc. They find that the Dally[KO] allele behaves like a Dally strong hypomorph Dally[MH32]. The Dally[KO] - but not the Dlp[KO] - caused reduced pMad in both anterior and posterior domains and reduced adult wing size (particularly in the Anterior-Posterior axis). These defects can be substantially corrected by supplying an endogenously tagged YFP-tagged Dally. By contrast, they were not rescued when a 3xHA Dlp was inserted in the Dally locus. These results support their conclusion that Dpp interacts with Dally but not Dlp.
They next wanted to determine the relative contributions of the Dally core or the HS chains to the Dpp distribution. To test this, they over-expressed UAS-Dally or UAS-Dally[deltaHS] (lacking the HS chains) in the dorsal wing. Dally[deltaHS] over-expression increased the distribution of OLLAS-Dpp but caused a reduction in pMad. They do a critical experiment, making the Dally[deltaHS] allele, they find that loss of the HS chains is nearly as severe as total loss of Dally (i.e., Dally[KO]). These results indicate that the HS are critical for Dally's role in Dpp distribution and signaling.
Prior work has shown that a stretch of 7 amino acids in the Dpp N-terminal domain is required to interact with heparin but not with Dpp receptors (Akiyama, 2008). The authors generated an HA-tagged Dpp allele lacking these residues (HA-dpp[deltaN]). It is an embryonic lethal allele, but they can get some animals to survive to larval stages if they also supply a transgene called "JAK" containing dpp regulatory sequences. In the JAK; HA-dpp[deltaN] mutant background, they find that the distribution and signaling of this Dpp molecule is largely normal. While over-expressed Dally can increase the distribution of HA-dpp[deltaN], over-expression of Dally[deltaHS] cannot. These latter results support the model that the HS chains in Dally are required for Dpp function but not because of a direct interaction with Dpp.
In the last part of the results, they attempt to determine if the Dpp receptor Thickveins (Tkv) is required for Dally-HS chains interaction. The 2008 (Akiyama) model posits that Tkv activates pMad downstream of Dpp and also internalizes and degrades Dpp. A 2022 (Romanova-Michaelides) model proposes that Dally (not Tkv) internalizes Dpp. To distinguish between these models, the authors deplete Tkv from the dorsal compartment of the wing disc and found that extracellular Dpp increased and expanded in that domain. These results support the model that Tkv is required to internalize Dpp. They then tested the model that Dally antagonizes Tkv-mediated Dpp internalization by determining whether the defective extracellular Dpp distribution in Dally[KO] mutants could be rescued by depleting Tkv. Extracellular Dpp did increase in the D vs V compartment, potentially providing some support for their model. The results are statistically significant but the statistics are buried in an excel file without a read-me page. The code for the statistics is available from Github. These p values should be made more readily accessible and/or intelligible to the reader.
Strengthens:<br /> 1. New genomically-engineered alleles<br /> A considerable strength of the study is the generation and characterization of new Dally, Dlp and Dpp alleles. These reagents will be of great use to the field.
2. Surveying multiple phenotypes<br /> The authors survey numerous parameters (Dpp distribution, Dpp signaling (pMad) and adult wing phenotypes) which provides many points of analysis.
Weaknesses (minor):<br /> 1. The results are statistically significant but the statistics are buried in a dense excel file without a read-me page. The code for the statistics is available from Github. These p values should be made more readily accessible to the reader.
An appraisal of whether the authors achieved their aims, and whether the results support their conclusions.<br /> The authors' model is that Dally (not Dlp) is required for Dpp distribution and signaling but that this is not due to a direct interaction with Dpp. Rather, they posit that Dally-HS antagonize Tkv-mediated Dpp internalization. Currently the results of the experiments could be considered consistent with their model. Finally, their results support the idea that one or more as-yet unidentified proteins interact with Dally-HS chains to control Dpp distribution and signaling in the wing disc.
There is much debate and controversy in the Dpp morphogen field. The generation of new, high quality alleles in this study will be useful to Drosophila community, and the results of this study support the concept that Tkv but not Dally regulate Dpp internalization. Thus the work could be impactful and fuel new debates among the morphogen researchers.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound, and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability, and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound (apart from a few points mentioned below) and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation have not been explored.
Strengths:<br /> The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound (apart from a few comments mentioned below). The results are clear and support the conclusions.
Weaknesses:<br /> 1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4-bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper, a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta/gamma is presented.
2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.
3) It is difficult to judge the error in the HDX experiments presented in Sup. data 1 and 2. In the method section, it is stated that the results represent the average from two samples. How is the SD error calculated in Fig.1B-C?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> During the last decades, extensive studies (mostly neglected by the authors), using in vitro and in vivo models, have elucidated the five-step mechanism of intoxication of botulinum neurotoxins (BoNTs). The binding domain (H chain) of all serotypes of BoNTs binds polysialogangliosides and the luminal domain of a synaptic vesicle protein (which varies among serotypes). When bound to the synaptic membrane of neurons, BoNTs are rapidly internalized by synaptic vesicles (SVs) via endocytosis. Subsequently, the catalytic domain (L chain) translocates, a process triggered by the acidification of these organelles. Following translocation, the disulfide bridge connecting the H chain with the L chain is reduced by the thioredoxin reductase/thioredoxin system, and it is refolded by the chaperone Hsp90 on SV's surface. Once released into the cytosol, the L chains of different serotypes cleave distinct peptide bonds of specific SNARE proteins, thereby disrupting neurotransmission.
In this study, Yeo et al. extensively revise the neuronal intoxication model, suggesting that BoNT/A follows a more complex intracellular route than previously thought. The authors propose that upon internalization, BoNT/A-containing endosomes are retro-axonally trafficked to the soma. At the level of the neuronal soma, this serotype then traffics to the endoplasmic reticulum (ER) via the Golgi apparatus. The ER SEC61 translocon complex facilitates the translocation of BoNT/A's LC from the ER lumen into the cytosol, where the thioredoxin reductase/thioredoxin system and HSP complexes release and refold the catalytic L chain. Subsequently, the L chain diffuses and cleaves SNAP25 first in the soma before reaching neurites and synapses.
Strengths:<br /> I appreciate the authors' efforts to confirm that the newly established methods somehow recapitulate aspects of the BoNTs mechanism of action, such as toxin binding and uptake occurring at the level of active synapses. Furthermore, even though I consider the SNAPR approach inadequate, the genome-wide RNAi screen has been well executed and thoroughly analyzed. It includes well-established positive and negative controls, making it a comprehensive resource not only for scientists working in the field of botulinum neurotoxins but also for cell biologists studying endocytosis more broadly.
Weaknesses:<br /> I have several concerns about the authors' main conclusions, primarily due to the lack of essential controls and validation for the newly developed methods used to assess toxin cleavage and trafficking into neurons. Furthermore, there is a significant discrepancy between the proposed intoxication model and existing studies conducted in more physiological settings. In my opinion, the authors have omitted over 20 years of work done in several labs worldwide (Montecucco, Montal, Schiavo, Rummel, Binz, etc.). I want to emphasize that I support changes in biological dogma only when these changes are supported by compelling experimental evidence, which I could not find in the present manuscript.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> This study examines the spatial and temporal patterns of occurrence and the interspecific associations within a terrestrial mammalian community along human disturbance gradients. They conclude that human activity leads to a higher incidence of positive associations.
Strengths:<br /> The theoretical framework of the study is brilliantly introduced. Solid data and sound methodology. This study is based on an extensive series of camera trap data. Good review of the literature on this topic.
Weaknesses:<br /> The authors use the terms associations and interactions interchangeably. It is not clear what the authors mean by "associations". A brief clarification would be helpful. Also, the authors do not delve into the different types of association found in the study. A more ecological perspective explaining why certain species tend to exhibit negative associations and why others show the opposite pattern (and thus, can be used as indicator species) is missing. Also, the authors do not distinguish between significant (true) non-random associations and random associations. In my opinion, associations are those in which two species co-occur more or less than expected by chance. This is not well addressed in the present version of the manuscript.
The obtained results support the conclusions of the study.
Anthropogenic pressures can shape species associations by increasing spatial and temporal co-occurrence, but above a certain threshold, the positive influence of human activity in terms of species associations could be reverted. This study can stimulate further work in this direction.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The manuscript has helped address a long-standing mystery in splicing regulation: whether splicing occurs co- or post-transcriptionally. Specifically, the authors (1) uniquely combined smFISH, expansion microscopy, and live cell imaging; (2) revealed the ordering and spatial distribution of splicing steps; and (3) discovered that nascent, not-yet-spliced transcripts move more slowly around the transcription site and undergo splicing as they move through the clouds. Based on the experimental results, the authors suggest that the observation of co-transcriptional splicing in previous literature could be due to the limitation of imaging resolution, meaning that the observed co-transcriptional splicing might actually be post-transcriptional splicing occurring in proximity to the transcription site. Overall, the work presented here clearly provides a comprehensive picture of splicing regulation.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary<br /> While DNA sequence divergence, differential expression, and differential methylation analysis have been conducted between humans and the great apes to study changes that "make us human", the role of lncRNAs and their impact on the human genome and biology has not been fully explored. In this study, the authors computationally predict HSlncRNAs as well as their DNA Binding sites using a method they have developed previously and then examine these predicted regions with different types of enrichment analyses. Broadly, the analysis is straightforward and after identifying these regions/HSlncRNAs the authors examined their effects using different external datasets.
I no longer have any concerns about the manuscript as the authors have addressed my comments in the first round of review.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
While the approach used in this study cannot identify cause and effect, the whole brain approach identified clusters representing circuits of potential importance and a series of new hypotheses to explore. The importance of the role of sexual behavior, specifically ejaculations rates, is worth emphasizing for the formation of pair bonds. It suggests that the role of sexual behavior in contributing to the strength of pair bonds should be explored more. It is also important to add that males and females in the study were screened for sexual receptivity. The identification of brain regions for pair bond maintenance centered around the amygdala was also intriguing.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this article, different machine learning models (pan-specific, peptide-specific, pre-trained, and ensemble models) are tested to predict TCR-specificity from a paired-chain peptide-TCR dataset. The data consists of 6,358 positive observations across 26 peptides (as compared to six peptides in NetTCR version 2.1) after several pre-processing steps (filtering and redundancy reduction). For each positive sample, five negative samples were generated by swapping TCRs of a given peptide with TCRs binding to other peptides. The weighted loss function is used to deal with the imbalanced dataset in pan-specific models.
The results demonstrate that the redundant data introduced during training did not lead to performance gain; rather, a decrease in performance was observed for the pan-specific model. The removal of outliers leads to better performance.<br /> <br /> To further improve the peptide-specific model performance, an architecture is created to combine pan-specific and peptide-specific models, where the pan-specific model is trained on pan-specific data while keeping the peptide-specific part of the model frozen, and the peptide-specific model is trained on a peptide-specific dataset while keeping the pan-specific part of the model frozen. This model surpassed the performance of individual pan-specific and peptide-specific models. Finally, sequence similarity-based predictions of TCRbase are integrated into the pre-trained CNN model, which further improved the model performance (mostly due to the better discrimination of binders and non-binders).<br /> <br /> The prediction for unseen peptides is still low in a pan-specific model; however, an improvement in prediction is observed for peptides with high similarity to the ones in the training dataset. Furthermore, it is shown that 15 observations show satisfactory performance as compared to the ~150 recommended in the literature.<br /> <br /> Models are evaluated on the external dataset (IMMREP benchmark). Peptide-specific models performed competitively with the best models in the benchmark. The pre-trained model performed worst, which the authors suggested could be because of positive and negative sample swapping across training and testing sets. To resolve this issue, they applied the redundancy removal technique to the IMMER dataset. The results agreed with the earlier conclusion that the pre-trained models surpassed peptide-specific models and the integration of similarity-based methods leads to performance boost. It highlights the need for the creation of a new benchmark without data redundancy or leakage problems.
The manuscript is well-written, clear, and easy to understand. The data is effectively presented. The results validate the drawn conclusions.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Here, the authors compare how different operationalizations of adverse childhood experience exposure related to patterns of skin conductance response during a fear conditioning task. They use a large dataset to definitively understand a phenomenon that, to date, has been addressed using a range of different definitions and methods, typically with insufficient statistical power. Specifically, the authors compared the following operationalizations: dichotomization of the sample into "exposed" and "non-exposed" categories, cumulative adversity exposure, specificity of adversity exposure, and dimensional (threat versus deprivation) adversity exposure. The paper is thoughtfully framed and provides clear descriptions and rationale for procedures, as well as package version information and code. The authors' overall aim of translating theoretical models of adversity into statistical models, and comparing the explanatory power of each model, respectively, is an important and helpful addition to the literature. However, the analysis would be strengthened by employing more sophisticated modelling techniques that account for between-subjects covariates and the presentation of the data needs to be streamlined to make it clearer for the broad audience for which it is intended.
Strengths<br /> Several outstanding strengths of this paper are the large sample size and its primary aim of statistically comparing leading theoretical models of adversity exposure in the context of skin conductance response. This paper also helpfully reports Cohen's d effect sizes, which aid in interpreting the magnitude of the findings. The methods and results are generally thorough.
Weaknesses<br /> The largest concern is that the paper primarily relies on ANOVAs and pairwise testing for its analyses and does not include between-subjects covariates. Employing mixed-effects models instead of ANOVAs would allow more sophisticated control over sources of random variance in the sample (especially important for samples from multi-site studies such as the present study), and further allow the inclusion of potentially relevant between-subjects covariates such as age (e.g. Eisenstein et al., 1990) and gender identity or sex assigned at birth (e.g. Kopacz II & Smith, 1971) (perhaps especially relevant due to possible to gender or sex-related differences in ACE exposure; e.g. Kendler et al., 2001). Also, proxies for socioeconomic status (e.g. income, education) can be linked with ACE exposure (e.g. Maholmes & King, 2012) and warrant consideration as covariates, especially if they differ across adversity-exposed and unexposed groups. On a related methodological note, the authors mention that scores representing threat and deprivation were not problematically collinear due to VIFs being <10; however, some sources indicate that VIFs should be <5 (e.g. Akinwande et al., 2015).
Additionally, the paper reports that higher trait anxiety and depression symptoms were observed in individuals exposed to ACEs, but it would be helpful to report whether patterns of SCR were in turn associated with these symptom measures and whether the different operationalizations of ACE exposure displayed differential associations with symptoms. Given the paper's framing of SCR as a potential mechanistic link between adversity and mental health problems, reporting these associations would be a helpful addition. These results could also have implications for the resilience interpretation in the discussion (lines 481-485), which is a particularly important and interesting interpretation.
Given that the manuscript criticizes the different operationalizations of childhood adversity, there should be greater justification of the rationale for choosing the model for the main analyses. Why not the 'cumulative risk' or 'specificity' model? Related to this, there should also be a stronger justification for selecting the 'moderate' approach for the main analysis. Why choose to cut off at moderate? Why not severe, or low? Related to this, why did they choose to cut off at all? Surely one could address this with the continuous variable, as they criticize cut-offs in Table 2.
In the Introduction, the authors predict less discrimination between signals of danger (CS+) and safety (CS-) in trauma-exposed individuals driven by reduced responses to the CS+. Given the potential impact of their findings for a larger audience, it is important to give greater theoretical context as to why CS discrimination is relevant here, and especially what a reduction in response specifically to danger cues would mean (e.g. in comparison to anxiety, where safety learning is impacted).
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
The present work establishes 14-3-3 proteins as binding partners of spastin and suggests that this binding is positively regulated by phosphorylation of spastin. The authors show evidence that 14-3-3 - spastin binding prevents spastin ubiquitination and final proteasomal degradation. By using drugs and peptides that separately inhibit 14-3-3 binding or spastin activity, they show that both proteins are necessary for axon regeneration in cell culture and in vivo models in rats.
Major strengths<br /> -The data establishing 14-3-3 and spastin as binding partners is convincing, as is its regulation by phosphorylation and its impact on protein levels related to the activity of the ubiquitin-proteasome system.
-The effects of FC-A on locomotor recovery after spinal cord contusion is very interesting.
Major weaknesses<br /> -Given that spastazoline has a major impact on neurite outgrowth suggests that cells simply cannot grow in the presence of the inhibitor and raises serious questions about any selectivity for the concomitant effect FC-A - dependent growth.
-The histological data and analyses following spinal cord injury are not convincing. For example, the colabeling of NF and 5-HT is not convincingly labeling fibers. Also, the quality, resolution and size of the images is insufficient to support the quantitative data and it is hence difficult to interpret the data. Reviewers recognize that during the review process, efforts were made to improve the quality of the images.
-Reviewers also observed that the data to infer Spastin actions on Microtubules across different experimental models is weak and that claims about "MT severing" and "microtubules dynamics" were wrongly used given the provided evidence.
-The manuscript lacks direct evidence that a 14-3-3 and spastin function as a complex in the same pathway to promote regeneration. It is recognized, however, that the authors had made changes in the manuscript title and claims not to imply that the current evidence is sufficient in that matter.
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www.youtube.com www.youtube.com
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The Angry River - The Hat ft. father John Misty (with lyrics) {YouTube}
site:: [[YouTube]] channel:: fatpetiobaby101 date:: 2014-04-16 url:: https://www.youtube.com/watch?v=hfzrRdarnEs accessed:: 2023-12-18
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The paper by Majeed et al has a valuable and worthwhile aim: to provide a set of tools to standardize the quantification of synapses using fluorescent markers in the nematode C. elegans. Using current approaches, the identification of synapses using fluorescent markers is tedious and subject to significant inter-experimenter variability. Majeed et al successfully develop and validate a computational pipeline called "WormPsyQi" that overcomes some of these obstacles and will be a powerful resource for many C. elegans neurobiologists.
Strengths:
The computational pipeline is rigorously validated and shown to accurately quantitate fluorescent puncta, at least as well as human experimenters. The inclusion of a mask - a region of interest defined by a cytoplasmic marker - is a powerful and useful approach. Users can take advantage of one of four pre-trained neural networks, or train their own. The software is freely available and appears to be user-friendly. A series of rigorous experiments demonstrates the utility of the pipeline for measuring differences in the number of synaptic puncta between sexes and across developmental stages. Neuron-to-neuron heterogeneity in patterns of synaptic growth during development are convincingly demonstrated. Weaknesses and caveats are realistically discussed.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The authors developed computational models that capture the electrical and Ca2+ signaling behavior in mesenteric arterial cells from male and female mice. Sex-specific differences in the L-type calcium channel and two voltage-gated potassium channels were carefully tuned based on experimental measurements. To incorporate the stochasticity of ion channel openings seen in smooth muscle cells under physiological conditions, noise was added to the membrane potential and the sarcoplasmic Ca2+ concentration equations. Finally, the models were assembled into 1D vessel representations and used to investigate the tissue-level electrical response to an L-type calcium channel blocker. This comprehensive computational framework helped provide nuanced insight into arterial myocyte function difficult to achieve through traditional experimental methods and can be further expanded into tissue-level studies that incorporate signaling pathways for blood pressure control.
Throughout the paper, model behavior was both validated by experimental recordings and well supported by previously published data. The main findings from the models suggested that sex-specific differences in membrane potential regulation and Ca2+ handling are attributable to variability in the gating of a small number of voltage-gated potassium channels and L-type calcium channels. This variability contributes to a higher Ca2+ channel blocker sensitivity in female arterial vessels. Overall, the study successfully presented novel sex-specific computational models of mesenteric arterial myocytes and demonstrated their use in drug-testing applications.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Otarigho et al. presented a convincing study revealing that in C. elegans, the neuropeptide Y receptor GPCR/NPR-15 mediates both molecular and behavioral immune responses to pathogen attack. Previously, three npr genes were found to be involved in worm defense. In this study, the authors screened mutants in the remaining npr genes against P. aeruginosa-mediated killing and found that npr-15 loss-of-function improved worm survival. npr-15 mutants also exhibited enhanced resistance to other pathogenic bacteria but displayed significantly reduced avoidance to S. aureus, independent of aerotaxis, pathogen intake and defecation. The enhanced resistance in npr-15 mutant worms was attributed to upregulation of immune and neuropeptide genes, many of which were controlled by the transcription factors ELT-2 and HLH-30. The authors found that NPR-15 regulates avoidance behavior via the TRPM gene, GON-2, which has a known role in modulating avoidance behavior through the intestine. The authors further showed that both NPR-15-dependent immune and behavioral responses to pathogen attack were mediated by the NPR-15-expressing neurons ASJ. Overall, the authors discovered that the NPR-15/ASJ neural circuit may regulate distinct defense mechanisms against pathogens under different circumstances. This study provides novel and useful information to researchers in the fields of neuroimmunology and C. elegans research.
Strengths:
1. This study uncovered specific molecules and neuronal cells that regulate both molecular immune defense and behavior defense against pathogen attack and indicate that the same neural circuit may regulate distinct defense mechanisms under different circumstances. This discovery is significant because it not only reveals regulatory mechanisms of different defense strategies but also suggests how C. elegans utilize its limited neural resources to accomplish complex regulatory tasks.
2. The conclusions in this study are supported by solid evidence, which are often derived from multiple approaches and/or experiments. Multiple pathogenic bacteria were tested to examine the effect of NPR-15 loss-of-function on immunity; the impacts of pharyngeal pumping and defecation on bacterial accumulation were ruled out when evaluating defense; RNA-seq and qPCR were used to measure gene expression; gene inactivation was done in multiple strains to assess gene function.
3. Gene differential expression, gene ontology and pathway analyses were performed to demonstrate that NPR-15 controls immunity through regulating immune pathways.
4. Elegant approaches were employed to examine avoidance behavior (partial lawn, full lawn, and lawn occupancy) and the involvement of neurons in regulating immunity and avoidance (the use of a diverse array of mutant strains).
5. Statistical analyses were appropriate and adequate.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> In this manuscript, Ruichen Yang et al. investigated the importance of BMP signaling in preventing microtia. The authors showed that Cre recombinase-mediated deletion of Bmpr1a using skeletal stem-specific Cre Prx1Cre leads to microtia in adult and young mice. In these mice, the distal auricle is more affected than the middle and proximal. In these Bmpr1a floxed Prx1Cre mice, auricle chondrocytes start to differentiate into osteoblasts through an increase in PKA signaling. The authors showed human single-cell RNA-Seq data sets where they observed increased PKA signaling in microtia patients which resembles their animal model experiments.
Strengths:<br /> Although the importance of BMP signaling in skeletal tissues has been previously reported, the importance of its role in microtia prevention is novel and very promising to study in detail. The authors satisfied the experimental questions by performing the correct methods and explaining the results in detail.
Weaknesses:<br /> There are minor concerns like typo mistakes and missing control data histology pictures which should be corrected.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Summary:
The major purpose of this manuscript is to examine whether leucine treatment would be a potential strategy to treat cytokine storm syndrome (CSS). CSS is a common symptom in multiple infectious diseases in clinic, gradually leads to multiple organ failure and high mortality. Strategies to treat CSS including pulse steroid therapy normally leads to severe side effects. Therefore, it is still required to develop safe strategy with high efficacy to treat CSS. In clinic, sepsis is well characterized to exhibit CSS and therefore multiple studies utilized LPS-induced sepsis model to evaluate CSS symptom. In this study, the authors examined whether leucine, an essential amino acid that has been absorbed daily in our body, could ameliorate CSS symptom in the LPS-induced sepsis mouse model. They found a potential protective effect of leucine in terms of the survival rate and inflammatory responses.
Strengths:
The study is overall well designed and the results are well analyzed with only minor issues. The methods they utilized is appropriate.
Weaknesses:
The mechanistical insights are not sufficient and could not fully explain the phenotype they found. Considering the importance of this study is to identify the potential protective role of leucine in CSS, the authors could also consider investigator-initiated clinical trials to further expand the significance of this study.
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
Summary:<br /> Seignette et al. investigated the potential roles of axo-axonic (chandelier) cells (ChCs) in a sensory system, namely visual processing. As introduced by the authors, the axo-axonic cell type has remained (and still is) somehow mysterious in its function. Seignette and colleagues leveraged the development of a transgenic mouse line selective for ChC, and applied a very wide range of techniques: transsynaptic rabies tracing, optogenetic input activation, in vitro electrophysiology, 2-photon recording in vivo, behavior and chemogenetic manipulations, to precisely determine the contribution of ChCs to the primary visual cortex network.
The main findings are 1) the identification of synaptic inputs to ChC, with a majority of local, deep layer principal neurons (PN), 2) the demonstration that ChC is strongly and synchronously activated by visual stimuli with low specificity in naive animals, 3) the recruitment of ChC by arousal/visuomotor mismatch, 4) the induction of functional and structural plasticity at the ChC-PN module, and, 5) the weak disinhibition of PNs induced by ChCs silencing. All these findings are strongly supported by experimental data and thoroughly compared to available evidence.
Strengths:<br /> This article reports an impressive range of very demanding experiments, which were well executed and analyzed, and are presented in a very clear and balanced manner. Moreover, the manuscript is well-written throughout, making it appealing to future readers. It has also been a pleasure to review this article.
In sum, this is an impressive study and an excellent manuscript, that presents no major flaws.
Notably, this study is one of the first studies to report on the activities and potential roles of axo-axonic cells in an active, integrated brain process, beyond locomotion as reported and published in V1. This type of research was much awaited in the fields of interneuron and vision research.
Weaknesses:<br /> There are no fundamental weaknesses; the latter mainly concern the presentation of the main results.
The main weakness may be that the different sections appear somehow disconnected conceptually.
Additionally, some parts deserve a more in-depth clarification/simplification of concepts and analytic methods for scientists outside the subfield of V1 research. Indeed, this paper will be of key interest to researchers of various backgrounds.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> In this paper, the authors show that the degree of pigmentation for RPE cells is not correlated with a level of maturation and function. They suggest that this status could be different in vitro than in vivo but do not provide proper experiments to validate this hypothesis. However, it is the first time that the absence of a correlation between pigmentation and function has been studied.
Strengths:<br /> The methods are good and the experiments are very rigorous.
Weaknesses:<br /> Demonstration of in vitro but no in vivo data.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> This work describes a simple mechanical model of worm locomotion, using a series of rigid segments connected by damped torsional springs and immersed in a viscous fluid. It uses this model to simulate forward crawling movement, as well as omega turns.
Strengths:<br /> The primary strength is in applying a biomechanical model to omega-turn behaviors. The biomechanics of nematode turning behaviors are relatively less well described and understood than forward crawling. The model itself may be a useful implementation to other researchers, particularly owing to its simplicity.
Weaknesses:<br /> The strength of the model presented in this work relative to prior approaches is not well supported, and in general, the paper would be improved with a better description of the broader context of existing modeling literature related to undulatory locomotion. This paper claims to improve on previous approaches to taking body shapes as inputs. However, the sole nematode model cited aims to do something different, and arguably more significant, which is to use experimentally derived parameters to model both the neural circuits that induce locomotion as well as the biomechanics and to subsequently compare the model to experimental data. Other modeling approaches do take experimental body kinematics as inputs and use them to produce force fields, however, they are not cited or discussed. Finally, the overall novelty of the approach is questionable. A functionally similar approach was developed in 2012 to describe worm locomotion in lattices (Majmudar, 2012, Roy. Soc. Int.), which is not discussed and would provide an interesting comparison and needed context.
The idea of applying biomechanical models to describe omega turns in C. elegans is a good one, however, the kinematic basis of the model as used in this paper (the authors do note that the control angle could be connected to a neural model, but don't do so in this work) limits the generation of neuromechanical control hypotheses. The model may provide insights into the biomechanics of such behaviors, however, the results described are very minimal and are purely qualitative. Overall, direct comparisons to the experiments are lacking or unclear. Furthermore, the paper claims the value of the model is to produce the force fields from a given body shape, but the force fields from omega turns are only pictured qualitatively. No comparison is made to other behaviors (the force experienced during crawling relative to turning for example might be interesting to consider) and the dependence of the behavior on the model parameters is not explored (for example, how does the omega turn change as the drag coefficients are changed). If the purpose of this paper is to recapitulate the swim-to-crawl transition with a simple model, and then apply the model to new behaviors, a more detailed analysis of the behavior of the model variables and their dependence on the variables would make for a stronger result. In some sense, because the model takes kinematics as an input and uses previously established techniques to model mechanics, it is unsurprising that it can reproduce experimentally observed kinematics, however, the forces calculated and the variation of parameters could be of interest.
Relatedly, a justification of why the drag coefficients had to be changed by a factor of 100 should be explored. Plate conditions are difficult to replicate and the rheology of plates likely depends on a number of factors, but is for example, changes in hydration level likely to produce a 100-fold change in drag? or something more interesting/subtle within the model producing the discrepancy?
Finally, the language used to distinguish different modeling approaches was often unclear. For example, it was unclear in what sense the model presented in Boyle, 2012 was a "kinetic model" and in many situations, it appeared that the term kinematic might have been more appropriate. Other phrases like "frictional forces caused by the tension of its muscles" were unclear at first glance, and might benefit from revision and more canonical usage of terms.
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Reviewer #1 (Public Review):
With genephys, the author provides a generative model of brain responses to stimulation. This generative model allows to mimic specific parameters of a brain response at the sensor level, to test the impact of those parameters on critical analytic methods utilized on real M/EEG data. Specifically, they compare the decoding output for differently set parameters to the decoding pattern observed in a classical passive viewing study in terms of the resulting temporal generalization matrix (TGM). They identify that the correspondence between the mimicked and the experimental TGM to depend on an oscillatory component that spans multiple channels, frequencies, and latencies of response; and an additive, slower response with a specific (cross-frequency) relation to the phase of the oscillatory, faster component.
A strength of the article is that it considers the complexity of neural data that contribute to the findings obtained in stimulation experiments. An additional strength is the provision of a Python package that allows scientists to explore the potential contribution of different aspects of neural signals to obtained experimental data and thereby to potentially test their theoretical assumptions critical parameters that contribute to their experimental data.
A weakness of the paper is that the power of the model is illustrated for only one specific set of parameters, added in a stepwise manner and the comparison to on specific empirical TGM, assumed to be prototypical; And that this comparison remains descriptive. (That is could a different selection of parameters lead to similar results and is there TGM data which matches these settings less well.) It further remained unclear to me, which implications may be drawn from the generative model, following from the capacities to mimic this specific TGM (i) for more complex cases, such as the comparison between experimental conditions, and (ii) about the complex nature of neural processes involved.
Towards this end I would appreciate (i) a more profound explanation of the conclusions that can be drawn from this specific showcase, including potential limitations, as well as wider considerations of how scientists may empower the generative model to (ii) understand their experimental data better and (iii) which added value the model may have in understanding the nature of underlaying brain mechanism (rather than a mere technical characterization of sensor data).
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Reviewer #1 (Public Review):
Force sensing and gating mechanisms of the mechanically activated ion channels is an area of broad interest in the field of mechanotransduction. These channels perform important biological functions by converting mechanical force into electrical signals. To understand their underlying physiological processes, it is important to determine gating mechanisms, especially those mediated by lipids. The authors in this manuscript describe a mechanism for mechanically induced activation of TREK-1 (TWIK-related K+ channel. They propose that force induced disruption of ganglioside (GM1) and cholesterol causes relocation of TREK-1 associated with phospholipase D2 (PLD2) to 4,5-bisphosphate (PIP2) clusters, where PLD2 catalytic activity produces phosphatidic acid that can activate the channel. To test their hypothesis, they use dSTORM to measure TREK-1 and PLD2 colocalization with either GM1 or PIP2. They find that shear stress decreases TREK-1/PLD2 colocalization with GM1 and relocates to cluster with PIP2. These movements are affected by TREK-1 C-terminal or PLD2 mutations suggesting that the interaction is important for channel re-location. The authors then draw a correlation to cholesterol suggesting that TREK-1 movement is cholesterol dependent. It is important to note that this is not the only method of channel activation and that one not involving PLD2 also exists. Overall, the authors conclude that force is sensed by ordered lipids and PLD2 associates with TREK-1 to selectively gate the channel.
The proposed mechanism is solid and the authors have revised the manuscript to address previous issues with the first version
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Reviewer #1 (Public Review):
Summary:<br /> The authors posed a research question about how an animal integrates sensory information to optimize its behavioral outputs and how this process evolved. Their data (behavioral output analysis with detailed categories in response to the different odors in different concentrations by comparing surface and cave populations and their hybrid) partially answer this tough question. They built a new low-disturbance system to answer the question. They also found that the personality of individual fish is a good predictor of behavioral outputs against odor response. They concluded that cavefish evolved to specialize their response to alanine and histidine while surface fish are more general responders, which was supported by their data.
Strengths:<br /> With their new system, the authors could generate clearer results without mechanical disturbances. The authors characterize multiple measurements to score the odor response behaviors, and also brought a new personality analysis. Their conclusion that cavefish evolved as a specialist to sense alanine and histidine among 6 tested amino acids was well supported by their data.
Weaknesses:<br /> The authors posed a big research question: How do animals evolve the processes of sensory integration to optimize their behavioral outputs? I personally feel that, to answer the questions about how sensory integration generates proper (evolved) behavior, the authors at least need to show the ecological relevance of their response. For the alanine/histidine preference in cavefish, they need data for the alanine and other amino acid concentrations in the local cave water and compare them with those of surface water.
Also, as for "personality matters", I read that personality explains a large variation in surface fish. Also, thigmotaxis or wall-following cavefish individuals are exceeded to respond well to odorants compared with circling and random swimming cavefish individuals. However, I failed to understand the authors' point about how much percentages of the odorant-response variations are explained (PVE) by personality. Association (= correlation) was good to show as the authors presented, but showing proper PVE or the effect size of personality to predict the behavioral outputs is important to conclude "personality is matter"; otherwise, the conclusion is not so supported.
From the above, I recommend the authors reconsider the title also their research questions well. At this moment, I feel that the authors' conclusions and their research questions are a little too exaggerated, with less supportive evidence.
Also, for the statistical method, Fisher's exact test is not appropriate for the compositional data (such as Figure 2B). The authors may quickly check it at https://en.wikipedia.org/wiki/Compositional_data or https://www.annualreviews.org/doi/pdf/10.1146/annurev-statistics-042720-124436.
The authors may want to use centered log transformation or other appropriate transformations (R-package could be: https://doi.org/10.1016/j.cageo.2006.11.017). According to changing the statistical tests, the authors' conclusion may not be supported.
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Reviewer #1 (Public Review):
Establishing direct links between the neuronal connectivity information of connectomics datasets with circuit physiology and behavior and exciting current research area in neurobiology. Until recently, studies of aggression in Drosophila had been conducted largely in males, and many of the neurons involved in this behavior are male-specific clusters. Since the currently available fly brain connectomes come from female brains, their applicability for the study of the circuitry underlying aggressive behavior is very limited.
The authors have previously used the Janelia hemibrain connectome paired with behavior analysis to show that activating either the aIPg or pC1d cell types can induce short term aggression in females, while activation of other PC1 clusters (a-c and e) does not. Here they expand on those findings, showing that optogenetic stimulation of aIPg neurons was sufficient to promote an aggressive internal state lasting at least 10 minutes following a 30 second activation. In addition, authors show that while stimulation of PC1d alone is not sufficient to induce this persistent aggressive state, simultaneous activation of PC1d + PC1e is, suggesting a synergistic effect. Connectomics analysis performed in the authors' previous study had shown that PC1d and aIPg are interconnected. However, silencing pC1d neuronal activity did not reduce aIPg-evoked persistent aggression, indicating that the aggressive state did not depend on pC1d-aIPg recurrent connectivity.
The conclusions are well supported by the data, and the results presented in this manuscript represent an important contribution to our understanding of the neuronal circuitry underlying female aggression.
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Reviewer #1 (Public Review):
The authors took advantage of a large dataset of transcriptomic information obtained from parasites recovered from 35 patients. In addition, parasites from 13 of these patients were reared for 1 generation in vivo, 10 for 2 generations, and 1 for a third generation. This provided the authors with a remarkable resource for monitoring how parasites initially adapt to the environmental change of being grown in culture. They focused initially on var gene expression due to the importance of this gene family for parasite virulence, then subsequently assessed changes in the entire transcriptome. Their goal was to develop a more accurate and informative computational pipeline for assessing var gene expression and secondly, to document the adaptation process at the whole transcriptome level.
Overall, the authors were largely successful in their aims. They provide convincing evidence that their new computational pipeline is better able to assemble var transcripts and assess the structure of the encoded PfEMP1s. They can also assess var gene switching as a tool for examining antigenic variation. They also documented potentially important changes in the overall transcriptome that will be important for researchers who employ ex vivo samples for assessing things like drug sensitivity profiles or metabolic states. These are likely to be important tools and insights for researchers working on field samples.
Interestingly, the conclusions about changes in var gene expression due to the transition to in vitro culture (one of the primary goals of the paper) were somewhat difficult to assess. The authors found that in most instances, var gene expression patterns changed only modestly. However, in a few cases, more substantial changes were observed. Thus, it is difficult to make firm conclusions about how one should interpret var gene expression profiles in parasites recently placed in culture. Changes in the core transcriptome however were more pronounced, justifying the authors recommendation for caution when interpreting the results of such experiments.
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Reviewer #1 (Public Review):
The contribution of Klughammer et al reports on the fabrication and functionalization of zero-mode waveguides of different diameters as a mimic system for nuclear pore complexes. Moreover, the researchers performed molecular transport measurements on these mimic systems (together with molecular dynamic simulations) to assess the contribution of pore diameter and Nsp functionalization on the translocation rates of BSA, the nuclear transport protein Kap95 and finally the impact of different Kap95 concentrations on BSA translocation and overall selectivity of the mimicked pores as function of their diameter. In order to assess the effect of the Nsp1 on the coated pores to the translocation rates and molecular selectivity they also conducted separated experiments on bare nano-pores, i.e., without coating, and of different diameters. One of the most novel aspects of this contribution is the detection scheme used to assess the translocation rates & selectivity, i.e., the use of an optical scheme based on single molecule fluorescence detection as compared to previous works that have mostly relied on conductance measurements. The results are convincing, the experiments carefully performed and the procedures explained in detail.
Importantly, this study provides new insights on the mechanisms of nuclear transport contributing to further our understanding on how real nuclear-pore complexes (i.e., in living cell) can regulate molecular transport. The recent findings that the nuclear pore complexes are sensitive to mechanical stimulation by modulating their effective diameters, adds an additional level of interest to the work reported here, since the authors thoroughly explored different nano-pore diameters and quantified their impact on translocation and selectivity. There are multiple avenues for future research based the system developed here, including higher throughput detection, extending to truly multicolor schemes or expanding the range of FG-Nups, nuclear transport proteins or cargos that need to be efficiently transported to the nucleus through the nuclear pore complexes. As a whole, this is an important contribution to the field.
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Reviewer #1 (Public Review):
Summary:<br /> Shibl et al., studied the possible role of dicarboxylate metabolite azelaic acid (Aze) in modulating the response of different bacteria, it was used as a carbon source by Phycobacter and possibly toxic for Alteromonas. The experiments were well conducted using transcriptomics, transcriptional factor coexpression networks, uptake experiments, and chemical methods to unravel the uptake, catabolism, and toxicity of Aze on these two bacteria. They identified a putative Aze TRAP transporter in bacteria and showed that Aze is assimilated through fatty acid degradation in Phycobacter. Meanwhile, in Alteromonas it is suggested that Aze inhibits the ribosome and/or protein synthesis, and that efflux pumps shuttles Aze outside the cytoplasm. Further on, they demonstrate that seawater amended with Aze selects for microbes that can catabolize Aze.
Major strengths:<br /> The manuscript is well written and very clear. Through the combination of gene expression, transcriptional factor co-expression networks, uptake experiments, and chemical methods Shibl et al., showed that Aze has a different response in two bacteria.
Major weakness:<br /> There is no phenotype confirmation of the Aze TRAP transporters through mutagenesis.
Impact on the field:<br /> Metabolites exert a significant influence on microbial communities in the ocean, playing a crucial role in their composition, dynamics, and biogeochemical cycles. This research highlights the intriguing capacity of a single metabolite to induce contrasting responses in distinct bacterial species, underscoring its role in shaping microbial interactions and ecosystem functions.
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Reviewer #1 (Public Review):
Peng et al develop a computational method to predict/rank transcription factors (TFs) according to their likelihood of being pioneer transcription factors--factors that are capable of binding nucleosomes--using ChIP-seq for 225 human transcription factors, MNase-seq and DNase-seq data from five cell lines. The authors developed relatively straightforward, easy to interpret computational methods that leverage the potential for MNase-seq to enable relatively precise identification of the nucleosome dyad. Using an established smoothing approach and local peak identification methods to estimate positions together with identification of ChIP-seq peaks and motifs within those peaks which they referred to as "ChIP-seq motifs", they were able to quantify "motif profiles" and their density in nucleosome regions (NRs) and nucleosome depleted regions (NDRs) relative to their estimated nucleosome dyad positions. Using these profiles, they arrived at an odd-ratio based motif enrichment score along with a Fisher's exact test to assess the odds and significance that a given transcription factor's ChIP-seq motifs are enriched in NRs compared to NDRs, hence, its potential to be a pioneer transcription factor. They showed that known pioneer transcription factors had among the highest enrichment scores, and they could identify a number of relatively novel pioneer TFs with high enrichment scores and relatively high expression in their corresponding cell line. They used multiple validation approaches including (1) calculating the ROC-AUC and Matthews correlation coefficient (MCC) and generating ROC and precision-recall curves associated with their enrichment score based on 32 known pioneer TFs among their 225 TFs which they used as positives and the remaining TFs (among the 225) as negatives; (2) use of the literature to note that known pioneer TFs that acted as key regulators of embryonic stem cell differentiation had a highest enrichment scores; (3) comparison of their enrichment scores to three classes of TFs defined by protein microarray and electromobility shift assays (1. strong binder to free and nucleosomal DNA, 2. weak binder to free and nucleosomal DNA, 3. strong binding to free but not nucleosomal DNA); and (4) correlation between their calculated TF motif nucleosome end/dyad binding ratio and relevant data from an NCAP-SELEX experiment. They also characterize the spatial distribution of TF motif binding relative to the dyad by (1) correlating TF motif density and nucleosome occupancy and (2) clustering TF motif binding profiles relative to their distance from the dyad and identifying 6 clusters.
The strengths of this paper are the use of MNase-seq data to define relatively precise dyad positions and ChIP-seq data together with motif analysis to arrive at relatively accurate TF binding profiles relative to dyad positions in NRs as well as in NDRs. This allowed them to use a relatively simple odds ratio based enrichment score which performs well in identifying known pioneer TFs. Moreover, their validation approaches either produced highly significant or reasonable, trending results.
The weaknesses of the paper are relatively minor, and the authors do a good job of describing the limitations of the data and approach.
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Reviewer #1 (Public Review):
Summary:
This revised study follows up on previous work showing a female-specific enhancer region of PAX1 is associated with adolescent idiopathic scoliosis (AIS). This new analysis combines human GWAS analysis from multiple countries to identify a new AIS-associated coding variant in the COL11A1 gene (COL11A1P1335L). Using a Pax1 knockout mouse they go on to find that PAX1 and Collagen XI protein are expressed in the intervertebral discs (IVDs) and robustly in the growth plate, showing that COL11A1 expression is reduced in Pax1 mutant growth plate. Moreover, other AIS-associated genes, Gpr126 and Sox6, were also reduced in Pax1 mutant mice, suggesting a common pathway is involved in AIS.
Using SV40 immortalized costal cartilage cells, derived from floxed Col11a1 mice primary rib cage cartilage, they go to show that removal of Col11a1 leads to reduction of Mmp3 expression. In this context, the expression of wild-type Col11a1 restored regular levels of Mmp3 expression, while expression of the AIS-associated Col11a1P1335L allele failed to restore normal Mmp3 expression. This supports a model that the AIS-associated Col11a1P1335L allele leads to the dysregulation of ECM in vivo.
Using this culture system, they go on to test the role of the estrogen receptor ESR2, showing that loss of this receptor leads to reduced Mmp3 and Pax1 expression, and increased Col11a1 expression. They support this by showing similar gene expression changes and estrogen receptor function in Rat cartilage endplate cell culture.
Altogether, this study nicely brings together an impressive number of human genetic data from multi-ethnic AIS cohorts and controls from across the globe and functionally tests these findings in cell culture and animal models. This study wonderfully integrates other findings from other human and mouse work in AIS and supports a new molecular mechanism by which estrogen can interact and synergize with COL11A1/PAX1/MMP3 signaling to change ECM development and dynamics, thus providing a tangible model for mutations and dysregulation of this pathway can increase the susceptibility of scoliosis.
Strengths:
This work integrates a large cohort of human genetic data from AIS patient and control from diverse ethnic backgrounds, across the globe. This work attempts to functionally test their findings in vivio and by use of cell culture.
Weaknesses:
Many of the main functional work was done in cell culture and not in vivo.
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Reviewer #1 (Public Review):
Over the last decade, numerous studies have identified adaptation signals in modern humans driven by genomic variants introgressed from archaic hominins such as Neanderthals and Denisovans. One of the most classic signals comes from a beneficial haplotype in the EPAS1 gene in Tibetans that is evidently of Denisovan origin and facilitated high altitude adaptation (HAA). Given that HAA is a complex trait with numerous underlying genetic contributions, in this paper Ferraretti et al. asked whether additional HAA-related genes may also exhibit a signature of adaptive introgression. Specifically, the authors considered that if such a signature exists, they most likely are only mild signals from polygenic selection, or soft sweeps on standing archaic variation, in contrast to a strong and nearly complete selection signal like in the EPAS1. Therefore, they leveraged two methods, including a composite likelihood method for detecting adaptive introgression and a biological network-based method for detecting polygenic selection, and identified two additional genes that harbor plausible signatures of adaptive introgression for HAA.
Strengths:<br /> The study is well motivated by an important question, which is, whether archaic introgression can drive polygenic adaptation via multiple small effect contributions in genes underlying different biological pathways regulating a complex trait (such as HAA). This is a valid question and the influence of archaic introgression on polygenic adaptation has not been thoroughly explored by previous studies
The authors reexamined previously published high-altitude Tibetan whole genome data and applied a couple of the recently developed methods for detecting adaptive introgression and polygenic selection.
Weaknesses:<br /> My main concern with this paper is that I am not too convinced that the reported genomic regions putatively under polygenic selection are indeed of archaic origin. Other than some straightforward population structure characterizations, the authors mainly did two analyses with regard to the identification of adaptive introgression: First, they used one composite likelihood-based method, the VolcanoFinder, to detect the plausible archaic adaptive introgression and found two candidate genes (EP300 and NOS2). Next, they attempted to validate the identified signal using another method that detects polygenic selection based on biological network enrichments for archaic variants.
In general, I don't see in the manuscript that the choice of methods here are well justified. VolcanoFinder is one among the several commonly used methods for detecting adaptive introgression (eg. the D, RD, U, and Q statistics, genomatnn, maldapt etc.). Even if the selection was mild and incomplete, some of these other methods should be able to recapitulate and validate the results, which are currently missing in this paper. Besides, some of the recent papers that studied the distribution of archaic ancestry in Tibetans don't seem to report archaic segments in the two gene regions. These all together made me not sure about the presence of archaic introgression, in contrast to just selection on ancestral variation.
Furthermore, the authors tried to validate the results by using signet, a method that detects enrichments of alleles under selection in a set of biological networks related to the trait. However, the authors did not provide sufficient description on how they defined archaic alleles when scoring the genes in the network. In fact, reading from the method description, they seemed to only have considered alleles shared between Tibetans and Denisovans, but not necessarily exclusively shared between them. If the alleles used for scoring the networks in Signet are also found in other populations such as Han Chinese or Africans, then that would make a substantial difference in the result, leading to potential false positives.
Overall, given the evidence provided by this article, I am not sure they are adequate to suggest archaic adaptive introgression. I recommend additional analyses for the authors to consider for rigorously testing their hypothesis. Please see the details in my review to the authors.
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Reviewer #1 (Public Review):
This study focuses on the defining cellular pathways critical for tRNA export from the nucleus. While a number of these pathways have been identified, the observation that the primary transport receptors identified thus far (Los1 and Msn5) are not essential and that cells are viable even when both the genes are deleted supports the idea that there are as yet unidentified mediators of tRNA export from the nucleus. This study implicates the helicase Dbp5 in one of these parallel pathways arguing that Dbp5 works in a pathway that is independent of Los1 and/or Msn5. The authors present genetic data to support this conclusion. At least one results suggests that the idea of these pathways in parallel may be too simplistic as deletion of the LOS1 gene, which is not essential decreases the interaction of tRNA export substrate with Dbp5 (Figure 2A). If the two pathways were working in parallel, one might have expected removing one pathways to lead to an increase in the use of the other pathway and hence the interaction with a receptor in that pathway. The authors provide solid evidence that Dbp5 interacts with tRNA directly and that addition of the factor Gle1 together with the previously identified co-factor InsP6 can trigger helicase activity and release of tRNA. The combination of in vivo studies and biochemistry provide evidence to consider how Dbp5 contributes to export of tRNA and more broadly adds to the conversation about how coding and non-coding RNA export from the nucleus might be coordinated to control cell physiology.
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Reviewer #1 (Public Review):
Summary:<br /> The authors present a detailed study of a nearly complete Entomophthora muscae genome assembly and annotation, along with comparative analyses among related and non-related entomopathogenic fungi. The genome is one of the largest fungal genomes sequenced, and the authors document the proliferation and evolution of transposons and the presence/absence of related genetic machinery to explore how this may have occurred. There has also been an expansion in gene number, which appears to contain many "novel" genes unique to E. muscae. Functionally, the authors were interested in CAZymes, proteases, circadian clock related genes (due to entomopathogenicity/ host manipulation), other insect pathogen-specific genes, and secondary metabolites. There are many interesting findings including expansions in trahalases, unique insulinase, and another peptidase, and some evidence for RIP in Entomophthoralean fungi. The authors performed a separate study examining E. muscae species complex and related strains. Specifically, morphological traits were measured for strains and then compared to the 28S+ITS-based phylogeny, showing little informativeness of these morpho characters with high levels of overlap.
This work represents a big leap forward in the genomics of non-Dikarya fungi and large fungal genomes. Most of the gene homologs have been studied in species that diverged hundreds of millions of years ago, and therefore using standard comparative genomic approaches is not trivial and still relatively little is known. This paper provides many new hypotheses and potential avenues of research about fungal genome size expansion, entomopathogenesis in zygomycetes, and cellular functions like RIP and circadian mechanisms.
Strengths:<br /> There are many strengths to this study. It represents a massive amount of work and a very thorough functional analysis of the gene content in these fungi (which are largely unsequenced and definitely understudied). Too often comparative genomic work will focus on one aspect and leave the reader wondering about all the other ways genome(s) are unique or different from others. This study really dove in and explored the relevant aspects of the E. muscae genome.
The authors used both a priori and emergent properties to shape their analyses (by searching for specific genes of interest and by analyzing genes underrepresented, expanded, or unique to their chosen taxa), enabling a detailed review of the genomic architecture and content. Specifically, I'm impressed by the analysis of missing genes (pFAMs) in E. muscae, none of which are enriched in relatives, suggesting this fungus is really different not by gene loss, but by its gene expansions.
Analyzing species-level boundaries and the data underlying those (genetic or morphological) is not something frequently presented in comparative genomic studies, however, here it is a welcome addition as the target species of the study is part of a species complex where morphology can be misleading and genetic data is infrequently collected in conjunction with the morphological data.
Weaknesses:<br /> The conclusions of this paper are mostly well supported by data, but a few points should be clarified.
In the analysis of Orthogroups (OGs), the claim in the text is that E. muscae "has genes in multi-species OGs no more frequently than Enotomophaga maimaiga. (Fig. 3F)" I don't see that in 3F. But maybe I'm really missing something.
Also related, based on what is written in the text of the OG section, I think portions of Figure 3G are incorrect/ duplicated. First, a general question, related to the first two portions of the graph. How do "Genes assigned to an OG" and "Genes not assigned to an OG" not equal 100% for each species? The graph as currently visualized does not show that. Then I think the bars in portion 3 "Genes in species-specific OG" are wrong (because in the text it says "N. thromboides had just 16.3%" species-specific OGs, but the graph clearly shows that bar at around 50%. I think portion 3 is just a duplicate of the bars in portion 4 - they look exactly the same - and in addition, as stated in the text portion 4 "Potentially species-specific genes" should be the simple addition of the bars in portion 2 and portion 3 for each species.
In the introduction, there is a name for the phenomenon of "clinging to or biting the tops of plants," it's called summit disease. And just for some context for the readers, summit disease is well-documented in many of these taxa in the older literature, but it is often ignored in modern studies - even though it is a fascinating effect seen in many insect hosts, caused by many, many fungi, nematodes (!), etc. This phenomenon has evolved many times. Nice discussions of this in Evans 1989 and Roy et al. 2006 (both of whom cite much of the older literature).
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Reviewer #1 (Public Review):
The authors set out to illuminate how legumes promote symbiosis with beneficial nitrogen-fixing bacteria while maintaining a general defensive posture towards the plethora of potentially pathogenic bacteria in their environment. Intriguingly, a protein involved in plant defence signalling, RIN4, is implicated as a type of 'gatekeeper' for symbiosis, connecting symbiosis signalling with defence signalling. Although questions remain about how exactly RIN4 enables symbiosis, the work opens an important door to new discoveries in this area.
Strengths:<br /> The study uses a multidisciplinary, state-of-the-art approach to implicate RIN4 in soybean nodulation and symbiosis development. The results support the authors' conclusions.
Weaknesses:<br /> No serious weaknesses, although the manuscript could be improved slightly from technical and communication standpoints.
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Reviewer #1 (Public Review):
Summary:<br /> The authors demonstrated that carbon depletion triggers the autophagy-dependent formation of Rubisco Containing Bodies, which contain chloroplast stroma material, but exclude thylakoids. The authors show that RCBs bud directly from the main body of chloroplasts rather than from stromules and that their formation is not dependent on the chloroplast fission factor DRP5. The authors also observed a transient engulfment of the RBCs by the tonoplast during delivery to the vacuolar lumen.
Strengths:<br /> The authors demonstrate that autophagy-related protein 8 (ATG8) co-localizes to the chloroplast demarking the place for RCB budding. The authors provide good-quality time-lapse images and co-localization of the markers corroborating previous observations that RCBs contain only stroma material and do not include thylakoid. The text is very well written and easy to follow.
Weaknesses:<br /> A significant portion of the results presented in the study comes across as a corroboration of the previous findings made under different stress conditions: autophagy-dependent formation of RCBs was reported by Ishida et all in 2009. Furthermore, some included results are not of particular relevance to the study's aim. For example, it is unclear what is the importance of the role of SA in the formation of stromules, which do not serve as an origin for the RCBs. Similarly, the significance of the transient engulfment of RCBs by the tonoplast remained elusive. Although it is indeed a curious observation, previously reported for peroxisomes, its presentation should include an adequate discussion maybe suggesting the involved mechanism. Finally, some conclusions are not fully supported by the data: the suggested timing of events poorly aligns between and even within experiments mostly due to high variation and low number of replicates. Most importantly, the discussion does not place the findings of this study into the context of current knowledge on chlorophagy and does not propose the significance of the piece-meal vs complete organelle sequestration into the vacuole under used conditions, and does not dwell on the early localization of ATG8 to the future budding place on the chloroplast.
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Reviewer #1 (Public Review):
Summary:<br /> The authors recently reported a scRNA-seq-based study focused on synovial fibroblasts using a mouse model of post-traumatic OA (Ref. 21). In the present manuscript, they reanalyzed the scRNA-seq data to investigate the diversity and roles of macrophages. In addition to their original scRNA-seq data (Ref. 21), they utilized the deposited data of other OA or RA models (Ref. 25-27) and compared cell types in the synovium. The authors extracted the macrophage/monocyte group, compared differentially expressed genes (DEGs) between OA and RA synovium, and analyzed macrophage subsets, including trajectory analysis. They further estimated the crosstalk between stromal and immune cells via M-CSF signaling, and transcription factors for monocyte differentiation.
Strengths:<br /> The descriptions are comprehensive, based on the scRNA-seq data including the original and other independent studies.
Weaknesses:<br /> Meanwhile, methods of sample preparation must be different, for example, the extent and location of excised synovium. The comparison with other studies is meaningful and informative; however, caution should be exercised regarding the potentially significant impact of methodological differences on the analysis results.
The various data obtained from these technologies are comprehensive and useful; however, they are just estimates. Without confirmation by experiments, it is impossible to determine how much of it can be believed. This issue is not limited to this paper.
Most of all signaling pathways and molecules described in the latter part of this study are previously known.
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Reviewer #1 (Public Review):
Summary: The goal of this study was to develop and validate novel molecules to selectively activate a cell signaling pathway, the Wnt pathway in this case, in target cells expressing a specific receptor. This was achieved through a two-component system that the authors call BRAID, where each component simultaneously binds the target cell-specific marker BKlotho and a Wnt co-receptor. These components, called SWIFT molecules, bring together the Wnt co-receptors LRP and FZD, activating the pathway specifically in cells that express BKlotho.
Results presented in the study demonstrate the desired activity of SWIFT molecules; the binding assays support simultaneous association of SWIFT with BKlotho and a Wnt co-receptor, and the Wnt reporter and qPCR assays support pathway activation in cell lines and primary cells in a BKlotho-dependent manner. In the future, the BRAID approach could be applied to activate Wnt signaling or another pathway initiated by a co-receptor complex in a cell type-specific manner, and/or in a FZD subtype-specific manner to activate distinct branches of Wnt signaling.
Strengths:
• This study successfully demonstrates a novel way to activate Wnt signaling in target cells expressing a specific marker. Given the role of the Wnt signaling pathway in key processes such as cell proliferation and tissue renewal and the value of modulating cell signaling in a cell type-specific manner, the cell targeting system developed here holds great therapeutic and research potential. It will be curious to see whether the BRAID design can be applied to other cell surface markers for Wnt activation, or for activation of other signaling pathways that require co-receptor association.
• Octet assay results show simultaneous binding of SWIFT molecules to both the Wnt co-receptor FZD/LRP and BKlotho, while negative control molecules without the FZD/LRP or BKlotho-binding module show neither receptor binding nor Wnt pathway activation. These results indicate that SWIFT molecules function through the intended mechanism.
• Exposure of two cell types simultaneously exposed to the SWIFT molecules in 2-layer cell culture demonstrated the ability of the molecules to activate Wnt signaling in a cell type- and BKlotho expression-specific manner.
Weaknesses:
• The study does not address whether the targeted cells express FGFR1c/2c/3c and whether the FGF21 full length moiety or the 39F7 IgG moiety of SWIFT molecules could unintentionally activate FGF signaling in these cells.
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Reviewer #1 (Public Review):
Summary:<br /> "Expanding the Drosophila toolkit for dual control of gene expression" by Zirin et al. aims to develop resources for simultaneous independent manipulation of multiple genes in Drosophila. The authors use CRISPR knock-ins to establish a collection of T2A-LexA and T2A-QF2 transgenes with expression patterns in a number of commonly studied organs and tissues. In addition to the transgenic lines that are established, the authors describe a number of plasmids that can be used to generate additional transgenes, including a plasmid to generate a dual insert of LexA and QF that can be resolved into a single insert using FLP/FRT-mediated recombination, and plasmids to generate RNAi reagents for the LexA and QF systems. Finally, the authors demonstrate that a subset of the LexA and QF lines that they generated can induce RNAi phenotypes when paired with LexAop or QUAS shRNA lines. In general, the claims of the paper are well supported by the evidence and the authors do a thorough job of validating the transgenic lines and characterizing their expression patterns.
Strengths:<br /> -Numerous Gal4 lines allow for highly specific genetic manipulation in a wide range of organs and tissues, however, similar tissue-specific drivers using alternative binary expression systems are not currently well developed. This study provides a large number of tissue and organ-specific LexA and QF2 driver lines that should be broadly useful for the Drosophila community.<br /> -While a minority of the driver lines do not express the expected pattern (likely due to cryptic regulatory elements in the LexA or QF2 sequences), the ability to generate drivers using two different Gal4 alternatives mitigates this issue (as in nearly all cases at least one of the two systems produces a clean driver line with the expected expression pattern).<br /> -The use of LexA-GAD provides an additional degree of control as it is subject to Gal80 repression. This could prove to be particularly useful in cases where a researcher wishes to manipulate multiple genes using Gal4 and LexA-GAD drivers as the Gal80(ts) system could be used for simultaneous temporal control of both constructs.<br /> -The use of Fly Cell Atlas information to generate novel oenocyte-specific driver lines provides a useful proof-of-concept for constructing additional highly tissue-specific drivers.
Weaknesses:<br /> -Since these reagents will most commonly be paired with existing Gal4 lines, adding information about corresponding Gal4 lines targeting these tissues and how faithfully the LexA and QF2 lines recapitulate these Gal4 patterns would be highly beneficial.<br /> -It is not stated in the manuscript if these transgenic lines and plasmids are currently publicly available. Information about how to obtain these reagents through Bloomington, Addgene, or TRiP should be added to the manuscript.
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Reviewer #1 (Public Review):
The findings in this paper can be split into three parts.
1) Processing of ITS2
Firstly, the authors identify two sites on ITS2 which are cleaved by the ScLas1-Grc3 complex, as part of 25S ribosomal RNA maturation.
For a smaller segment of ITS2 (33nt), the two sites separate out 3 parts with sizes of 10nt, 14nt, and 9nt (Figure 1C). However, bands in mass spec. occur at 23nt (14nt+9nt) and 14nt, but not 9nt alone. Additional bands can be seen at 22nt and 21nt. Hence the evidence for these two specific sites seems somewhat uncertain. It is not clear if there is an experimental limitation in terms of accuracy, or that the cleavage is perhaps somewhat approximate at the two sites. The authors may try to clarify these results a bit further.
For a larger ITS2 (81nt), similar support is found for the two cleavage sites, but now the possible fragments are 14nt, 30nt, 37nt, and 44nt (14nt+30nt) (Figure 1E). The observed bands match these fragments at 44nt, 37nt, 30nt, but again there are additional bands at 36nt, 28nt, and 13nt, which are not fully explained. It may be useful for explain or discuss these discrepancies.
Another useful result of these experiments is to confirm that Las1 alone has only weak activity against ITS2, but very strong activity when it is part of the Las1-Grc3 complex.
2) Structure of Las1, and Las1-Grc3 complexes
A second important contribution of this work are X-ray and cryoEM structures of Las1-Grc3 from Sc and Cj. It is interesting that even though the complexes are very similar, CjGrc3 shows weak activation of CjLas1, whereas ScGrc3 more strongly activates ScLas1. The X-ray and cryoEM structures are very similar. However, the X-ray structures also show an additional (CC) domain from Las1 not resolved in the cryoEM map. This difference is significant, because it suggests the CC domain may remain more flexible in solution, but stabilizes in the crystal. Also interestingly, the CC domains have different structures and are in different positions in ScLas1-Grc3 vs CjLas1-Grc3, again hinting that they are more dynamic. Further experiments described by the authors confirm the CC domain is indeed important in RNA binding and activity. Whether they are only implicated in binding RNA or both binding and cleavage is somewhat unclear.
The structure of Las1-Grc3 is described as resembling a butterfly, with Las1 being the body and Grc3 the wings. While this is a useful description, it may be a bit misleading. The complex has C2 symmetry, with one Las1-Grc3 unit related to the other by about ~180 rotation around a vertical axis parallel to the body of the butterfly as proposed. To use the butterfly analogy, one half of the body and one wing faces the opposite way as the other, not a mirror symmetry as a real butterfly would have.
Both Cj and Sc structures show the C-terminal of Grc3 binding to the active pocket of Las1, explaining its effect on activity. Mutation experiments also further show the importance of these residues on activity. Reciprocally, a region in Las1, LCT, inserts into Grc3, forming a stable complex. Again mutating these residues affects activity, strengthening their importance and the evidence for how the stable complex forms.
Finally, an X-ray structure of dimeric Las1 in Cj, without Grc3 is presented. Truncating CC and LCT appeared to be necessary to allow the dimer to crystallize. Superposition with Las1 dimer in Las1-Grc3 shows a conformational difference, and different distances between residues in the active pocket, explaining the change in activity with and without Grc3. Interestingly, the Las1 domains themselves do not change too much, i.e. both domains can be matched with less than 1Å Ca-RMSD, so the difference may be more of a repositioning of the two domains for the active conformation.
One notable strength of this study is the use of both X-ray and cryoEM to obtain structures of the Las1-Grc3 and dimeric Las1 complexes. Typically structures of cryoEM at ~3Å are sufficient for reliable modeling; for example, the backbone and side chains of residues in the active site are well resolved. However, in this case, a cryoEM model of the Las1 dimer was not obtained, so it was important to show first that the Las1-Las1 conformation in the Las1-Grc3 complex is the same in both X-ray and cryoEM models. Otherwise, there may remain doubt whether the X-ray model of Las1 dimer could be compared to the cryoEM map of Las1-Grc3, as crystallization conditions could potentially influence conformation and arrangement. It would be interesting to know whether a cryoEM structure of the Las1 dimer alone was attempted - perhaps it was too small to be reliably seen in micrographs. Having had such a model could avoid the need of X-ray structures, although of course more experimental data are always useful.
3) Mechanism of ITS2 cleavage
The proposed mechanism shown in Figure 8 seems to be well supported by the obtained structures and biochemical experiments. A question that remains is why it is proposed that both C2 and C2' cleavage can be performed upon a single binding of the ITS2 RNA, i.e. seeming to suggest there are two binding sites. This would seem to directly generate 3 fragments, without any other intermediate products. Mass spec. seemed to show the intermediate products, perhaps indicating two binding events for each cleavage process. Perhaps the authors could discuss this more. Also perhaps can be good to discuss whether it would be possible to obtain a structure with the bound RNA, further giving structural information of how the exact cleavage process is performed.
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Reviewer #1 (Public Review):
Summary:<br /> Ellis et al. investigated the functional and topographical organization of the visual cortex in infants and toddlers, as evidenced by movie-viewing data. They build directly on prior research that revealed topographic maps in infants who completed a retinotopy task, claiming that even a limited amount of rich, naturalistic movie-viewing data is sufficient to reveal this organization, within and across participants. Generating this evidence required methodological innovations to acquire high-quality fMRI data from awake infants (which have been described by this group, and elsewhere) and analytical creativity. The authors provide evidence for structured functional responses in infant visual cortex at multiple levels of analyses; homotopic brain regions (defined based on a retinotopy task) responded more similarly to one another than to other brain regions in visual cortex during movie-viewing; ICA applied to movie-viewing data revealed components that were identifiable as spatial frequency, and to a lesser degree, meridian maps, and shared response modeling analyses suggested that visual cortex responses were similar across infants/toddlers, as well as across infants/toddlers and adults. These results are suggestive of fairly mature functional response profiles in the visual cortex in infants/toddlers and highlight the potential of movie-viewing data for studying finer-grained aspects of functional brain responses, but further evidence is necessary to support their claims and the study motivation needs refining, in light of prior research.
Strengths:<br /> - This study links the authors' prior evidence for retinotopic organization of visual cortex in human infants (Ellis et al., 2021) and research by others using movie-viewing fMRI experiments with adults to reveal retinotopic organization (Knapen, 2021).
- Awake infant fMRI data are rare, time-consuming, and expensive to collect; they are therefore of high value to the community. The raw and preprocessed fMRI and anatomical data analyzed will be made publicly available.
Weaknesses:<br /> - The Methods are at times difficult to understand and in some cases seem inappropriate for the conclusions drawn. For example, I believe that the movie-defined ICA components were validated using independent data from the retinotopy task, but this was a point of confusion among reviewers. In either case: more analyses should be done to support the conclusion that the components identified from the movie reproduce retinotopic maps (for example, by comparing the performance of movie-viewing maps to available alternatives (anatomical ROIs, group-defined ROIs). Also, the ROIs used for the homotopy analyses were defined based on the retinotopic task rather than based on movie-viewing data alone - leaving it unclear whether movie-viewing data alone can be used to recover functionally distinct regions within the visual cortex.
- The authors previously reported on retinotopic organization of the visual cortex in human infants (Ellis et al., 2021) and suggest that the feasibility of using movie-viewing experiments to recover these topographic maps is still in question. They point out that movies may not fully sample the stimulus parameters necessary for revealing topographic maps/areas in the visual cortex, or the time-resolution constraints of fMRI might limit the use of movie stimuli, or the rich, uncontrolled nature of movies might make them inferior to stimuli that are designed for retinotopic mapping, or might lead to variable attention between participants that makes measuring the structure of visual responses across individuals challenging. This motivation doesn't sufficiently highlight the importance or value of testing this question in infants. Further, it's unclear if/how this motivation takes into account prior research using movie-viewing fMRI experiments to reveal retinotopic organization in adults (e.g., Knapen, 2021). Given the evidence for retinotopic organization in infants and evidence for the use of movie-viewing experiments in adults, an alternative framing of the novel contribution of this study is that it tests whether retinotopic organization is measurable using a limited amount of movie-viewing data (i.e., a methodological stress test). The study motivation and discussion could be strengthened by more attention to relevant work with adults and/or more explanation of the importance of testing this question in infants (is the reason to test this question in infants purely methodological - i.e., as a way to negate the need for retinotopic tasks in subsequent research, given the time constraints of scanning human infants?).
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Reviewer #1 (Public Review):
Summary:
The paper of Mao et al. expands the genetic toolset that was previously developed by the Rao lab (Denfg et al 2019) to introduce the conditional KO or downregulation of neurotransmission components in Drosophila. The authors then use these tools to investigate neurotransmission in the clock neurons of the Drosophila brain. They first test some known components and then analyze the contribution of the CNMa neuropeptide and its receptor to the circadian behavior. The results indicate that CNMA acts from a subset of DN1ps (dorsal clock neurons) to set the phase of the morning peak of locomotor activity in light:dark cycles, with an advanced morning activity in the absence of the neuropeptide. Interestingly, the receptor for the PDF neuropeptide appears to be acting in some of the CNMa neurons to control morning activity.
Strengths/weaknesses:
This is clearly a very useful new set of tools to restrict the manipulation of these components to specific neuronal populations, and overall (see specific points below), the paper is convincing to show that the tools indeed allow to efficiently and specifically eliminate neuropeptides/receptors from subsets of neurons. The analysis of the CNMa function in the clock network reveals a new and interesting function for CNMa. but this part needs to be improved. Some of the behavioral data (PDF/PDFR) do not fit with published work with the mutants. This should be clarified by providing more data comparing the described genotypes with the classical mutants. Some conclusions also need to be toned down.
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Reviewer #1 (Public Review):
Characterizing gene-by-environment interactions has been of great interest for quite some time, as these effects are believed (based on plausible hypotheses and some data) to have importance for the interpretation of complex disease risk. Here, a major class of variants of interest is genetic regulatory variants where e.g. binding of context-specific regulators (TFs, etc) provides a plausible mechanism. However, these variants have been difficult to identify in eQTL and other studies.
This study leverages the MPRA approach to screen for many thousands of constructs of putative regulatory variants for their effects on vascular endothelial cells with and without caffeine. They identify thousands of sequences that are differentially regulated between the conditions, and with motif enrichment approaches and comparisons to prior studies, identify some TFs (including novel ones) that may have a role in how these cells respond to caffeine. Next, by allele-specific expression analysis, they identify thousands of variants that are not only regulatory (having a different activity from the two allelic versions of the construct) but also a major subset that has different regulatory activity following the caffeine treatment. Again, motif analysis indicates potential mechanisms, and the eQTL comparison nicely demonstrates the value of these discoveries. The MRPA approach is clearly fruitful and informative, and identifying many context-specific regulatory variants is informative for people working on genetic regulatory variation.
The part of the paper that felt underwhelming and not so well-founded was the link to complex disease. I was somewhat surprised to see caffeine experiments in vascular endothelial cells being so strongly framed in terms of CAD. This cell type (and potentially also caffeine) is relevant in many biological processes and diseases. More importantly, given the strongly disease-focused framing, I was surprised to find few results that would actually link the regulatory variant data here to CAD via GWAS overlap or other analyses. Maybe the results were slim here with little overlap, but the results provided do not really justify the implications that disease-relevant findings are being made.
Specifically, the evidence of PIP4K2B let alone the studied cASE variant having a causal role in CAD is weak. This is based on a previously published pTWAS paper, but the variant itself is not a significant GWAS variant. TWAS is known to easily suffer from non-causal hits due to LD and other complications, and hence this link should be taken with a heavy grain of salt. I would be more convinced if the variant was a significant GWAS hit, and even more so if it was a fine-mapped variant, but it is neither of them. As such, the language (Discussion) is not justified by the data: "By studying different environmental contexts, we can identify that, in this instance, caffeine can reduce the risk of poor cardiovascular health outcomes. If the environmental context was not considered and this work was conducted solely in the control condition, the decreased risk induced by caffeine would not have been observed." the decreased [CAD] risk induced by caffeine would not have been observed." Has to be softened. Furthermore, Figure 5 is an illustration but very little data (cASE p-values, etc) is provided here and in the text.
Furthermore, I find some of the suggested links to CAD via lipid biology and related TFs quite speculative; are these processes really taking place in vascular endothelian cells? The paper that is being referred to seems to focus on the liver.
Finally, the analyses seem carefully done, but in Figure 2A the systematic inflation of p-values seems concerning. This could be the real biology of broadly distributed response to caffeine, but it's also consistent with a bias that is unaccounted for and inflates p-values across the board. And do we really expect all these elements to respond to caffeine (more or less)? It is difficult to say what exactly this might be, but the caffeine libraries seem to have a higher sequencing coverage (SFig 5). How does this affect the results? Can it bias the DE results via different overdispersion, or the ASE or cASE estimation when the caffeine condition is a higher power (which ASE analysis is typically sensitive to)?
The library included negative controls of variants that are not believed to be regulatory variants, but I don't see a systematic presentation of the null obtained from these presented in the paper.
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Reviewer #1 (Public Review):
In this manuscript, the authors investigate whether the effects of the BCG vaccine on immunity to Mtb infection could be improved by inhibiting amidation of the peptidoglycan sidechains to allow for recognition by NOD-1. This is a very important area and an interesting new approach to improve vaccination for TB. The authors find that CRISPRi knockdown of murT-gatD causes rather dramatic cell wall defects, more accessible cell wall labeling, and results in attenuated growth in macrophages and mice. There is some data presented to support that the murT-gatD KD strain may be more protective in the animal model, but most comparisons made are not significant and some interpretations stated in the results section do not reflect the data in the figures. It seems that the most important comparisons are between WT BCG+Dox and rBCG+Dox, and the manuscript would be clearer if this comparison was focused on specifically.
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Reviewer #1 (Public Review):
Predator-prey interactions often involve one predator and one prey. Where more than one predator hunts a single prey, a key question is whether the predators involved are cooperating in some manner. Where this has been observed in biology, it has been suggested that complex cognitive processes may be needed to support the cooperation, such as each predator representing the intention of other predators. In this study the authors ask whether cooperation can emerge in a highly idealized scenario with little more than a basic reinforcement learning approach. Due to the size of the resulting state space, computing the value function becomes computationally cumbersome, so a function approximation method using a variant of a deep Q-network (DQN) is used. The authors have successfully shown that cooperation, here operationalized as a higher success rate with two predators in the context of sharing of the reward (prey that's captured), can emerge in this context. Further, they show that a cluster-based analysis of the DQN can guide the generation of a short description length rule-based approach that they also test and show qualitative agreement with the original DQN results.
Strengths of the work include providing a demonstration proof that cooperation can emerge with simple rules in a predator-prey context, suggesting that its emergence over phylogenetic time within certain clades of animals may not require the complex cognitive processes that prior work has suggested may be needed. Given the simplicity of the rules, one possible outcome could be a widening of investigation into cooperative hunting beyond the usual small number of species in which this has been observed, such as chimpanzees, seals, dolphins, whales, wild dogs, and big cats. The authors have done well to show how, with a variety of adjustments, a DQN network can be used to gain insights into a complex ethological phenomena.
One weakness of the work is the simplicity of the environment, a 2D plane that is 10 body lengths in each dimension, with full observability and no limitations to movement besides the boundaries of the space. Recent literature suggests that more complex phenomena such as planning may only evolve in the context of partial observability in predator-prey interactions. Thus the absence of more advanced tactics on the part of the predator agents may reflect limitations due to the simplicity of the behavioral arena, or limitations of associative learning alone to drive the emergence of these tactics. Another is that although correlations between network activity are discussed, and used to generate a rule-based approach that succeeds in replicating some of the results, there is no further analysis that may go beyond correlation to a causal analysis.
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Joint Public Review:
LD Score regression (LDSC) is a software tool widely used in the field of genome-wide association studies (GWAS) for estimating heritabilities, genetic correlations, the extent of confounding, and biological enrichment. LDSC is for the most part not regarded as an accurate estimator of \emph{absolute} heritability (although useful for relative comparisons). It is relied on primarily for its other uses (e.g., estimating genetic correlations). The authors propose a new method called \texttt{i-LDSC}, extending the original LDSC in order to estimate a component of genetic variance in addition to the narrow-sense heritability---epistatic genetic variance, although not necessarily all of it. Epistasis in quantitative genetics refers to the component of genetic variance that cannot be captured by a linear model regressing total genetic values on single-SNP genotypes. \texttt{i-LDSC} seems aimed at estimating that part of the epistatic variance residing in statistical interactions between pairs of SNPs. To simplify, the basic model of \texttt{i-LDSC} for two SNPs $X_1$ and $X_2$ is<br /> \begin{equation}\label{eq:twoX}<br /> Y = X_1 \beta_1 + X_2 \beta_2 + X_1 X_2 \theta + E,<br /> \end{equation}<br /> and estimation of the epistatic variance associated with the product term proceeds through a variant of the original LD Score that measures the extent to which a SNP tags products of genotypes (rather than genotypes themselves). The authors conducted simulations to test their method and then applied it to a number of traits in the UK Biobank and Biobank Japan. They found that for all traits the additive genetic variance was larger than the epistatic, but for height the absolute size of the epistatic component was estimated to be non-negligible. An interpretation of the authors' results that perhaps cannot be ruled out, however, is that pairwise epistasis overall does not make a detectable contribution to the variance of quantitative traits.
Major Comments
This paper has a lot of strong points, and I commend the authors for the effort and ingenuity expended in tackling the difficult problem of estimating epistatic (non-additive) genetic variance from GWAS summary statistics. The mere possibility of the estimated univariate regression coefficient containing a contribution from epistasis, as represented in the manuscript's Equation~3 and elsewhere, is intriguing in and of itself.
Is \texttt{i-LDSC} Estimating Epistasis?
Perhaps the issue that has given me the most pause is uncertainty over whether the paper's method is really estimating the non-additive genetic variance, as this has been traditionally defined in quantitative genetics with great consequences for the correlations between relatives and evolutionary theory (Fisher, 1930, 1941; Lynch & Walsh, 1998; Burger, 2000; Ewens, 2004).
Let us call the expected phenotypic value of a given multiple-SNP genotype the \emph{total genetic value}. If we apply least-squares regression to obtain the coefficients of the SNPs in a simple linear model predicting the total genetic values, then the partial regression coefficients are the \emph{average effects of gene substitution} and the variance in the predicted values resulting from the model is called the \emph{additive genetic variance}. (This is all theoretical and definitional, not empirical. We do not actually perform this regression.) The variance in the residuals---the differences between the total genetic values and the additive predicted values---is the \emph{non-additive genetic variance}. Notice that this is an orthogonal decomposition of the variance in total genetic values. Thus, in order for the variance in $\mathbf{W}\bm{\theta}$ to qualify as the non-additive genetic variance, it must be orthogonal to $\mathbf{X} \bm{\beta}$.
At first, I very much doubted whether this is generally true. And I was not reassured by the authors' reply to Reviewer~1 on this point, which did not seem to show any grasp of the issue at all. But to my surprise I discovered in elementary simulations of Equation~\ref{eq:twoX} above that for mean-centered $X_1$ and $X_2$, $(X_1 \beta_1 + X_2 \beta_2)$ is uncorrelated with $X_1 X_2 \theta$ for seemingly arbitrary correlation between $X_1$ and $X_2$. A partition of the outcome's variance between these two components is thus an orthogonal decomposition after all. Furthermore, the result seems general for any number of independent variables and their pairwise products. I am also encouraged by the report that standard and interaction LD Scores are ``lowly correlated' (line~179), meaning that the standard LDSC slope is scarcely affected by the inclusion of interaction LD Scores in the regression; this behavior is what we should expect from an orthogonal decomposition.
I have therefore come to the view that the additional variance component estimated by \texttt{i-LDSC} has a close correspondence with the epistatic (non-additive) genetic variance after all.
In order to make this point transparent to all readers, however, I think that the authors should put much more effort into placing their work into the traditional framework of the field. It was certainly not intuitive to multiple reviewers that $\mathbf{X}\bm{\beta}$ is orthogonal to $\mathbf{W}\bm{\theta}$. There are even contrary suggestions. For if $(\mathbf{X}\bm{\beta})^\intercal \mathbf{W} \bm{\theta} = \bm{\beta}^\intercal \mathbf{X}^\intercal \mathbf{W} \bm{\theta} $ is to equal zero, we know that we can't get there by $\mathbf{X}^\intercal \mathbf{W}$ equaling zero because then the method has nothing to go on (e.g., line~139). We thus have a quadratic form---each term being the weighted product of an average (additive) effect and an interaction coefficient---needing to cancel out to equal zero. I wonder if the authors can put forth a rigorous argument or compelling intuition for why this should be the case.
In the case of two polymorphic sites, quantitative genetics has traditionally partitioned the total genetic variance into the following orthogonal components:<br /> \begin{itemize}<br /> \item additive genetic variance, $\sigma^2_A$, the numerator of the narrow-sense heritability;<br /> \item dominance genetic variance, $\sigma^2_D$;<br /> \item additive-by-additive genetic variance, $\sigma^2_{AA}$;<br /> \item additive-by-dominance genetic variance, $\sigma^2_{AD}$; and<br /> \item dominance-by-dominance genetic variance, $\sigma^2_{DD}$.<br /> \end{itemize}<br /> See Lynch and Walsh (1998, pp. 88-92) for a thorough numerical example. This decomposition is not arbitrary or trivial, since each component has a distinct coefficient in the correlations between relatives. Is it possible for the authors to relate the variance associated with their $\mathbf{W}\bm{\theta}$ to this traditional decomposition? Besides justifying the work in this paper, the establishment of a relationship can have the possible practical benefit of allowing \texttt{i-LDSC} estimates of non-additive genetic variance to be checked against empirical correlations between relatives. For example, if we know from other methods that $\sigma^2_D$ is negligible but that \texttt{i-LDSC} returns a sizable $\sigma^2_{AA}$, we might predict that the parent-offspring correlation should be equal to the sibling correlation; a sizable $\sigma^2_D$ would make the sibling correlation higher. Admittedly, however, such an exercise can get rather complicated for the variance contributed by pairs of SNPs that are close together (Lynch & Walsh, 1998, pp. 146-152).
I would also like the authors to clarify whether LDSC consistently overestimates the narrow-sense heritability in the case that pairwise epistasis is present. The figures seem to show this. I have conflicting intuitions here. On the one hand, if GWAS summary statistics can be inflated by the tagging of epistasis, then it seems that LDSC should overestimate heritability (or at least this should be an upwardly biasing factor; other factors may lead the net bias to be different). On the other hand, if standard and interaction LD Scores are lowly correlated, then I feel that the inclusion of interaction LD Score in the regression should not strongly affect the coefficient of the standard LD Score. Relatedly, I find it rather curious that \texttt{i-LDSC} seems increasingly biased as the proportion of genetic variance that is non-additive goes up---but perhaps this is not too important, since such a high ratio of narrow-sense to broad-sense heritability is not realistic.
How Much Epistasis Is \texttt{i-LDSC} Detecting?
I think the proper conclusion to be drawn from the authors' analyses is that statistically significant epistatic (non-additive) genetic variance was not detected. Specifically, I think that the analysis presented in Supplementary Table~S6 should be treated as a main analysis rather than a supplementary one, and the results here show no statistically significant epistasis. Let me explain.
Most serious researchers, I think, treat LDSC as an unreliable estimator of narrow-sense heritability; it typically returns estimates that are too low. Not even the original LDSC paper pressed strongly to use the method for estimating $h^2$ (Bulik-Sullivan et al., 2015). As a practical matter, when researchers are focused on estimating absolute heritability with high accuracy, they usually turn to GCTA/GREML (Evans et al., 2018; Wainschtein et al., 2022).
One reason for low estimates with LDSC is that if SNPs with higher LD Scores are less likely to be causal or to have large effect sizes, then the slope of univariate LDSC will not rise as much as it ``should' with increasing LD Score. This was a scenario actually simulated by the authors and displayed in their Supplementary Figure~S15. [Incidentally, the authors might have acknowledged earlier work in this vein. A simulation inducing a negative correlation between LD Scores and $\chi^2$ statistics was presented by Bulik-Sullivan et al. (2015, Supplementary Figure 7), and the potentially biasing effect of a correlation over SNPs between LD Scores and contributed genetic variance was a major theme of Lee et al. (2018).] A negative correlation between LD Score and contributed variance does seem to hold for a number of reasons, including the fact that regions of the genome with higher recombination rates tend to be more functional. In short, the authors did very well to carry out this simulation and to show in their Supplementary Figure~S15 that this flaw of LDSC in estimating narrow-sense heritability is also a flaw of \texttt{i-LDSC} in estimating broad-sense heritability. But they should have carried the investigation at least one step further, as I will explain below.
Another reason for LDSC being a downwardly biased estimator of heritability is that it is often applied to meta-analyses of different cohorts, where heterogeneity (and possibly major but undetected errors by individual cohorts) lead to attenuation of the overall heritability (de Vlaming et al., 2017).
The optimal case for using LDSC to estimate heritability, then, is incorporating the LD-related annotation introduced by Gazal et al. (2017) into a stratified-LDSC (s-LDSC) analysis of a single large cohort. This is analogous to the calculation of multiple GRMs defined by MAF and LD in the GCTA/GREML papers cited above. When this was done by Gazal et al. (2017, Supplementary Table 8b), the joint impact of the improvements was to increase the estimated narrow-sense heritability of height from 0.216 to 0.534.
All of this has at least a few ramifications for \texttt{i-LDSC}. First, the authors do not consider whether a relationship between their interaction LD Scores and interaction effect sizes might bias their estimates. (This would be on top of any biasing relationship between standard LD Scores and linear effect sizes, as displayed in Supplementary Figure~S15.) I find some kind of statistical relationship over the whole genome, induced perhaps by evolutionary forces, between \emph{cis}-acting epistasis and interaction LD Scores to be plausible, albeit without intuition regarding the sign of any resulting bias. The authors should investigate this issue or at least mention it as a matter for future study. Second, it might be that the authors are comparing the estimates of broad-sense heritability in Table~1 to the wrong estimates of narrow-sense heritability. Although the estimates did come from single large cohorts, they seem to have been obtained with simple univariate LDSC rather than s-LDSC. When the estimate of $h^2$ obtained with LDSC is too low, some will suspect that the additional variance detected by \texttt{i-LDSC} is simply additive genetic variance missed by the downward bias of LDSC. Consider that the authors' own Supplementary Table~S6 gives s-LDSC heritability estimates that are consistently higher than the LDSC estimates in Table~1. E.g., the estimated $h^2$ of height goes from 0.37 to 0.43. The latter figure cuts quite a bit into the estimated broad-sense heritability of 0.48 obtained with \texttt{i-LDSC}.
Here we come to a critical point. Lines 282--286 are not entirely clear, but I interpret them to mean that the manuscript's Equation~5 was expanded by stratifying $\ell$ into the components of s-LDSC and this was how the estimates in Supplementary Table~S6 were obtained. If that interpretation is correct, then the scenario of \texttt{i-LDSC} picking up missed additive genetic variance seems rather plausible. At the very least, the increases in broad-sense heritability reported in Supplementary Table~S6 are smaller in magnitude and \emph{not statistically significant}. Perhaps what this means is that the headline should be a \emph{negligible} contribution of pairwise epistasis revealed by this novel and ingenious method, analogous to what has been discovered with respect to dominance (Hivert et al., 2021; Pazokitoroudi et al., 2021; Okbay et al., 2022; Palmer et al., 2023).
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Bulik-Sullivan, B., Loh, P.-R., Finucane, H. K., Ripke, S., Yang, J., Schizophrenia Working Group of the Psychiatric Genomics Consortium, Patterson, N., Daly, M. J., Price, A. L., & Neale, B. M. (2015). LD Score regression distinguishes confounding from polygenicity in genome-wide association studies. Nature Genetics, 47, 291-295.
Burger, R. (2000). The mathematical theory of selection, recombination, and mutation. Wiley.
de Vlaming, R., Okbay, A., Rietveld, C. A., Johannesson, M., Magnusson, P. K. E., Uitterlinden, A. G., van Rooij, F. J. A., Hofman, A., Groe- nen, P. J. F., Thurik, A. R., & Koellinger, P. D. (2017). Meta-GWAS Accuracy and Power (MetaGAP) calculator shows that hiding heritability is partially due to imperfect genetic correlations across studies. PLoS Genetics, 13, e1006495.
Evans, L. M., Tahmasbi, R., Vrieze, S. I., Abecasis, G. R., Das, S., Gazal, S., Bjelland, D. W., de Candia, T. R., Haplotype Reference Consortium, Goddard, M. E., Neale, B. M., Yang, J., Visscher, P. M., & Keller, M. C. (2018). Comparison of methods that use whole genome data to estimate the heritability and genetic architecture of complex traits. Nature Genetics, 50, 737-745.
Ewens, W. J. (2004). Mathematical population genetics I. Theoretical introduction (2nd ed.). Springer.
Fisher, R. A. (1930). The genetical theory of natural selection. Oxford University Press.
Fisher, R. A. (1941). Average excess and average effect of a gene substitution. Annals of Eugenics, 11, 53-63.
Gazal, S., Finucane, H. K., Furlotte, N. A., Loh, P.-R., Palamara, P. F., Liu, X., Schoech, A., Bulik-Sullivan, B., Neale, B. M., Gusev, A., & Price, A. L. (2017). Linkage disequilibrium-dependent architecture of human complex traits shows action of negative selection. Nature Genetics, 49, 1421-1427.
Hivert, V., Sidorenko, J., Rohart, F., Goddard, M. E., Yang, J., Wray, N. R., Yengo, L., & Visscher, P. M. (2021). Estimation of non-additive genetic variance in human complex traits from a large sample of unrelated individuals. American Journal of Human Genetics, 108, 786- 798.
Lee, J. J., McGue, M., Iacono, W. G., & Chow, C. C. (2018). The accuracy of LD Score regression as an estimator of confounding and genetic correlations in genome-wide association studies. Genetic Epidemiology, 42, 783-795.
Lynch, M., & Walsh, B. (1998). Genetics and the analysis of quantitative traits. Sinauer.
Okbay, A., Wu, Y., Wang, N., Jayashankar, H., Bennett, M., Nehzati, S. M., Sidorenko, J., Kweon, H., Goldman, G., Gjorgjieva, T., Jiang, Y., Hicks, B., Tian, C., Hinds, D. A., Ahlskog, R., Magnusson, P. K. E., Oskarsson, S., Hayward, C., Campbell, A., ... Young, A. I. (2022). Polygenic prediction of educational attainment within and between families from genome-wide association analyses in 3 million individu- als. Nature Genetics, 54, 437-449.
Palmer, D. S., Zhou, W., Abbott, L., Wigdor, E. M., Baya, N., Churchhouse, C., Seed, C., Poterba, T., King, D., Kanai, M., Bloemendal, A., & Neale, B. M. (2023). Analysis of genetic dominance in the UK Biobank. Science, 379, 1341-1348.
Pazokitoroudi, A., Chiu, A. M., Burch, K. S., Pasaniuc, B., & Sankararaman, S. (2021). Quantifying the contribution of dominance deviation effects to complex trait variation in biobank-scale data. American Journal of Human Genetics, 108, 799-808.
Wainschtein, P., Jain, D., Zheng, Z., TOPMed Anthropometry Working Group, NHLBI Trans-Omics for Precision Medicine Consoritum, Cupples, L. A., Shadyab, A. H., McKnight, B., Shoemaker, B. M., Mitchell, B. D., Psaty, B. M., Kooperberg, C., Liu, C.-T., Albert, C. M., Roden, D., Chasman, D. I., Darbar, D., Lloyd-Jones, D. M., Arnett, D. K., . . . Visscher, P. M. (2022). Assessing the contribution of rare variants to complex trait heritability from whole-genome sequence data. Nature Genetics, 54, 263-273.
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Reviewer #1 (Public Review):
In this work, 40 healthy volunteers underwent a placebo followed by a ketamine infusion during a resting state fMRI scan. The authors use principal components analysis (PCA) of the difference in global brain connectivity (GBC) between the ketamine and placebo infusions as their summary neural measure. First, a GBC map is computed after processing with the HCP minimal pipeline and removal of the global brain signal for each scan (~4.5 min, TR=700ms). Then the significant PCA components of difference between ketamine and placebo GBC maps are taken as the neural effect of interest and compared to the mean delta GBC. The first two principal components account for 24.5% of the variance of the data and had correlations with SST and PVALB cortical gene expression patterns that were above chance. No significant correlations were found between mean change in GBC and these genes. Additionally, in comparison with the mean GBC the PCs were found to better correlate with behavioral measures.
To further support their aim to establish the multi-dimensionality of the ketamine response using their neural measure, the PCA dimensionality was estimated in external datasets that used psilocybin and LSD with sample size matching using identical processing and found lower dimensionality in these datasets.
Effective dimensionality was calculated using the participation ratio and dataset re-sampling was used to control for sample size in this calculation, but dimensionality is also affected by motion within the sample among other noise sources, which are not well discussed. In particular, each drug may affect physiological noise in different ways and this may in turn affect their dimensionality measurement.
A PCA decomposition of the changes (ketamine-placebo) in 31 measured behavioral variables was also performed which resulted in two major PCs which accounted for 41.4% of the variance. Following prior work, behavioral PCs were mapped onto the neural PCs to create neuro-behavioral PCs. The weighing of the PCs at the individual level was explored to compare inter-individual variability.
In an earlier fMRI study of the timeseries response to ketamine (De Simoni, 2013) it was clear that there are both individual and regional brain response differences. Behaviorally, there is known individual variability in the response to ketamine insofar as only about 60% of depressed people will experience symptom improvement and even then to varying levels. Thus, it is good to see that the compound summary measure of the PCA of the change in GBC after ketamine follows this pattern and shows inter-individual differences.
A strength of this paper is that it brings together multimodal and external datasets and combines them in a linked analysis to support their investigational aims. Several sets of analyses are used to draw relations between fMRI results, genetics, and behavioral measures but the range of conclusions is limited by the understandably small sample size for this kind of drug challenge study. A weakness is that the chosen summary measure (delta GBC of ketamine-placebo, followed by a group-level PCA) that has been principally developed by this lab and has not seen wide replication. The presentation of the analyses could be simplified to increase readability and impact. Nevertheless, this study provides informative steps toward the development of markers for individualized drug response.
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Reviewer #1 (Public Review):
This thorough study expands our understanding of BMP signaling, a conserved developmental pathway, involved in processes diverse such as body patterning and neurogenesis. The authors applied multiple, state-of-art strategies to the anthozoan Nematostella vectensis in order to first identify the direct BMP signaling targets - bound by the activated pSMAD1/5 protein - and then dissect the role of a novel pSMAD1/5 gradient modulator, zwim4-6. The list of target genes features multiple developmental regulators, many of which are bilaterally expressed, and which are notably shared between Drosophila and Xenopus. The analysis identified in particular zswim4-6 a novel nuclear modulator of the BMP pathway conserved also in vertebrates. A combination of both loss-of-function (injection of antisense morpholino oligonucleotide, CRISPR/Cas9 knockout, expression of dominant negative) and gain-of-function assays, and of transcriptome sequencing identified that zwim acts as a transcriptional repression of BMP signaling. Functional manipulation of zswim5 in zebrafish shows a conserved role in modulating BMP signaling in a vertebrate.<br /> The particular strength of the study lies in the careful and thorough analysis performed. This is solid developmental work, where one clear biological question is progressively dissected, with the most appropriate tools. The functional results are further validated by alternative approaches. Data is clearly presented and methods are detailed.
I have a couple of comments.<br /> 1) I was intrigued - as the authors - by the fact that the ChiP-Seq did not identify any known BMP ligand bound by pSMAD1/5. Are these genes found in the published ChiP-Seq data of the other species used for the comparative analysis? One hypothesis could be that there is a change in the regulatory interactions and that the initial set-up of the gradient requires indeed a feedback loop, which is then turned off at later gastrula. In this case, immunoprecipitation at early gastrula, prior to the set-up of the pSMAD1/5 gradient, could reveal a different scenario. Alternately, the regulation could be indirect, for example, through RGM, an additional regulator of BMP signaling expressed on the side of lower BMP activity, which is among the targets of the ChiP-Seq. This aspect could be discussed. Additionally, even if this is perhaps outside the scope of this study, I think it would be informative to further assess the effect of ZSWIM manipulation on RGM (and vice versa).<br /> 2) I do not fully understand the rationale behind the choice of performing the comparative assays in zebrafish: as the conservation was initially identified in Xenopus, I would have expected the experiment to be performed in frog. Furthermore, reading the phylogeny (Figure 4A), it is not obvious to me why ZSWIM5 was chosen for the assay (over the other paralog ZSWIM6). Could the Authors comment on this experiment further?
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Reviewer #1 (Public Review):
In several developmental systems, the core Planar Cell Polarity (PCP) pathway organises the dynamics of cellular behaviours underlying morphogenesis. During pupal stages, the Drosophila wing undergoes a complex morphogenetic process that results in the simultaneous elongation and narrowing of the wing blade along the proximal-distal and anterior-posterior axes, respectively. It was proposed that this dynamic process is driven by mechanical stress that results in cell deformations and cell rearrangements. However, prior work by Etournay et al. (eLife 2015) shows that mutants that reduce of mechanical stresses do not completely eliminate oriented cell rearrangements. Here, Piscitello-Gomez et al. use imaging techniques previously developed by them and others, combined with a computational analysis of a rheological model, to evaluate the role of the core-PCP pathway as a possible patterning cue that could orient cell rearrangements in this system. Surprisingly, the authors found that core-PCP mutants only affect an early retraction velocity upon laser ablation, but do not seem to drive overall morphogenesis in this system. Therefore, the original question of the work, namely, identifying the patterning cues that establish oriented cell rearrangements in this system, remains unanswered.
The work exemplifies how the integration of mechanical perturbations, image analysis, and computational modelling could be used to investigate the contribution of a specific patterning cue in morphogenesis. While the conclusions of the manuscript are solid and the data support the conclusion that core-PCP pathway mutants do not display an altered cell dynamic or cell elongation phenotype relative to wild-type controls, one challenge of the approach is that the time-lapse imaging technique is done only in a handful of pupal wings. This does not permit to conclude whether subtle changes in cell elongation or cell rearrangements could account for observed changes in the shape of adult wings (that are more round in these mutants). Other patterning and polarity cues such as Fat-Daschous or Toll-like signalling are suggested by the authors, but their examination is left for future studies.
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Reviewer #1 (Public Review):
This study uses electrophysiological techniques in vitro to address the role of the Na+ leak channel NALCN in various physiological functions in cartwheel interneurons of the dorsal cochlear nucleus. Comparing wild type and glycinergic neuron-specific knockout mice for NALCN, the authors show that these channels 1) are required for spontaneous firing, 2) are modulated by noradrenaline (NA, via alpha2 receptors) and GABA (through GABAB receptors), 3) how the modulation by NA enhances IPSCs in these neurons.
This work builds on previous results from the Trussell's lab in terms of the physiology of cartwheel cells, and from other labs in terms of the role of NALCN channels, that have been characterized in more and more brain areas somewhat recently; for this reason, this study could be of interest for researchers that work in other preparations as well. The general conclusions are strongly supported by results that are clearly and elegantly presented.
In this revised submission, the authors addressed all my questions. This is very interesting work that could be of interest for researchers working in other brain areas as well.
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Reviewer #1 (Public Review):
In the manuscript "Long‐read single‐cell sequencing reveals expressions of hypermutation clusters of isoforms in human liver cancer cells", S. Liu et al present a protocol combining 10x Genomics single-cell assay with Element LoopSeq synthetic long-read sequencing to study single nucleotide variants (SNVs) and gene fusions in Hepatocellular carcinoma (HCC) at single‐cell level. The authors were the first to combine LoopSeq synthetic long‐read sequencing technology and 10x Genomics barcoding for single cell sequencing. For each cell and each somatic mutation, they obtain fractions of mutated transcripts per gene and per each transcript isoform. The manuscript states that these values (as well as gene fusion information) provide better features for tumor-normal classification than gene expression levels. The authors identified many SNVs in genes of the human major histocompatibility complex (HLA) with up to 25 SNVs in the same molecule of HLA‐DQB1 transcript. The analysis shows that most mutations occur in HLA genes and suggests evolution pathways that led to these hypermutation clusters.
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Reviewer #1 (Public Review):
Summary:<br /> The paper investigates the physiological and neural processes that relate to infants' attention allocation in a naturalistic setting. Contrary to experimental paradigms that are usually employed in developmental research, this study investigates attention processes while letting the infants be free to play with three toys in the vicinity of their caregiver, which is closer to a common, everyday life context. The paper focuses on infants at 5 and 10 months of age and finds differences in what predicts attention allocation. At 5 months, attention episodes are shorter and their duration is predicted by autonomic arousal. At 10 months, attention episodes are longer, and their duration can be predicted by theta power. Moreover, theta power predicted the proportion of looking at the toys, as well as a decrease in arousal (heart rate). Overall, the authors conclude that attentional systems change across development, becoming more driven by cortical processes.
Strengths:<br /> I enjoyed reading the paper, I am impressed with the level of detail of the analyses, and I am strongly in favour of the overall approach, which tries to move beyond in-lab settings. The collection of multiple sources of data (EEG, heart rate, looking behaviour) at two different ages (5 and 10 months) is a key strength of this paper. The original analyses, which build onto robust EEG preprocessing, are an additional feat that improves the overall value of the paper. The careful consideration of how theta power might change before, during, and in the prediction of attention episodes is especially remarkable. However, I have a few major concerns that I would like the authors to address, especially on the methodological side.
Points of improvement<br /> 1. Noise<br /> The first concern is the level of noise across age groups, periods of attention allocation, and metrics. Starting with EEG, I appreciate the analysis of noise reported in supplementary materials. The analysis focuses on a broad level (average noise in 5-month-olds vs 10-month-olds) but variations might be more fine-grained (for example, noise in 5mos might be due to fussiness and crying, while at 10 months it might be due to increased movements). More importantly, noise might even be the same across age groups, but correlated to other aspects of their behaviour (head or eye movements) that are directly related to the measures of interest. Is it possible that noise might co-vary with some of the behaviours of interest, thus leading to either spurious effects or false negatives? One way to address this issue would be for example to check if noise in the signal can predict attention episodes. If this is the case, noise should be added as a covariate in many of the analyses of this paper.<br /> Moving onto the video coding, I see that inter-rater reliability was not very high. Is this due to the fine-grained nature of the coding (20ms)? Is it driven by differences in expertise among the two coders? Or because coding this fine-grained behaviour from video data is simply too difficult? The main dependent variable (looking duration) is extracted from the video coding, and I think the authors should be confident they are maximising measurement accuracy.
2. Cross-correlation analyses<br /> I would like to raise two issues here. The first is the potential problem of using auto-correlated variables as input for cross-correlations. I am not sure whether theta power was significantly autocorrelated. If it is, could it explain the cross-correlation result? The fact that the cross-correlation plots in Figure 6 peak at zero, and are significant (but lower) around zero, makes me think that it could be a consequence of periods around zero being autocorrelated. Relatedly: how does the fact that the significant lag includes zero, and a bit before, affect the interpretation of this effect?
A second issue with the cross-correlation analyses is the coding of the looking behaviour. If I understand correctly, if an infant looked for a full second at the same object, they would get a maximum score (e.g., 1) while if they looked at 500ms at the object and 500ms away from the object, they would receive a score of e.g., 0.5. However, if they looked at one object for 500ms and another object for 500ms, they would receive a maximum score (e.g., 1). The reason seems unclear to me because these are different attention episodes, but they would be treated as one. In addition, the authors also show that within an attentional episode theta power changes (for 10mos). What is the reason behind this scoring system? Wouldn't it be better to adjust by the number of attention switches, e.g., with the formula: looking-time/(1+N_switches), so that if infants looked for a full second, but made 1 switch from one object to the other, the score would be .5, thus reflecting that attention was terminated within that episode?
3. Clearer definitions of variables, constructs, and visualisations<br /> The second issue is the overall clarity and systematicity of the paper. The concept of attention appears with many different names. Only in the abstract, it is described as attention control, attentional behaviours, attentiveness, attention durations, attention shifts and attention episode. More names are used elsewhere in the paper. Although some of them are indeed meant to describe different aspects, others are overlapping. As a consequence, the main results also become more difficult to grasp. For example, it is stated that autonomic arousal predicts attention, but it's harder to understand what specific aspect (duration of looking, disengagement, etc.) it is predictive of. Relatedly, the cognitive process under investigation (e.g., attention) and its operationalization (e.g., duration of consecutive looking toward a toy) are used interchangeably. I would want to see more demarcation between different concepts and between concepts and measurements.
General Remarks<br /> In general, the authors achieved their aim in that they successfully showed the relationship between looking behaviour (as a proxy of attention), autonomic arousal, and electrophysiology. Two aspects are especially interesting. First, the fact that at 5 months, autonomic arousal predicts the duration of subsequent attention episodes, but at 10 months this effect is not present. Conversely, at 10 months, theta power predicts the duration of looking episodes, but this effect is not present in 5-month-old infants. This pattern of results suggests that younger infants have less control over their attention, which mostly depends on their current state of arousal, but older infants have gained cortical control of their attention, which in turn impacts their looking behaviour and arousal.
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Reviewer #1 (Public Review):
In this study, authors performed multiple sets of mesoscale chromatin simulations at nucleosome resolution to study the effects of TF binding on chromatin structures. Through simulations at various conditions, authors performed systemically analysis to investigate how linker histone, tail acetylation, and linker DNA length can operate together with TFs to regulate chromatin architecture. Using gene Eed as one example, authors found that binding of Myc:Max could repress the gene expression by increasing fiber folding and compaction and this repression can be reversed by the linker histone. Understanding how transcription factors bind to regulatory DNA elements and modulate chromatin structure and accessibility is an essential question in epigenetics. Through modelling of TF binding to chromatin structures at nucleosome levels, authors demonstrated that TF binding could create microdomains that are visible in the ensemble-based contact maps and short DNA linkers prevent the formation microdomains. It has also been shown that tail acetylation and TF binding have opposite effects on chromatin compaction and linker histone can compete for the linker DNA with TF binding to impair the effect of TF binding. This study improves our knowledge on how TFs collaborate with different epigenetic marks and chromatin features to regulate chromatin structure and accessibility, which will be of broad interest to the community.
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Reviewer #1 (Public Review):
In this very strong and interesting paper the authors present a convincing series of experiments that reveal molecular mechansism of neuronal cell type diversification in the nervous system of Drosophila. The authors show that a homeodomain transcription factor, Bsh, fulfills several critical functions - repressing an alternative fate and inducing downstream homeodomain transcription factors with whom Bsh may collaborate to induce L4 and L5 fates (the author's accompanying paper reveals how Bsh can induce two distinct fates). The authors make elegant use of powerful genetic tools and an arsenal of satisfying cell identity markers.
I believe that this is an important study because it provides some fundamental insights into the conservation of neuronal diversification programs. It is very satisfying to see that similar organizational principles apply in different organism to generate cell type diversity. The authors should also be commended for contextualizing their work very well, giving a broad, scholarly background to the problem of neuronal cell type diversification.
My one suggestion for the authors is to perhaps address in the Discussion (or experimentally address if they wish) how they reconcile that Bsh is on the one hand: (a) continuously expressed in L4/L4, (b) binding directly to a cohort of terminal effectors that are also continuously expressed but then, on the other hand, is not required for their maintaining L4 fate? A few questions: Is Bsh only NOT required for maintaining Ap expression or is it also NOT required for maintaining other terminal markers of L4? The former could be easily explained - Bsh simply kicks of Ap, Ap then autoregulates, but Bsh and Ap then continuously activate terminal effector genes. The second scenario would require a little more complex mechanism: Bsh binding of targets (with Notch) may open chromatin, but then once that's done, Bsh is no longer needed and Ap alone can continue to express genes. I feel that the authors should be at least discussing this. The postmitotic Bsh removal experiment in which they only checked Ap and depression of other markers is a little unsatisfying without further discussion (or experiments, such as testing terminal L4 markers). I hasten to add that this comment does not take away from my overall appreciation for the depth and quality of the data and the importance of their conclusions.
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Reviewer #1 (Public Review):
Like the "preceding" co-submitted paper, this is again a very strong and interesting paper in which the authors address a question that is raised by the finding in their co-submitted paper - how does one factor induce two different fates. The authors provide an extremely satisfying answer - only one subset of the cells neighbors a source of signaling cells that trigger that subset to adopt a specific fate. The signal here is Delta and the read-out is Notch, whose intracellular domain, in conjunction with, presumably, SuH cooperates with Bsh to distinguish L4 from L5 fate (L5 is not neighbored by signal-providing cells). Like the back-to-back paper, the data is rigorous, well-presented and presents important conclusions. There's a wealth of data on the different functions of Notch (with and without Bsh). All very satisfying.
I have again one suggestion that the authors may want to consider discussing. I'm wondering whether the open chromatin that the author convincingly measure is the CAUSE or the CONSEQUENCE of Bsh being able to activate L4 target genes. What I mean by this is that currently the authors seem to be focused on a somewhat sequential model where Notch signaling opens chromatin and this then enables Bsh to activate a specific set of target genes. But isn't it equally possible that the combined activity of Bsh/Notch(intra)/SuH opens chromatin? That's not a semantic/minor difference, it's a fundamentally different mechanism, I would think. This mechanism also solves the conundrum of specificity - how does Notch know which genes to "open" up? It would seem more intuitive to me to think that it's working together with Bsh to open up chromatin, with chromatin accessibility than being a "mere" secondary consequence. If I'm not overlooking something fundamental here, there is actually also a way to distinguish between these models - test chromatin accessibility in a Bsh mutant. If the author's model is true, chromatin accessibility should be unchanged.
I again finish by commending the authors for this terrific piece of work.
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Reviewer #1 (Public Review):
Summary:
This study aims to provide imaging methods for users of the field of human layer-fMRI. This is an emerging field with 240 papers published so far. Different than implied in the manuscript, 3T is well represented among those papers. E.g. see the papers below that are not cited in the manuscript. Thus, the claim on the impact of developing 3T methodology for wider dissemination is not justified. Specifically, because some of the previous papers perform whole brain layer-fMRI (also at 3T) in more efficient, and more established procedures.
The authors implemented a sequence with lots of nice features. Including their own SMS EPI, diffusion bipolar pulses, eye-saturation bands, and they built their own reconstruction around it. This is not trivial. Only a few labs around the world have this level of engineering expertise. I applaud this technical achievement. However, I doubt that any of this is the right tool for layer-fMRI, nor does it represent an advancement for the field. In the thermal noise dominated regime of sub-millimeter fMRI (especially at 3T), it is established to use 3D readouts over 2D (SMS) readouts. While it is not trivial to implement SMS, the vendor implementations (as well as the CMRR and MGH implementations) are most widely applied across the majority of current fMRI studies already. The author's work on this does not serve any previous shortcomings in the field.
The mechanism to use bi-polar gradients to increase the localization specificity is doubtful to me. In my understanding, killing the intra-vascular BOLD should make it less specific. Also, the empirical data do not suggest a higher localization specificity to me.
Embedding this work in the literature of previous methods is incomplete. Recent trends of vessel signal manipulation with ABC or VAPER are not mentioned. Comparisons with VASO are outdated and incorrect.
The reproducibility of the methods and the result is doubtful (see below).
I don't think that this manuscript is in the top 50% of the 240 layer-fmri papers out there.
3T layer-fMRI papers that are not cited:<br /> Taso, M., Munsch, F., Zhao, L., Alsop, D.C., 2021. Regional and depth-dependence of cortical blood-flow assessed with high-resolution Arterial Spin Labeling (ASL). Journal of Cerebral Blood Flow and Metabolism. https://doi.org/10.1177/0271678X20982382
Wu, P.Y., Chu, Y.H., Lin, J.F.L., Kuo, W.J., Lin, F.H., 2018. Feature-dependent intrinsic functional connectivity across cortical depths in the human auditory cortex. Scientific Reports 8, 1-14. https://doi.org/10.1038/s41598-018-31292-x
Lifshits, S., Tomer, O., Shamir, I., Barazany, D., Tsarfaty, G., Rosset, S., Assaf, Y., 2018. Resolution considerations in imaging of the cortical layers. NeuroImage 164, 112-120. https://doi.org/10.1016/j.neuroimage.2017.02.086
Puckett, A.M., Aquino, K.M., Robinson, P.A., Breakspear, M., Schira, M.M., 2016. The spatiotemporal hemodynamic response function for depth-dependent functional imaging of human cortex. NeuroImage 139, 240-248. https://doi.org/10.1016/j.neuroimage.2016.06.019
Olman, C.A., Inati, S., Heeger, D.J., 2007. The effect of large veins on spatial localization with GE BOLD at 3 T: Displacement, not blurring. NeuroImage 34, 1126-1135. https://doi.org/10.1016/j.neuroimage.2006.08.045
Ress, D., Glover, G.H., Liu, J., Wandell, B., 2007. Laminar profiles of functional activity in the human brain. NeuroImage 34, 74-84. https://doi.org/10.1016/j.neuroimage.2006.08.020
Huber, L., Kronbichler, L., Stirnberg, R., Ehses, P., Stocker, T., Fernández-Cabello, S., Poser, B.A., Kronbichler, M., 2023. Evaluating the capabilities and challenges of layer-fMRI VASO at 3T. Aperture Neuro 3. https://doi.org/10.52294/001c.85117
Scheeringa, R., Bonnefond, M., van Mourik, T., Jensen, O., Norris, D.G., Koopmans, P.J., 2022. Relating neural oscillations to laminar fMRI connectivity in visual cortex. Cerebral Cortex. https://doi.org/10.1093/cercor/bhac154
Strengths:
See above. The authors developed their own SMS sequence with many features. This is important to the field. And does not leave sequence development work to view isolated monopoly labs. This work democratises SMS.<br /> The questions addressed here are of high relevance to the field: getting tools with good sensitivity, user-friendly applicability, and locally specific brain activity mapping is an important topic in the field of layer-fMRI.
Weaknesses:
1. I feel the authors need to justify why flow-crushing helps localization specificity. There is an entire family of recent papers that aim to achieve higher localization specificity by doing the exact opposite. Namely, MT or ABC fRMRI aims to increase the localization specificity by highlighting the intravascular BOLD by means of suppressing non-flowing tissue. To name a few:
Priovoulos, N., de Oliveira, I.A.F., Poser, B.A., Norris, D.G., van der Zwaag, W., 2023. Combining arterial blood contrast with BOLD increases fMRI intracortical contrast. Human Brain Mapping hbm.26227. https://doi.org/10.1002/hbm.26227.
Pfaffenrot, V., Koopmans, P.J., 2022. Magnetization Transfer weighted laminar fMRI with multi-echo FLASH. NeuroImage 119725. https://doi.org/10.1016/j.neuroimage.2022.119725
Schulz, J., Fazal, Z., Metere, R., Marques, J.P., Norris, D.G., 2020. Arterial blood contrast ( ABC ) enabled by magnetization transfer ( MT ): a novel MRI technique for enhancing the measurement of brain activation changes. bioRxiv. https://doi.org/10.1101/2020.05.20.106666
Based on this literature, it seems that the proposed method will make the vein problem worse, not better. The authors could make it clearer how they reason that making GE-BOLD signals more extra-vascular weighted should help to reduce large vein effects.
The empirical evidence for the claim that flow crushing helps with the localization specificity should be made clearer. The response magnitude with and without flow crushing looks pretty much identical to me (see Fig, 6d).<br /> It's unclear to me what to look for in Fig. 5. I cannot discern any layer patterns in these maps. It's too noisy. The two maps of TE=43ms look like identical copies from each other. Maybe an editorial error?
The authors discuss bipolar crushing with respect to SE-BOLD where it has been previously applied. For SE-BOLD at UHF, a substantial portion of the vein signal comes from the intravascular compartment. So I agree that for SE-BOLD, it makes sense to crush the intravascular signal. For GE-BOLD however, this reasoning does not hold. For GE-BOLD (even at 3T), most of the vein signal comes from extravascular dephasing around large unspecific veins, and the bipolar crushing is not expected to help with this.
2. The bipolar crushing is limited to one single direction of flow. This introduces a lot of artificial variance across the cortical folding pattern. This is not mentioned in the manuscript. There is an entire family of papers that perform layer-fmri with black-blood imaging that solves this with a 3D contrast preparation (VAPER) that is applied across a longer time period, thus killing the blood signal while it flows across all directions of the vascular tree. Here, the signal cruising is happening with a 2D readout as a "snap-shot" crushing. This does not allow the blood to flow in multiple directions.<br /> VAPER also accounts for BOLD contaminations of larger draining veins by means of a tag-control sampling. The proposed approach here does not account for this contamination.
Chai, Y., Li, L., Huber, L., Poser, B.A., Bandettini, P.A., 2020. Integrated VASO and perfusion contrast: A new tool for laminar functional MRI. NeuroImage 207, 116358. https://doi.org/10.1016/j.neuroimage.2019.116358
Chai, Y., Liu, T.T., Marrett, S., Li, L., Khojandi, A., Handwerker, D.A., Alink, A., Muckli, L., Bandettini, P.A., 2021. Topographical and laminar distribution of audiovisual processing within human planum temporale. Progress in Neurobiology 102121. https://doi.org/10.1016/j.pneurobio.2021.102121
If I would recommend anyone to perform layer-fMRI with blood crushing, it seems that VAPER is the superior approach. The authors could make it clearer why users might want to use the unidirectional crushing instead.
3. The comparison with VASO is misleading.<br /> The authors claim that previous VASO approaches were limited by TRs of 8.2s. The authors might be advised to check the latest literature of the last years.<br /> Koiso et al. performed whole brain layer-fMRI VASO at 0.8mm at 3.9 seconds (with reliable activation), 2.7 seconds (with unconvincing activation pattern, though), and 2.3 (without activation).<br /> Also, whole brain layer-fMRI BOLD at 0.5mm and 0.7mm has been previously performed by the Juelich group at TRs of 3.5s (their TR definition is 'fishy' though).
Koiso, K., Müller, A.K., Akamatsu, K., Dresbach, S., Gulban, O.F., Goebel, R., Miyawaki, Y., Poser, B.A., Huber, L., 2023. Acquisition and processing methods of whole-brain layer-fMRI VASO and BOLD: The Kenshu dataset. Aperture Neuro 34. https://doi.org/10.1101/2022.08.19.504502
Yun, S.D., Pais‐Roldán, P., Palomero‐Gallagher, N., Shah, N.J., 2022. Mapping of whole‐cerebrum resting‐state networks using ultra‐high resolution acquisition protocols. Human Brain Mapping. https://doi.org/10.1002/hbm.25855
Pais-Roldan, P., Yun, S.D., Palomero-Gallagher, N., Shah, N.J., 2023. Cortical depth-dependent human fMRI of resting-state networks using EPIK. Front. Neurosci. 17, 1151544. https://doi.org/10.3389/fnins.2023.1151544
The authors are correct that VASO is not advised as a turn-key method for lower brain areas, incl. Hippocampus and subcortex. However, the authors use this word of caution that is intended for inexperienced "users" as a statement that this cannot be performed. This statement is taken out of context. This statement is not from the academic literature. It's advice for the 40+ user base that wants to perform layer-fMRI as a plug-and-play routine tool in neuroscience usage. In fact, sub-millimeter VASO is routinely being performed by MRI-physicists across all brain areas (including deep brain structures, hippocampus etc). E.g. see Koiso et al. and an overview lecture from a layer-fMRI workshop that I had recently attended: https://youtu.be/kzh-nWXd54s?si=hoIJjLLIxFUJ4g20&t=2401
Thus, the authors could embed this phrasing into the context of their own method that they are proposing in the manuscript. E.g. the authors could state whether they think that their sequence has the potential to be disseminated across sites, considering that it requires slow offline reconstruction in Matlab?<br /> Do the authors think that the results shown in Fig. 6c are suggesting turn-key acquisition of a routine mapping tool? In my humble opinion, it looks like random noise, with most of the activation outside the ROI (in white matter).
4. The repeatability of the results is questionable.<br /> The authors perform experiments about the robustness of the method (line 620). The corresponding results are not suggesting any robustness to me. In fact, the layer profiles in Fig. 4c vs. Fig 4d are completely opposite. The location of peaks turns into locations of dips and vice versa.<br /> The methods are not described in enough detail to reproduce these results.<br /> The authors mention that their image reconstruction is done "using in-house MATLAB code" (line 634). They do not post a link to github, nor do they say if they share this code.
It is not trivial to get good phase data for fMRI. The authors do not mention how they perform the respective coil-combination.<br /> No data are shared for reproduction of the analysis.
5. The application of NODRIC is not validated.<br /> Previous applications of NORDIC at 3T layer-fMRI have resulted in mixed success. When not adjusted for the right SNR regime it can result in artifactual reductions of beta scores, depending on the SNR across layers. The authors could validate their application of NORDIC and confirm that the average layer-profiles are unaffected by the application of NORDIC. Also, the NORDIC version should be explicitly mentioned in the manuscript.
Akbari, A., Gati, J.S., Zeman, P., Liem, B., Menon, R.S., 2023. Layer Dependence of Monocular and Binocular Responses in Human Ocular Dominance Columns at 7T using VASO and BOLD (preprint). Neuroscience. https://doi.org/10.1101/2023.04.06.535924
Knudsen, L., Guo, F., Huang, J., Blicher, J.U., Lund, T.E., Zhou, Y., Zhang, P., Yang, Y., 2023. The laminar pattern of proprioceptive activation in human primary motor cortex. bioRxiv. https://doi.org/10.1101/2023.10.29.564658
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Reviewer #1 (Public Review):
Summary:<br /> Based on a "dichoptic-background-movie" paradigm that modulates ocular dominance, the present study combines fMRI and TMS to examine the role of the frontoparietal attentional network in ocular dominance shifts. The authors claimed a causal role of FEF in generating the attention-induced ocular dominance shift.
Strengths:<br /> A combination of fMRI, TMS, and "dichoptic-background-movie" paradigm techniques is used to reveal the causal role of the frontoparietal attentional network in ocular dominance shifts. The conclusions of this paper are mostly well supported by data.
Weaknesses:<br /> The relationship between eye dominance, eye-based attention shift, and cortical functions remains unclear and merits further delineation. The rationale of the experimental design related to the hemispheric asymmetry in the FEF and other regions should be clarified.
Theoretically, how the eye-related functions in this area could be achieved, and how it interacts with the ocular representation in V1 warrant further clarification.
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Reviewer #1 (Public Review):
Summary:<br /> The authors of this study investigated the development of interoceptive sensitivity in the context of cardiac and respiratory interoception in 3-, 9-, and 18-month-old infants using a combination of both cross-sectional and longitudinal designs. They utilised the cardiac interoception paradigm developed by Maister et al (2017) and also developed a new paradigm to investigate respiratory interoception in infants. The main findings of this research are that 9-month-old infants displayed a preference for stimuli presented synchronously with their own heartbeat and respiration. The authors found less reliable effects in the 18-month-old group, and this was especially true for the respiratory interoceptive data. The authors replicated a visual preference for synchrony over asynchrony for the cardiac domain in 3-month-old infants, while they found inconclusive evidence regarding the respiratory domain. Considering the developmental nature of the study, the authors also investigated the presence of developmental trajectories and associations between the two interoceptive domains. They found evidence for a relationship between cardiac and respiratory interoceptive sensitivity at 18 months only and preliminary evidence for an increase in respiratory interoception between 9 and 18 months.
Strengths: The conclusions of this paper are mostly well supported by data, and the data analysis procedures are rigorous and well-justified. The main strengths of the paper are:<br /> - A first attempt to explore the association between two different interoceptive domains. How different organ-specific axes of interoception relate to each other is still open and exploring this from a developmental lens can help shed light into possible relationships. The authors have to be commended for developing novel interoceptive tasks aimed at assessing respiratory interoceptive sensitivity in infants and toddlers, and for trying to assess the relationship between cardiac and respiratory interoception across developmental time.<br /> - A thorough justification of the developmental ages selected for the study. The authors provide a rationale behind their choice to examine interoceptive sensitivity at 3, 9, and 18 months of age. These are well justified based on the literature pertaining to self- and social development. Sometimes, I wondered whether explaining the link between these self and social processes and interoception would have been beneficial as a reader not familiar with the topics may miss the point.<br /> - An explanation of the direction of looking behaviour using latent curve analysis. I found this additional analysis extremely helpful in providing a better understanding of the data based on previous research and analytical choices (though see comment under weaknesses). As the authors explain in the manuscript, it is often difficult to interpret the direction of infant-looking behaviour as novelty and familiarity preferences can also be driven by hidden confounders (e.g. task difficulty). The authors provide some evidence that analytical choices can explain some of these effects. Beyond the field of interoception, these findings will be relevant to development psychologists and will inform future studies using looking time as a measure of infants' ability to discriminate among stimuli.<br /> - The use of simulation analysis to account for the small sample size. The authors acknowledge that some of the effects reported in their study could be explained by a small sample size (i.e. the 3-month-olds and 18-month-olds data). Using a simulation approach, the authors try to overcome some of these limitations and provide convincing evidence of interoceptive abilities in infancy and toddlerhood (but see also my next point).
Weaknesses:<br /> - The authors should carefully address the potential confounding of not counterbalancing the conditions of the first trial in both interoceptive tasks for the 9-month and 18-month age groups. The results of these groups could indeed be driven by having seen the synchronous trial first.<br /> - The conclusion that cardiac interoception remains stable across infancy is not fully warranted by the data. Given the small sample size of 18-month-old toddlers included in the final analyses, it might be misleading to state this without including the caveat that the study may be underpowered. In other words, the small sample size could explain the direction of the results for this age group.
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Reviewer #1 (Public Review):
This study examined a universal fractal primate brain shape. However, the paper does not seem well structured and is not well written. It is not clear what the purpose of the paper is. And there is a lack of explanation for why the proposed analysis is necessary. As a result, it is challenging to clearly understand what novelty in the paper is and what the main findings are. Additionally, several terms are introduced without adequate explanation and contextualization, further complicating comprehension. Does the second section, "2. Coarse-graining procedure", serve as an introduction or a method? Moreover, the rationale behind the use of the coarse-graining procedure is not adequately elucidated. Overall, it is strongly recommended that the paper undergoes significant improvements in terms of its structure, explanatory depth, and overall clarity to enhance its comprehensibility.
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Reviewer #1 (Public Review):
The major aim of the paper was a method for determining genetic associations between two traits using common variants tested in genome-wide association studies. The work includes a software implementation and application of their approach. The results of the application of their method generally agree with what others have seen using similar AD and UKB data.
The paper has several distinct portions. The first is a method for testing genetic associations between two or more traits using genome-wide association tests statistics. The second is a python implementation of the method. The last portion is the results of their method using GWAS from AD and UK Biobank.
Regarding the method, it seems like it has similarities to LDSC, and it is not clear how it differs from LDSC or other similar methods. The implementation of the method used python 2.7 (or at least was reportedly tested using that version) that was retired in 2020. The implementation was committed between Wed Oct 3 15:21:49 2018 to Mon Jan 28 09:18:09 2019 using data that existed at the time so it was a bit surprising it used python 2.7 since it was initially going to be set for end-of-life in 2015. Anyway, trying to run the package resulted in unmet dependency errors, which I think are related to an internal package not getting installed. I would expect that published software could be installed using standard tooling for the language, and, ideally, software should have automated testing of key portions.
Regarding the main results, they find what has largely been shown by others using the same data or similar data, which add prima facie validity to the work The portions of the work dealing with AD subgroups, pathology, biomarkers, and cognitive traits of interest. I was puzzled why the authors suggested surprise regarding parental history and high cholesterol not associated with MCI or cognitive composite scores since the this would seem like the likely fallout of selection of the WRAP cohort. The discussion paragraph that started "What's more, environmental factors may play a big role in the identified associations." confused me. I think what the authors are referring to are how selection, especially in a biobank dataset, can induce correlations, which is not what I think of as an environmental effect.
Overall, the work has merit, but I am left without a clear impression of the improvement in the approach over similar methods. Likewise, the results are interesting, but similar findings are described with the data that was used in the study, which are over 5 years old at the time of this review.
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Reviewer #1 (Public Review):
Summary:<br /> Frey et al. report the structures of aSyn fibrils that were obtained under a variety of conditions. These include the generation of aSyn fibrils without seeds, but in different buffers and at different pH values. These also include the generation of aSyn fibrils in the presence of seeding fibrils, again performed in different buffers and at different pH values, while the seeds were generated at different conditions. The authors find that fibril polymorphs primarily correlate with fibril growth buffer conditions, and not such much with the type of seed. However, the presence of a seed is still required, likely because fibrils can also seed along their lateral surfaces, not only at the blunt ends.
Strengths:<br /> The manuscript includes an excellent review of the numerous available structures of aSyn. As the authors state, "it seems that there are about as many unique atomic-resolution structures of these aggregates as there are publications describing them."
The text is interesting to read, figures are clear and not redundant.
Weaknesses:<br /> The manuscript is excellently written, but sometimes a few commas are lacking.
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Reviewer #1 (Public Review):
Summary:<br /> UGGTs are involved in the prevention of premature degradation for misfolded glycoproteins, by utilizing UGGT-KO cells and a number of different ERAD substrates. They proposed a concept by which the fate of glycoproteins can be determined by a tug-of-war between UGGTs and EDEMs.
Strengths:<br /> The authors provided a wealth of data to indicate that UGGT1 competes with EDEMs, which promotes glycoprotein degradation.
Weaknesses:<br /> Less clear, though, is the involvement of UGGT2 in the process. Also, to this reviewer, some data do not necessarily support the conclusion.
Major criticisms:
1. One of the biggest problems I had on reading through this manuscript is that, while the authors appeared to generate UGGTs-KO cells from HCT116 and HeLa cells, it was not clearly indicated which cell line was used for each experiment. I assume that it was HCT116 cells in most cases, but I did not see that it was clearly mentioned. As the expression level of UGGT2 relative to UGGT1 is quite different between the two cell lines, it would be critical to know which cells were used for each experiment.
2. While most of the authors' conclusion is sound, some claims, to this reviewer, were not fully supported by the data. Especially I cannot help being puzzled by the authors' claim about the involvement of UGGT2 in the ERAD process. In most of the cases, KO of UGGT2 does not seem to affect the stability of ERAD substrates (ex. Fig. 1C, 2A, 3D). When the author suggests that UGGT2 is also involved in the ERAD, it is far from convincing (ex. Fig. 2D/E). Especially because now it has been suggested that the main role of UGGT2 may be distinct from UGGT1, playing a role in lipid quality control (Hung, et al., PNAS 2022), it is imperative to provide convincing evidence if the authors want to claim the involvement of UGGT2 in a protein quality control system.
In fact, it was not clear at all whether even UGGT1 is also involved in the process in Fig. 2D/E, as the difference, if any, is so subtle. How the authors can be sure that this is significant enough? While the authors claim that the difference is statistically significant (n=3), this may end up with experimental artifacts. To say the least, I would urge the authors to try rescue experiments with UGGT1 or 2, to clarify that the defect in UGGT-DKO cells can be reversed. It may also be interesting to see that the subtle difference the authors observed is indeed N-glycan-dependent by testing a non-glycosylated version of the protein (just like NHK-QQQ mutants in Fig. 2C).
To this reviewer, it is still possible that the involvement of UGGT1 (or 2, if any) could be totally substrate-dependent, and the substrates used in Fig 2D or E happen not to be dependent to the action of UGGTs. To the reviewer, without the data of Fig. 2D and E the authors provide enough evidence to demonstrate the involvement of UGGT1 in preventing premature degradation of glycoprotein ERAD substrates. I am just afraid that the authors may have overinterpreted the data, as if the UGGTs are involved in stabilization of all glycoproteins destined for ERAD.
3. I am a bit puzzled by the DNJ treatment experiments. First, I do not see the detailed conditions of the DNJ treatment (concentration? Time?). Then, I was a bit surprised to see that there were so little G3M9 glycans formed, and there was about the same amount of G2M9 also formed (Figure 1 Figure supplement 4B-D), despite the fact that glucose trimming of newly syntheized glycoproteins are expected to be completely impaired (unless the authors used DNJ concentration which does not completely impair the trimming of the first Glc). Even considering the involvement of Golgi endo-alpha-mannosidase, a similar amount of G3M9 and G2M9 may suggest that the experimental conditions used for this experiment (i.e. concentration of DNJ, duration of treatment, etc) is not properly optimized.
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Reviewer #1 (Public Review):
It is known that aberrant habit formation is a characteristic of obsessive-compulsive disorder (OCD). Habits can be defined according to the following features (Balleine and Dezfouli, 2019): rapid execution, invariant response topography, action 'chunking' and resistance to devaluation.<br /> The extent to which OCD behavior is derived from enhanced habit formation relative to deficits in goal-directed behavior is a topic of debate in the current literature. This study examined habit-learning specifically (cf. deficits in goal-directed behavior) by regularly presenting, via smartphone, sequential learning tasks to patients with OCD and healthy controls. Participants engaged in the tasks every day over the course of a month. Automaticity, including the extent to which individual actions in the sequence become part of a unified 'chunk', was an important outcome variable. Following the 30 days of training, in-laboratory tasks were then administered to examine 1) if performing the learned sequences themselves had become rewarding 2) differences in goal-directed vs. habitual behavior.
Several hypotheses were tested, including:<br /> Patients would have impaired procedural learning vs. healthy volunteers (this was not supported, possibly because there were fewer demands on memory in the task used here)<br /> Once the task had been learned, patients would display automaticity faster (unexpectedly, patients were slower to display automaticity)<br /> Habits would form faster under a continuous (vs. variable) reinforcement schedule
Exploratory analyses were also conducted: an interesting finding was that OCD patients with higher self-reported symptoms voluntarily completed more sessions with the habit-training app and reported a reduction in symptoms.
Strengths
This paper is well situated theoretically within the habit learning/OCD literature.<br /> Daily training in a motor-learning task, delivered via smartphone, was innovative, ecologically valid and more likely to assay habitual behaviors specifically. Daily training is also more similar to studies with non-humans, making a better link with that literature. The use of a sequential-learning task (cf. tasks that require a single response) is also more ecologically valid.<br /> The in-laboratory tests (after the 1 month of training) allowed the researchers to test if the OCD group preferred familiar, but more difficult, sequences over newer, simpler sequences.
Weaknesses
The authors were not able to test one criterion of habits, namely resistance to devaluation, due to the nature of the task.<br /> The sample size was relatively small. Some potentially interesting individual differences within the OCD group could have been examined more thoroughly with a bigger sample (e.g., preference for familiar sequences). A larger sample may have allowed the statistical testing of any effects due to medication status.
The authors achieved their aims in that two groups of participants (patients with OCD and controls) engaged with the task over the course of 30 days. The repeated nature of the task meant that 'overtraining' was almost certainly established, and automaticity was demonstrated. This allowed the authors to test their hypotheses about habit learning. The results are supportive of the author's conclusions.
This article is likely to be impactful -- the delivery of a task across 30 days to a patient group is innovative and represents a new approach for the study of habit learning that is superior to an in-laboratory approach.
An interesting aspect of this manuscript is that it prompts a comparison with previous studies of goal-directed/habitual responding in OCD that used devaluation protocols, and which may have had their effects due to deficits in goal-directed behavior and not enhanced habit learning per se.
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Reviewer #1 (Public Review):
Summary:<br /> Ger and colleagues address an issue that often impedes computational modeling: the inherent ambiguity between stochasticity in behavior and structural mismatch between the assumed and true model. They propose a solution to use RNNs to estimate the ceiling on explainable variation within a behavioral dataset. With this information in hand, it is possible to determine the extent to which "worse fits" result from behavioral stochasticity versus failures of the cognitive model to capture nuances in behavior (model misspecification). The authors demonstrate the efficacy of the approach in a synthetic toy problem and then use the method to show that poorer model fits to 2-step data in participants with low IQ are actually due to an increase in inherent stochasticity, rather than systemic mismatch between model and behavior.
Strengths:<br /> Overall I found the ideas conveyed in the paper interesting and the paper to be extremely clear and well-written. The method itself is clever and intuitive and I believe it could be useful in certain circumstances, particularly ones where the sources of structure in behavioral data are unknown. In general, the support for the method is clear and compelling. The flexibility of the method also means that it can be applied to different types of behavioral data - without any hypotheses about the exact behavioral features that might be present in a given task.
Weaknesses:<br /> That said, I have some concerns with the manuscript in its current form, largely related to the applicability of the proposed methods for problems of importance in computational cognitive neuroscience. This concern stems from the fact that the toy problem explored in the manuscript is somewhat simple, and the theoretical problem addressed in it could have been identified through other means (for example through the use of posterior predictive checking for model validation), and the actual behavioral data analyzed were interpreted as a null result (failure to reject that the behavioral stochasticity hypothesis), rather than actual identification of model-misspecification. I expand on these primary concerns and raise several smaller points below.
A primary question I have about this work is whether the method described would actually provide any advantage for real cognitive modeling problems beyond what is typically done to minimize the chance of model misspecification (in particular, post-predictive checking). The toy problem examined in the manuscript is pretty extreme (two of the three synthetic agents are very far from what a human would do on the task, and the models deviate from one another to a degree that detecting the difference should not be difficult for any method). The issue posed in the toy data would easily be identified by following good modeling practices, which include using posterior predictive checking over summary measures to identify model insufficiencies, which in turn would call for the need for a broader set of models (See Wilson & Collins 2019). Thus, I am left wondering whether this method could actually identify model misspecification in real world data, particularly in situations where standard posterior predictive checking would fall short. The conclusions from the main empirical data set rest largely on a null result, and the utility of a method for detecting model misspecification seems like it should depend on its ability to detect its presence, not just its absence, in real data.
Beyond the question of its advantage above and beyond data- and hypothesis-informed methods for identifying model misspecification, I am also concerned that if the method does identify a model-insufficiency, then you still would need to use these other methods in order to understand what aspect of behavior deviated from model predictions in order to design a better model. In general, it seems that the authors should be clear that this is a tool that might be helpful in some situations, but that it will need to be used in combination with other well-described modeling techniques (posterior predictive checking for model validation and guiding cognitive model extensions to capture unexplained features of the data). A general stylistic concern I have with this manuscript is that it presents and characterizes a new tool to help with cognitive computational modeling, but it does not really adhere to best modeling practices (see Collins & Wilson, eLife), which involve looking at data to identify core behavioral features and simulating data from best-fitting models to confirm that these features are reproduced. One could take away from this paper that you would be better off fitting a neural network to your behavioral data rather than carefully comparing the predictions of your cognitive model to your actual data, but I think that would be a highly misleading takeaway since summary measures of behavior would just as easily have diagnosed the model misspecification in the toy problem, and have the added advantage that they provide information about which cognitive processes are missing in such cases.
As a more minor point, it is also worth noting that this method could not distinguish behavioral stochasticity from the deterministic structure that is not repeated across training/test sets (for example, because a specific sequence is present in the training set but not the test set). This should be included in the discussion of method limitations. It was also not entirely clear to me whether the method could be applied to real behavioral data without extensive pretraining (on >500 participants) which would certainly limit its applicability for standard cases.
The authors focus on model misspecification, but in reality, all of our models are misspecified to some degree since the true process-generating behavior almost certainly deviates from our simple models (ie. as George Box is frequently quoted, "all models are wrong, but some of them are useful"). It would be useful to have some more nuanced discussion of situations in which misspecification is and is not problematic.
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Reviewer #1 (Public Review):
The authors present a model for multisensory correlation detection that is based on the neurobiologically plausible Hassenstein Reichardt detector. It modifies their previously reported model (Parise & Ernst, 2016) in two ways: a bandpass (rather than lowpass) filter is initially applied and the filtered signals are then squared. The study shows that this model can account for synchrony judgment, temporal order judgment, etc in two new data sets (acquired in this study) and a range of previous data sets.
Strengths:<br /> 1. The model goes beyond descriptive models such as cumulative Gaussians for TOJ and differences in cumulative Gaussians for SJ tasks by providing a mechanism that builds on the neurobiologically plausible Hassenstein-Reichardt detector.<br /> 2. This modified model can account for results from two new experiments that focus on the detection of correlated transients and frequency doubling. The model also accounts for several behavioural results from experiments including stochastic sequences of A/V events and sinewave modulations.
Additional thoughts:<br /> 1. The model introduces two changes: bandpass filtering and squaring of the inputs. The authors emphasize that these changes allow the model to focus selectively on transient rather than sustained channels. But shouldn't the two changes be introduced separately? Transients may also be detected for signed signals.
2. Because the model is applied only to rather simple artificial signals, it remains unclear to what extent it can account for AV correlation detection for naturalistic signals. In particular, speech appears to rely on correlation detection of signed signals. Can this modified model account for SJ or TOJ judgments for naturalistic signals?
Even Nidiffer et al. (2018) which is explicitly modelled by the authors report a significant difference in performance for correlated and anti-correlated signals. This seems to disagree with the results of study 1 which is reported in the current paper and the model's predictions. How can these contradicting results be explained? In case the brain performs correlation detection on signed and unsigned signals, is a more complex mechanism needed to arbitrate between those two mechanisms?
3. The number of parameters seems quite comparable for the authors' model and descriptive models (e.g. PSF models). This is because time constants require refitting (at least for some experimental data sets) and the correlation values need to be passed through a response mode (i.e. probit function) to account for behavioural data. It remains unclear how the brain adjusts the time constants to different sensory signals.
4. Fujisaki and Nishida (2005, 2006) proposed mechanisms for AV correlation detection based on the Hassenstein-Reichardt motion detector (though not formalized as a computational model).
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> This paper identifies GABA cells in the preoptic hypothalamus which are involved in REM sleep rebound (the increase in REM sleep) after selective REM sleep deprivation. By calcium photometry, these cells are most active during REM, and show more claim signals during REM deprivation, suggesting they respond to "REM pressure". Inhibiting these cells ontogenetically diminishes REM sleep. The optogenetic and photometry work is carried out to a high standard, the paper is well-written, and the findings are interesting.
Points that could be addressed or discussed:<br /> 1. The circuit mechanism for REM rebound is not defined. How do the authors see REM rebound as working from the POAGAD2 cells? Although the POAGAD2 does project to the TMN, the actual REM rebound could be mediated by a projection of these cells elsewhere. This could be discussed.
2. The "POAGAD2 to TMN" name for these cells is somewhat confusing. The authors chose this name because they approach the POAGAD2 cells via retrograde AAV labelling (rAAV injected into the TMN). However, the name also seems to imply that neurons (perhaps histamine neurons) in the TMN are involved in the REM rebound, but there is no evidence in the paper that this is the case. Although it is nice to see from the photometry studies that the histamine cells are selectively more active (as expected) in NREM sleep (Fig. S2), I could not logically see how this was a relevant finding to REM rebound or the subject of the paper. There are many other types of cells in the TMN area, not just histamine cells, so are the authors suggesting that these non-histamine cells in the TMN could be involved?
3. It is a puzzle why most of the neurons in the POA seem to have their highest activity in REM, as also found by Miracca et al 2022, yet presumably some of these cells are going to be involved in NREM sleep as well. Could the same POAGAD2-TMN cells identified by the authors also be involved in inducing NREM sleep-inhibiting histamine neurons (Chung et al). And some of these POA cells will also be involved in NREM sleep homeostasis (e.g. Ma et al Curr Biol)? Is NREM sleep rebound necessary before getting REM sleep rebound? Indeed, can these two things (NREM and REM sleep rebound) be separated?
4. Is it possible to narrow down the POA area where the GAD2 cells are located more precisely?
5. It would be ideal to further characterize these particular GAD2 cells by RT-PCR or RNA seq. Which other markers do they express?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The study by Liff et al significantly advances our understanding of transgenerational olfactory changes resulting from fear conditioning, particularly in revealing elevated odor-encoding neurons in both conditioned mice (F0) and their progeny (F1). The authors attribute F0 increases to biased stem cell receptor selection, building upon the seminal work of Dias and Ressler (2014). While the dedication and use of novel histological techniques add strength to the study, there are notable weaknesses, including the need for clarification on discrepancies with previous findings, the decision to modify paradigms, and the presentation of behavioral data in supplementary materials.
Overall, the manuscript has strong potential but would benefit from addressing these weaknesses and minor recommendations to enhance its quality and contribution to the field.
Strengths:<br /> - Significant contribution to understanding transgenerational olfactory changes induced by fear conditioning.<br /> - Use of novel histological techniques and exploration of stem cell involvement adds depth to the study.
Weaknesses:<br /> Discrepancies with previous findings need clarification, especially regarding the absence of similar behavioral effects in F1. Lack of discussion on the decision to modify paradigms instead of using the same model. Presentation of behavioral data in supplementary materials, with a recommendation to include behavioral quantification in main figures. Absence of quantification for freezing behavior, a crucial measure in fear conditioning.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The authors examined whether archerfish have the capacity for motor adaptation in response to airflow perturbations. Through two experiments, they demonstrated that archerfish could adapt. Moreover, when the fish flipped its body position with the perturbation remaining constant, it did not instantaneously counteract the error. Instead, the archerfish initially persisted in correcting for the original perturbation before eventually adapting, consistent with the notion that the archerfish's internal model has been adapted in egocentric coordinates.
Evaluation:<br /> The results of both experiments were convincing, given the observable learning curve and the clear aftereffect. The ability of these fish to correct their errors is also remarkable. Nonetheless, certain aspects of the experiment's motivation and conclusions temper my enthusiasm.
1. The authors motivated their experiments with two hypotheses, asking whether archerfish can adapt to light refractions using an innate look-up table as opposed to possessing a capacity to adapt. However, the present experiments are not designed to arbitrate between these ideas. That is, the current experiments do not rule out the look-up table hypothesis, which predicts, for example, that motor adaptation may not generalize to de novo situations with arbitrary action-outcome associations. Such look-up table operations may also show set-size effects, whereas other mechanisms might not. Whether their capacity to adapt is innate or learned was also not directly tested, as noted by the authors in the discussion. Could the authors clarify how they see their results positioned in light of the two hypotheses noted in the Introduction?
2. The authors claim that archerfish use egocentric coordinates rather than allocentric coordinates. However, the current experiments do not make clear whether the archerfish are "aware" that their position was flipped (as the authors noted, no visual cues were provided). As such, for example, if the fish were "unaware" of the switch, can the authors still assert that generalization occurs in egocentric coordinates? Or simply that, when archerfish are ostensibly unaware of changes in body position, they continue with previously successful actions.
3. The experiments offer an opportunity to examine whether archerfish demonstrate any savings from one session to another. Savings are often attributed to a faster look-up table operation. As such, if archerfish do not exhibit savings, it might indicate a scenario where they do not possess a refined look-up table and must rely on implicit mechanisms to relearn each time.
4. The authors suggest that motor adaptation in response to wind may hint at mechanisms used to adapt to light refraction. However, how strong of a parallel can one draw between adapting to wind versus adapting to light refraction? This seems important given the claims in this paper regarding shared mechanisms between these processes. As a thought experiment, what would the authors predict if they provided a perturbation more akin to light refraction (e.g., a film that distorts light in a new direction, rather than airflow)?
5. The number of fish excluded was greater than those included. This raises the question as to whether these fish are merely elite specimens or representative of the species in general.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> Huang and Luo investigated whether regularities between stimulus features can be exploited to facilitate the encoding of each set of stimuli in visual working memory, improving performance. They recorded both behavioural and neural (EEG) data from human participants during a sequential delayed response task involving three items with two properties: location and colour. In the key condition ('aligned trajectory'), the distance between locations of successively presented stimuli was identical to their 'distance' in colour space, permitting a compression strategy of encoding only the location and colour of the first stimulus and the relative distance of the second and third stimulus (as opposed to remembering 3 locations and 3 colours, this would only require remembering 1 location, 1 colour, and 2 distances). Participants recalled the location and colour of each item after a delay.
Consistent with the compression account, participants' location and colour recall errors were correlated and were overall lower compared to a non-compressible condition ('misaligned trajectory'). Multivariate analysis of the neural data permitted decoding of the locations and colours during encoding. Crucially, the relative distance could also be decoded - a necessary ingredient for the compression strategy.
Strengths:<br /> The main strength of this study is a novel experimental design that elegantly demonstrates how we exploit stimulus structure to overcome working memory capacity limits. The behavioural results are robust and support the main hypothesis of compressed encoding across a number of analyses. The simple and well-controlled design is suited to neuroimaging studies and paves the way for investigating the neural basis of how environmental structure is detected and represented in memory. Prior studies on this topic have primarily studied behaviour only (e.g., Brady & Tenenbaum, 2013).
Weaknesses:<br /> The main weakness of the study is that the EEG results do not make a clear case for compression or demonstrate its neural basis. If the main aim of this strategy is to improve memory maintenance, it seems that it should be employed during the encoding phase. From then on, the neural representation in memory should be in the compressed format. The only positive evidence for this occurs in the late encoding phase (the re-activation of decoding of the distance between items 1 and 2, Fig. 5A), but the link to behaviour seems fairly weak (p=0.068). Stronger evidence would be showing decoding of the compressed code during memory maintenance or recall, but this is not presented. On the contrary, during location recall (after the majority of memory maintenance is already over), colour decoding re-emerges, but in the un-compressed item-by-item code (Fig. 4B). The authors suggest that compression is consolidated at this point, but its utility at this late stage is not obvious.
Impact:<br /> This important study elegantly demonstrates that the use of shared structure can improve capacity-limited visual working memory. The paradigm and approach explicitly link this field to recent findings on the role of replay in structure learning and will therefore be of interest to neuroscientists studying both topics.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Padamsey et al. followed up on their previous study in which they found that male mice sacrifice visual cortex computation precision to save energy in periods of food restriction (Padamsey et al. 2021, Neuron). In the present study, the authors find that female mice show much lower levels of adaptation in response to food restriction on the level of metabolic signaling and visual cortex computation. This is an important finding for understanding sex differences in adaptation to food scarcity and also impacts the interpretation of studies employing food restriction in behavioral analyses and learning paradigms.
The manuscript is, in general, very clear and the conclusions are straightforward. The main limitation, that the number of experiments is insufficient to compare the effects of food restriction in males and females directly, is discussed by the authors: to address this point they use Bayes factor analysis to provide an estimate of the likelihood that females and males indeed differ in terms of energy metabolism and sensory processing adaptions during food restriction.
The following points are not entirely clear yet.<br /> 1. For a number of experiments the authors use their new data set on females and compare that with the data set previously published on males. In how far are these data sets comparable? Have they been performed originally in parallel for example using siblings of different sexes or have the experiments been conducted several years apart from each other? What is the expected variability, if one repeated these experiments with the same sex considering the differences/similarities between experimental setups, housing conditions, interindividual differences, etc.?
2. Energy consumption and visual processing may differ between periods in which animals are in different behavioral states. Is there a possibility that male and female mice differed in behavioral state during measurements? Were animals running or resting during visual stimulation and during ATP measurements?
3. Related to the previous point: the authors show that ATP consumption was reduced in male mice during visual stimulation. What about visual cortex ATP consumption in the absence of visual stimulation? Do food-deprived males and/or females show lower ATP consumption in the visual cortex e.g. during sleep?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> Zhang et al. provide valuable data for understanding molecular features of the human spinal cord. The authors made considerable efforts to acknowledge and objectively address the limitations of Visium while attempting to overcome them by utilizing single-nucleus RNA sequencing (snRNA-seq) from the same tissue. By mapping snRNA-seq clusters to Visium data, they offer spatial information, complemented by RNA-ISH and immunofluorescence (IF) validation. They also discuss gender-related differences and the similarities between human and mouse data, aiming to establish a crucial foundation for experimental research. However, I have some comments below.
1. The observation of gender-related differences is interesting. The authors reported that SCN10A, associated with nociceptos, exhibited stronger expression in females. While they intend to validate this finding through IF, the quantitative difference is not clearly observed in the IF data (Figure 5f). It would be essential to provide validation through DAPI-based cell counts, demonstrating the difference in CHAT/SCNA10A co-expression.
2. It is meritorious that in novel features of the transcriptomic study, the authors considered gender-related differences and similarities between humans and mice. Nevertheless, despite the extensive bioinformatics-based analyses performed, the results mostly confirm what has been previously reported (Nguyen et al. 2021; Yadav et al. 2023; Jung et al. 2023).
3. The study did not perform snRNA-seq in the DRG. The limitations of Visium in cell type separation are acknowledged, and the authors are aware that Visium alone has limitations in describing cell expression patterns. The authors need to validate their findings via analyses of public DRG snRNA-seq data (Jung et al. 2023 Ncom; Nguyen et al. 2021eLife) before drawing broad conclusions.
4. Figure 7's comparison between human Visium spot data and Renthal et al.'s mouse snRNA-seq may have limitations as Visium spot data could not provide a transcriptional profile at the single cell resolution. The authors need to clarify this point.
5. Recent findings indicate that type 2 cytokines can directly stimulate sensory neurons. This includes the expression of IL-4RA, IL31RA, and IL13RA in DRG. These findings support the role of JAK kinase inhibitors in mediating chronic itch. Demonstrating the expression of these itch receptors in DRG would be valuable.
6. Given that juxtacrine and paracrine signals operate from 0 to 200 um, spatial information is vital to understanding intercellular communication. The presentation of spatial information using Visium is meaningful, and more comprehensive analyses of potential interaction based on distance should be provided, beyond the top 10 interactions (Figure 8).
7. The gender-related differences are interesting and, if possible, it would be interesting to explore whether age-related differences or degeneration-related factors exist. Using public data could allow the examination of age-related changes.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary and strengths<br /> The authors tried to address why only a subset of genes are highlighted in many publications. Is it because these highlighted genes are more important than others? Or is it because there are non-genetic reasons? This is a critical question because in the effort to discover new genes for drug targets and clinical benefit, we need to expand a pool of genes for deep analyses. So I appreciate the authors' efforts in this study, as it is timely and important. They also provided a framework called FMUG (short for Find My Understudied Gene) to evaluate genes for a number of features for subsequent analyses.
Weaknesses<br /> Many of the figures are hard to comprehend, and the figure legends do not sufficiently explain them.<br /> # For example, what was plotted in Fig 1b? The number of articles increased from results -> write-ups -> follow-ups in all four categories with different degrees. But it does not seem to match what the authors meant to deliver.<br /> # Fig 4 is also confusing. It appears that the genes were clustered by many features that the authors developed. But does it have any relationship with genes being under- or over-studied?
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eduscol.education.fr eduscol.education.fr
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deux jours ouvrables correspondantau délai accordé à l’élève pour présenter sa défense (art. R. 421-10-1 du code del’éducation) dans le cadre du respect du principe du contradictoire.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The type I ABC importer OpuA from Lactococcus lactis is the best-studied transporter involved in osmoprotection. In contrast to most ABC import systems, the substrate binding protein is fused via a short linker to the transmembrane domain of the transporter. Consequently, this moiety is called the substrate binding domain (SBD). OpuA has been studied in the past in great detail and we have a very detailed knowledge about function, mechanisms of activation and deactivation as well as structure.
Strengths:<br /> Application of smFRET to unravel transient interactions of the SBDs. The method is applied at a superb quality and the data evaluation is excellent.
Weaknesses:<br /> The proposed model is not directly supported by experimental data. Rather all alternative models are excluded as they do not fit the obtained data.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This is an interesting manuscript that extends prior work from this group identifying that a chemovar of Cannabis induces apoptosis of T-ALL cells by preventing NOTCH1 cleavage. Here the authors isolate specific components of the chemovar responsible for this effect to CBD and CBDV. They identify the mechanism of action of these agents as occurring via the integrated stress response. Overall the work is well performed but there are two lingering questions that would be helpful to address as follows:
-Exactly how CBD and CBDV result in the upregulation of the TRPV1/integrated stress response is unclear. What is the most proximal target of these agents that results in these changes?
-Related to the above, all experiments to confirm the mechanism of action of CBD/CBDV rely on chemical agents, whose precise targets are not fully clear in some cases. Thus, some use of genetic means (such as by knockout of TRPV1, ATF4) to confirm the dependency of these pathways on drug response and NOTCH cleavage would be very helpful.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Summary:
The manuscript of Heydasch et al. addresses the spatiotemporal regulation of Rho GTPase signaling in living cells and its coupling to the mechanical state of the cell. They focus on a GAP of RhoA, the Rho-specific GAP Deleted in Liver Cancer 1 (DLC1). They first show that removing DLC1 either by a CRISPR KO or by downregulation using siRNA leads to increased contractility and globally elevated RhoA activity, as revealed by a FRET biosensor. This result was expected, since DLC1 is deactivating RhoA its absence should lead to increasing amounts of active RhoA. To go beyond global and steady levels of RhoA activity, the authors developed an acute optogenetic system to study transient RhoA activity dynamics in different genetic and subcellular contexts. In WT cells, they found that pulses of activation lead to an increased RhoA activity at focal adhesions (FA) compared to plasma membrane (PM), which suggests that FAs contain less RhoA GAPs, more RhoA, or that FAs involve positive feedback implying other GEFs for example. In DLC1 KO cells, they found that the RhoA response upon pulses of optogenetic activation was increased (higher peak) both at FA and PM, which could be expected since less GAP should increase the amount of active RhoA. But surprisingly, they observed a higher rate of RhoA deactivation in DLC1 KO cells, which is counterintuitive: less GAP should result in a slower rate of deactivation. Less GAP should also lead to a lower rate of observed RhoA activation (smaller koff) and delayed peak. From the data, it seems hard to conclude on these two expectations since the initial rates (slopes right after the activation) and times at peak appear similar in both WT and DLC1 KO cells. Further on, the authors study the dynamics of DLC1 on FAs depending on the mechanical state and nicely show a causal decrease of DLC1 enrichment at FA upon FA reinforcement, hereby probing a positive feedback where RhoA activation is further amplified as the force exerted at FA is increasing.
Strengths:
- Experiments are precise and well done.<br /> - Technically, the work brings original and interesting data. The use of transient optogenetic activation within focal adhesions together with a biosensor of activity is new and elegant.<br /> - The link between DLC1 and global contractility/RhoA activity is clear and convincing.<br /> - The surprisingly higher rate of RhoA deactivation in DLC1 KO cells is convincing, as well as the differences in the dynamics of RhoA between focal adhesions and plasma membrane.<br /> - The correlation between DLC1 enrichment and focal adhesion dynamics is very clear.
Weaknesses:
- There is no explanation for the higher rate of RhoA deactivation in DLC1 KO cells.<br /> - For the optogenetic experiments, it is not clear if we are looking at the actual RhoA dynamics of the activity or at the dynamics of the optogenetic tool itself.<br /> - There is no model to analyze transient RhoA responses, however, the quantitative nature of the data calls for it. Even a simple model with linear activation-deactivation kinetics fitted on the data would be of benefit for the conclusions on the observed rates and absolute amounts.
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> This study uses whole genome sequencing to characterise the population structure and genetic diversity of a collection of 58 isolates of E. coli associated with neonatal meningitis (NMEC) from seven countries, including 52 isolates that the authors sequenced themselves and a further 6 publicly available genome sequences. Additionally, the study used sequencing to investigate three case studies of apparent relapse. The data show that in all three cases, the relapse was caused by the same NMEC strain as the initial infection. In two cases they also found evidence for gut persistence of the NMEC strain, which may act as a reservoir for persistence and reinfection in neonates. This finding is of clinical importance as it suggests that decolonisation of the gut could be helpful in preventing relapse of meningitis in NMEC patients.
Strengths:<br /> The study presents complete genome sequences for n=18 diverse isolates, which will serve as useful references for future studies of NMEC. The genomic analyses are high quality, the population genomic analyses are comprehensive and the case study investigations are convincing.
Weaknesses:<br /> The NMEC collection described in the study includes isolates from just seven countries. The majority (n=51/58, 88%) are from high-income countries in Europe, Australia, or North America; the rest are from Cambodia (n=7, 12%). Therefore it is not clear how well the results reflect the global diversity of NMEC, nor the populations of NMEC affecting the most populous regions.
The virulence factors section highlights several potentially interesting genes that are present at apparently high frequency in the NMEC genomes; however, without knowing their frequency in the broader E. coli population it is hard to know the significance of this.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Summary:
Cincotta et al set out to investigate the presence of glucocorticoid receptors in the male and female embryonic germline. They further investigate the impact of tissue specific genetically induced receptor absence and/or systemic receptor activation on fertility and RNA regulation. They are motivated by several lines of research that report inter and transgenerational effects of stress and or glucocorticoid receptor activation and suggest that their findings provide an explanatory mechanism to mechanistically back parental stress hormone exposure induced phenotypes in the offspring.
Strengths:
- A chronological immunofluorescent assessment of GR in fetal and early life oocyte and sperm development.<br /> - RNA seq data that reveal novel cell type specific isoforms validated by q-RT PCR E15.5 in the oocyte.<br /> - 2 alternative approaches to knock out GR to study transcriptional outcomes. Oocytes: systemic GR KO (E17.5) with low input 3-tag seq and germline specific GR KO (E15.5) on fetal oocyte expression via 10X single cell seq and 3-cap sequencing on sorted KO versus WT oocytes - both indicating little impact on polyadenylated RNAs -<br /> - 2 alternative approaches to assess the effect of GR activation in vivo (systemic) and ex vivo (ovary culture): here the RNA seq did show again some changes in germ cells and many in the soma.<br /> - They exclude oocyte specific GR signaling inhibition via beta isoforms<br /> - Perinatal male germline shows differential splicing regulation in response to systemic Dex administration, results were backed up with q-PCR analysis of splicing factors.
Weaknesses:
- Sequencing techniques used are not Total RNA but either are focused on all polyA transcripts (10x) - effects on non-polyA-transcripts are left unexplored.<br /> The number of replicates in the low input seq is very low and hence this might be underpowered. Since Dex treatment showed some (modest) changes in oocyte RNA effects of GR depletion might only become apparent upon Dex treatment as an interaction. Meaning GR activation in the presence of GR shows changes, upon GR depletion those changes are abolished --> statistically speaking an interaction --> conclusion: there are germline GR effects that get abolished when there is no GR hinting on germline GR autonomous effects.<br /> - Effects in oocytes following systemic Dex might be indirect due to GR activation in the soma. The changes observed might be irrelevant to meiosis and thus in the manuscript are deemed irrelevant, but they could still lead to settle consequences. in other terms.
Even though ex vivo culture of ovaries shows GR translocation to nucleus it is not sure whether the in vivo systemic administration does the same. The authors argue in their rebuttal that GR is already nuclear in fetal oocytes hence the<br /> conclusion that fetal oocytes are resistant to GR manipulation is understandable, at least for the readouts that were considered. Yet the question arises: If GR is already nuclear (active) in the absence of additional Dex treatment why does GR knock out not elicit any changes in the considered readouts -> what are we missing.
This work is a good reference point for researchers interested in glucocorticoid hormone signaling fertility and RNA splicing. It might spark further studies on germline-specific GR functions and the impact of GR activation on alternative splicing.<br /> The study provides a characterization of GR and some aspects of GR perturbation, and the negative findings in this study do help to rule out a range of specific roles of GR in the germline. This will help the study of unexplored options. The authors do acknowledge the unexplored options in their discussion.<br /> The intro of the study eludes to implications for intergenerational effects via epigenetic modifications in the germline and points out additional potential indirect effects of reproductive tissue GR signaling on the germline. Future studies might hence focus on further exploration of epigenetic modifications and/or indirect effects.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The work by Debashish U. Menon, Noel Murcia, and Terry Magnuson brings important knowledge about histone H3.3 dynamics involved in meiotic sex chromosome inactivation (MSCI). MSCI is unique to gametes and failure during this process can lead to infertility. Classically, MSCI has been studied in the context of DNA Damage repair pathways and little is known about the epigenetic mechanisms behind maintenance of the sex body as a silencing platform during meiosis. One of the major strengths of this work is the evidence provided on the role of ARID1A, a BAF subunit, in MSCI through the regulation of H3.3 occupancy in specific genic regions.
Using RNA seq and CUT&RUN and ATAC-seq, the authors show that ARID1A regulates chromatin accessibility of the sex chromosomes and XY gene expression. Loss of ARID1A increases promoter accessibility of XY linked genes with concomitant influx of RNA pol II to the sex body and up regulation of XY-linked genes. This work suggests that ARID1A regulates chromatin composition of the sex body since in the absence of ARID1A, spermatocytes show less enrichment of H3.3 in the sex chromosomes and stable levels of the canonical histones H3.1/3.2. By overlapping CUT&RUN and ATAC-seq data, authors show that changes in chromatin accessibility in the absence of ARID1A are given by redistribution of occupancy of H3.3. Gained open chromatin in mutants corresponds to up regulation of H3.3 occupancy at transcription start sites of genes mediated by ARID1A.
Interestingly, ARID1A loss caused increased promoter occupancy by H3.3 in regions usually occupied by PRDM9. PRDM9 catalyzes histone H3 lysine 4 trimethylation during meiotic prophase I, and positions double strand break (DSB) hotspots. Lack of ARID1A causes reduction in occupancy of DMC1, a recombinase involved in DSB repair, in non-homologous sex regions. These data suggest that ARID1A might indirectly influence DNA DSB repair on the sex chromosomes by regulating the localization of H3.3. This is very interesting given the recently suggested role for ARID1A in genome instability in cancer cells. It raises the question of whether this role is also involved in meiotic DSB repair in autosomes and/or how this mechanism differs in sex chromosomes compared to autosomes.
The fact that there are Arid1a transcripts that escape the Cre system in the Arid1a KO mouse model might difficult the interpretation of the data. The phenotype of the Arid1a knockout is probably masked by the fact that many of the sequencing techniques used here are done on a heterogeneous population of knockout and wild type spermatocytes. In relation to this, I think that the use of the term "pachytene arrest" might be overstated, since this is not the phenotype truly observed. Knockout mice produce sperm, and probably litters, although a full description of the subfertility phenotype is lacking, along with identification of the stage at which cell death is happening by detection of apoptosis.<br /> It is clear from this work that ARID1a is part of the protein network that contribute to silencing of the sex chromosomes. However, it is challenging to understand the timing of the role of ARID1a in the context of the well-known DDR pathways that have been described for MSCI. Staining of chromosome spreads with Arid1a antibody showed localization at the sex chromosomes by diplonema, however, analysis of gene expression in Arid1a ko was performed on pachytene spermatocytes. Therefore, is not very clear how the chromatin remodeling activity of Arid1a in diplonema is affecting gene expression of a previous stage. CUTnRUN showed that ARID1a is present at the sex chromatin in earlier stages, leading to hypothesize that immunofluorescence with ARID1a antibody might not reflect ARID1a real localization.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In the submitted manuscript, Port et al. investigated the host and viral factors influencing the airborne transmission of SARS-CoV-2 Alpha and Delta variants of concern (VOC) using a Syrian hamster model. The authors analyzed the viral load profiles of the animal respiratory tracts and air samples from cages by quantifying gRNA, sgRNA, and infectious virus titers. They also assessed the breathing patterns, exhaled aerosol aerodynamic profile, and size distribution of airborne particles after SARS-CoV-2 Alpha and Delta infections. The data showed that male sex was associated with increased viral replication and virus shedding in the air. The relationship between co-infection with VOCs and the exposure pattern/timeframe was also tested. This study appears to be an expansion of a previous report (Port et al., 2022, Nature Microbiology). The experimental designs were rigorous, and the data were solid. These results will contribute to the understanding of the roles of host and virus factors in the airborne transmission of SARS-CoV-2 VOCs.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary and strengths<br /> This is an interesting, timely and informative article. The authors used publicly available data (made available by a funding agency) to examine some of the academic characteristics of the individuals recipients of the National Institutes of Health (NIH) k99/R00 award program during the entire history of this funding mechanism (17 years, total ~ 4 billion US dollars (annual investment of ~230 million USD)). The analysis focuses on the pedigree and the NIH funding portfolio of the institutions hosting the k99 awardees as postdoctoral researchers and the institutions hiring these individuals. The authors also analyze the data by gender, by whether the R00 portion of the awards eventually gets activated and based on whether the awardees stayed/were hired as faculty at their k99 (postdoctoral) host institution or moved elsewhere. The authors further sought to examine the rates of funding for those in systematically marginalized groups by analyzing the patterns of receiving k99 awards and hiring k99 awardees at historically black colleges and universities.
The goals and analysis are reasonable and the limitations of the data are described adequately. It is worth noting that some of the observed funding and hiring traits are in line with the Matthew effect in science (Merton, 1968: https://www.science.org/doi/10.1126/science.159.3810.56) and in science funding (Bol et al., 2018: https://www.pnas.org/doi/10.1073/pnas.1719557115). Overall, the article is a valuable addition to the research culture literature examining the academic funding and hiring traits in the United States. The findings can provide further insights for the leadership at funding and hiring institutions and science policy makers for individual and large-scale improvements that can benefit the scientific community.
Weaknesses<br /> The authors have addressed my recommendations in the previous review round in a satisfactory way.
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braveneweurope.com braveneweurope.com
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This dangerous framing is compounded by a generally supine media owned or controlled by the 1%
- for: 1% - media control
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This 1% of humanity uses its awesome power to manipulate societal aspirations and the narratives around climate change.
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for: quote - Kevin Anderson, quote - 1% manipulation
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quote
- This 1% of humanity uses its awesome power to manipulate
- societal aspirations and
- the narratives around climate change.
- These extend from
- well-funded advertising to
- pseudo-technical solutions,
- the financialisation of carbon emissions (and increasingly, nature) to
- labelling extreme any meaningful narrative that questions inequality and power.
- This 1% of humanity uses its awesome power to manipulate
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study by He, Liu, and He et al. investigated the fundamental role of microglia in modulating general anesthesia. While microglia have been previously shown to regulate neuronal network activity, their role in the induction of (i.e., LORR) and emergence from (i.e., RORR) anesthesia has only recently been explored. Recently published work by Cao et al. reported that microglia modulate general anesthesia via P2Y12 receptor. The present study largely reproduces those findings and does so using an impressive array of techniques and clever approaches. Following the serendipitous discovery that microglia-depleted mice exhibit increased LORR and decreased RORR, the authors go on to demonstrate that microglia regulate neuronal activity in a region-specific manner during anesthesia via purinergic receptor-mediated calcium signaling. The manuscript is well written and the data are convincing, elegantly validated using several different methods and controls, and largely complete. Nevertheless, this Reviewer has a few minor comments and suggestions to further strengthen the manuscript.
Strengths:
Impressive number of genetic mouse models, techniques, controls, and methods of validation.
Weaknesses:
Some of the novelty of these findings may be reduced based on the recent publication of a similar study.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Given knowledge of the amino acid sequence and of some version of the 3D structure of two monomers that are expected to form a complex, the authors investigate whether it is possible to accurately predict which residues will be in contact in the 3D structure of the expected complex. To this effect, they train a deep learning model that takes as inputs the geometric structures of the individual monomers, per-residue features (PSSMs) extracted from MSAs for each monomer, and rich representations of the amino acid sequences computed with the pre-trained protein language models ESM-1b, MSA Transformer, and ESM-IF. Predicting inter-protein contacts in complexes is an important problem. Multimer variants of AlphaFold, such as AlphaFold-Multimer, are the current state of the art for full protein complex structure prediction, and if the three-dimensional structure of a complex can be accurately predicted then the inter-protein contacts can also be accurately determined. By contrast, the method presented here seeks state-of-the-art performance among models that have been trained end-to-end for inter-protein contact prediction.
Strengths:
The paper is carefully written and the method is very well detailed. The model works both for homodimers and heterodimers. The ablation studies convincingly demonstrate that the chosen model architecture is appropriate for the task. Various comparisons suggest that PLMGraph-Inter performs substantially better, given the same input than DeepHomo, GLINTER, CDPred, DeepHomo2, and DRN-1D2D_Inter. As a byproduct of the analysis, a potentially useful heuristic criterion for acceptable contact prediction quality is found by the authors: namely, to have at least 50% precision in the prediction of the top 50 contacts.
Weaknesses:
My biggest issue with this work is the evaluations made using *bound* monomer structures as inputs, coming from the very complexes to be predicted. Conformational changes in protein-protein association are the key element of the binding mechanism and are challenging to predict. While the GLINTER paper (Xie & Xu, 2022) is guilty of the same sin, the authors of CDPred (Guo et al., 2022) correctly only report test results obtained using predicted unbound tertiary structures as inputs to their model. Test results using experimental monomer structures in bound states can hide important limitations in the model, and thus say very little about the realistic use cases in which only the unbound structures (experimental or predicted) are available. I therefore strongly suggest reducing the importance given to the results obtained using bound structures and emphasizing instead those obtained using predicted monomer structures as inputs.
In particular, the most relevant comparison with AlphaFold-Multimer (AFM) is given in Figure S2, *not* Figure 6. Unfortunately, it substantially shrinks the proportion of structures for which AFM fails while PLMGraph-Inter performs decently. Still, it would be interesting to investigate why this occurs. One possibility would be that the predicted monomer structures are of bad quality there, and PLMGraph-Inter may be able to rely on a signal from its language model features instead. Finally, AFM multimer confidence values ("iptm + ptm") should be provided, especially in the cases in which AFM struggles.
Besides, in cases where *any* experimental structures - bound or unbound - are available and given to PLMGraph-Inter as inputs, they should also be provided to AlphaFold-Multimer (AFM) as templates. Withholding these from AFM only makes the comparison artificially unfair. Hence, a new test should be run using AFM templates, and a new version of Figure 6 should be produced. Additionally, AFM's mean precision, at least for top-50 contact prediction, should be reported so it can be compared with PLMGraph-Inter's.
It's a shame that many of the structures used in the comparison with AFM are actually in the AFM v2 training set. If there are any outside the AFM v2 training set and, ideally, not sequence- or structure-homologous to anything in the AFM v2 training set, they should be discussed and reported on separately. In addition, why not test on structures from the "Benchmark 2" or "Recent-PDB-Multimers" datasets used in the AFM paper?
It is also worth noting that the AFM v2 weights have now been outdated for a while, and better v3 weights now exist, with a training cutoff of 2021-09-30.
Another weakness in the evaluation framework: because PLMGraph-Inter uses structural inputs, it is not sufficient to make its test set non-redundant in sequence to its training set. It must also be non-redundant in structure. The Benchmark 2 dataset mentioned above is an example of a test set constructed by removing structures with homologous templates in the AF2 training set. Something similar should be done here.
Finally, the performance of DRN-1D2D for top-50 precision reported in Table 1 suggests to me that, in an ablation study, language model features alone would yield better performance than geometric features alone. So, I am puzzled why model "a" in the ablation is a "geometry-only" model and not a "LM-only" one.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> This paper uses a high-throughput assay of transcription levels to (i) assess the potential of large numbers of Escherichia coli genomic sequences to function as promoters, and (ii) identify regulatory sequences in some of those promoters. This is a substantial undertaking, and while much of the work supports principles of transcription and transcription regulation described by many prior studies, there is considerable value in assessing promoters on such a large scale. The identification of putative regulatory sequences in larger numbers of promoters will likely be valuable to other groups studying transcription regulation in E. coli. And the analysis of antisense promoters provides some interesting new insight that goes beyond previous anecdotal studies.
Strengths:<br /> - The presentation of the work is very clear, and the conclusions are mostly well supported by the data.<br /> - The assays are rigorously controlled and analyzed.<br /> - Conclusions regarding the impact of antisense transcription on sense transcript levels provide new insight. While these data are consistent with previous anecdotal studies, to my knowledge this is the first large-scale analysis supporting a negative regulatory role for antisense transcription.<br /> - The putative regulatory elements mapped in the high-throughput mutagenesis experiments will be a valuable resource for the scientific community.
Weaknesses:<br /> (all minor)<br /> - There are some parts where the authors could clarify their arguments.<br /> - I'm not convinced that intragenic promoters impact codon usage rather than the other way around.<br /> - The authors should present a more nuanced discussion of promoters that avoids making yes/no calls (i.e., characterize sequences by promoter strength rather than a binary yes/no call of being a promoter).<br /> - Data relating to intragenic promoters should be presented and discussed for sense and antisense promoters separately.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this work, Xie et al. developed SCA-seq, which is a multiOME mapping method that can obtain chromatin accessibility, methylation, and 3D genome information at the same time. SCA-seq first uses M.CviPI DNA methyltransferase to treat chromatin, then perform proximity ligation followed by long-read sequencing. This method is highly relevant to a few previously reported long read sequencing technologies. Specifically, NanoNome, SMAC-seq, and Fiber-seq have been reported to use m6A or GpC methyltransferase accessibility to map open chromatin, or open chromatin together with CpG methylation; Pore-C and MC-3C have been reported to use long read sequencing to map multiplex chromatin interactions, or together with CpG methylation. Therefore, as a combination of NanoNome/SMAC-seq/Fiber-seq and Pore-C/MC-3C, SCA-seq is one step forward. The authors tested SCA-seq in 293T cells and performed benchmark analyses testing the performance of SCA-seq in generating each data module (open chromatin and 3D genome). The QC metrics appear to be good and I am convinced that this is a valuable addition to the toolsets of multi-OMIC long-read sequencing mapping.
The revised manuscript addressed most of my questions except my concern about Fig. S9. This figure is about a theory that a chromatin region can become open due to interaction with other regions, and the author propose a mathematic model to compute such effects. I was concerned about the errors in the model of Fig. S9a, and I was also concerned about the lack of evidence or validation. In their responses, the authors admitted that they cannot provide biological evidence or validations but still chose to keep the figure and the text.
The revised Fig. S9a now uses a symmetric genome interaction matrix as I suggested. But Figure S9a still have a lot of problems. Firstly, the diagonal of the matrix in Fig. S9a still has many 0's, which I asked in my previous comments without an answer. The legend mentioned that the contacts were defined as 2, 0 or -2 but the revised Fig. S9a only shows 1,0, or -1 values. Furthermore, Fig. S9b,9c,9d all added a panel of CTCF+/- but there is no explanation in text or figure legend about these newly added panels. Given many unaddressed problems, I would still suggest deleting this figure.
In my opinion, this paper does not need Fig. S9 to support its major story. The model in this figure is independent of SCA-seq. I think it should be spinoff as an independent paper if the authors can provide more convincing analysis or experiments. I understand eLife lets authors to decide what to include in their paper. If the authors insist to include Fig. S9, I strongly suggest they should at least provide adequate explanation about all the figure panels. At this point, the Fig. S9 is not solid and clearly have many errors. The readers should ignore this part.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 Public Review
Anobile and colleagues present a manuscript detailing an account of numerosity processing with an appeal to a two-channel model. Specifically, the authors propose that the perception of numerosity relies on (at least) two distinct channels for small and large numerosities, which should be evident in subject reports of perceived numerosity. To do this, the authors had subjects reproduce visual dot arrays of numerosities ranging from 8 to 32 dots, by having subjects repetitively press a response key at a pre-instructed rate (fast or slow) until the number of presses equaled the number of perceived dots. The subjects performed the task remarkably well, yet with a general bias to overestimate the number of presented dots. Further, no difference was observed in the precision of responses across numerosities, providing evidence for a scalar system. No differences between fast and slow tapping were observed. For behavioral analysis, the authors examined correlations between the Weber fractions for all presented numerosities. Here, it was found that the precision at each numerosity was similar to that at neighboring numerosities, but less similar to more distant ones. The authors then went on to conduct PCA and clustering analyses on the weber fractions, finding that the first two components exhibited an interaction with the presented numerosity, such that each was dominant at distinct lower and upper ranges and further well-fit by a log-Gaussian model consistent with the channel explanation proposed at the beginning.
Overall, the authors provide compelling evidence for a two-channel system supporting numerosity processing that is instantiated in sensorimotor processes. A strength of the presented work is the principled approach the authors took to identify mechanisms, as well as the controls put in place to ensure adequate data for analysis. Some questions do remain in the data, and there are aspects of the presentation that could be adjusted.
-The use of a binary colormap for the correlation matrix seems unnecessary. Binary colormaps between two opposing colors (with white in the middle) are best for results spanning positive and negative values (say, correlation values between -1 and +1), but the correlations here are all positive, so a uniform colormap should be applied. I can appreciate that the authors were trying to emphasize that a 2+ channel system would lead to lower correlations at larger ratios, but that's emphasized better in the numerical ratio line plots.
-In Figure 1, the correlation matrices in Figure 1 appear blurred out. I am not sure if this was intentional but suspect it was not, and so they should appear like those presented in Figure 3.
-It's notable that the authors also collected data on a timing task to rule out a duration-based strategy in the numerosity task. If possible, it would be great to have the author also conduct the rest of the analyses on the duration task as well; that is, to look at WF correlation matrices/ratios as well as PCA. There is evidence that duration processing is also distinctly sensorimotor, and may also rely on similar channels. Evidence either for or against this would likely be of great interest.
-For the duration task, there was no fast tapping condition. Why not? Was this to keep the overall task length short?
-The number of subjects/trials seems a bit odd. Why did some subjects perform both and not others? The targets say they were presented "between 25 and 30 times", but why was this variable at all?
-For the PCA analysis, my read of the methods and results is that this was done on all the data, across subjects. If the data were run on individual subjects and the resulting PCA components averaged, would the same results be found?
-For the data presented in Figure 2, it would be helpful to also see individual subject data underlaid on the plots to get a sense of individual differences. For the reproduced number, these will likely be clustered together given how small the error bars are, but for the WF data it may show how consistently "flat" the data are. Indeed, in other magnitude reproduction tasks, it is not uncommon to see the WF decrease as a function of target magnitude (or even increase). It may be possible that the reason for the observed findings is that some subjects get more variable (higher WFs) with larger target numbers and others get less variable (lower WFs).
-Regarding the two-channel model, I wonder how much the results would translate to different ranges of numerosities? For example, are the two channels supported here specific to these ranges of low and high numbers, or would there be a re-mapping to a higher range (say, 32 to 64 dots) or to a narrower range (say 16 to 32 dots). It would be helpful to know if there is any evidence for this kind of remapping.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The study presents an extensive computational approach to identify the motor neuron input from the characteristics of single motor neuron discharge patterns during a ramp up/down contraction. This reverse engineering approach is relevant due to limitations in our ability to estimate this input experimentally. Using well-established models of single motor neurons, a (very) large number of simulations were performed that allowed identification of this relation. In this way, the results enable researchers to measure motor neuron behavior and from those results determine the underlying neural input scheme. Overall, the results are very convincing and represent an important step forward in understanding the neural strategies for controlling movement.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> In this study, Basha and colleagues aim to test whether the thalamic nucleus reuniens can facilitate the hippocampus/prefrontal cortex coupling during sleep. Considering the importance of sleep in memory consolidation, this study is important to understand the functional interaction between these three majorly involved regions. This work suggests that the thalamic nucleus reuniens has a functional role in synchronizing the hippocampus and prefrontal cortex.
Strengths:<br /> The authors performed recordings in naturally sleeping cats, and analysed the correlation between the main slow wave sleep oscillatory hallmarks: slow waves, spindles, and hippocampal ripples, and with reuniens' neurons firing. They also associated intracellular recordings to assess the reuniens-prefrontal connectivity, and computational models of large networks in which they determined that the coupling of oscillations is modulated by the strength of hippocampal-thalamic connections.
Weaknesses:<br /> The authors' main claim is made on slow waves and spindle coupling, which are recorded both in the prefrontal cortex and surprisingly in reuniens. Known to be generated in the cortex by cortico-thalamic mechanisms, the slow waves and spindles recorded in reuniens show no evidence of local generation in the reuniens, which is not anatomically equipped to generate such activities. Until shown differently, these oscillations recorded in reuniens are most likely volume-conducted from nearby cortices. Therefore, such a caveat is a major obstacle to analysing their correlation (in time or frequency domains) with oscillations in other regions.
Finally, the choice of the animal model (cats) is the best suited one, as too few data, particularly anatomical ones regarding reuniens connectivity, are available to support functional results.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary: By elevating Ca influx and inducing PTP, the authors have maximised the release probability. In this condition, the release probability is nearly one. Under such a condition, the release site can release another vesicle in a short time. By analyzing mean, variance and covariance, the authors propose a release model that each release site contains a docking site and a replacement site. They excluded the LS-TS model (Neher and Brose) based on discrepancy between model and the data (mean and covariance).
Strengths: The authors have used a minimal stimulation and modelling nicely to look into stochastic nature of release sites with good resolution. This cannot be done at other synapses. Overall conclusions are reasonable and convincing.
Weaknesses: The interpretation is somewhat model-dependent, and it is unclear if the interpretation is unique. For example, it is unclear if the heterogeneous release probability among sites, silent sites, can explain the results. However, the authors discuss these potential caveats in a fair manner and argue that their model is very likely to be the best so far.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Activity has effects on the development of neural circuitry during almost any step of neuronal differentiation. In particular during specific time periods of circuit development, so called critical periods (CP), altered neural activity can induce permanent changes of neuronal and network excitability. In complex neural networks it is often difficult to pinpoint the specific network components that are permanently altered by activity, and it often remains unclear how activity is integrated during the CP to set mature network excitability. This study combines electrophysiology with pharmacological and optogenetic manipulation in the Drosophila genetic model system to pinpoint the neural substrate that is influenced by altered activity during a critical period (CP) of larval locomotor circuit development. Moreover, it is then tested whether and how different manipulations of synaptic input are integrated during the CP to tune network excitability.
Strengths: Based on previous work, during the CP network activity is increased by feeding the GABA-AR antagonist PTX. This results in permanent network activity changes as highly convincingly assayed by a prolonged recovery period following induced seizure and by altered intersegmental locomotor network coordination. This is then used to provide two important findings: First, compelling electro- and optophysiological as well as anatomical experiments track the site of network change down to the level of single neurons and pre- versus postsynaptic specializations. In short, increased activity during the CP increases both, the magnitude of excitatory and inhibitory synaptic transmission to the aCC motoneuron, but excitation is affected more strongly. This results in altered excitation inhibition ratios. Fine electrophysiology shows that excitatory synapse strengthening occurs postsynaptically. High quality anatomy shows that dendrite size and numbers of synaptic contacts remain unaltered. It is a major accomplishment to track the tuning of network excitability during the CP down to the physiology of specific synapses at identified neurons.<br /> Second, additional experiments with single neuron resolution demonstrate that during the CP different forms of activity manipulation are integrated so that opposing manipulations can rescue altered setpoints. This provides novel insight into how developing neural network excitability is tuned, and it indicates that during the CP training can rescue the effects of hyperactivity.
Weaknesses: There are no major weaknesses to the findings presented, but the molecular cause that underlies increased motoneuron postsynaptic responsiveness as well as the mechanism that integrates different forms of activity during the CP remain unknown. However, the discussion addresses this point adequately.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> Peterson et al., present a series of experiments in which the Pavlovian performance (i.e. time spent at a food cup/port) of male and female rats is assessed in various tasks in which context/cue/outcome relationships are altered. The authors find no sex differences in context-irrelevant tasks and no such differences in tasks in which the context signals that different cues will earn different outcomes. They do find sex differences, however, when a single outcome is given and context cues must be used to ascertain which cue will be rewarded with that outcome (Ctx-dep O1 task). Specifically, they found that males acquired the task faster, but that once acquired, the performance of the task was more resilient in female rats against exposure to a stressor. Finally, they show that these sex differences are reflected in differential rates of c-fos expression in all three subregions of rat OFC, medial, lateral, and ventral, in the sense that it is higher in females than males, and only in the animals subject to the Ctx-dep O1 task in which sex differences were observed.
Strengths:<br /> • Well-written.<br /> • Experiments elegantly designed.<br /> • Robust statistics.<br /> • Behaviour is the main feature of this manuscript, rather than any flashy techniques or fashionable lab methodologies, and luckily the behaviour is done really well.<br /> • For the most part I think the conclusions were well supported, although I do have some slightly different interpretations to the authors in places.
Weaknesses:<br /> 1. With regards to the claim (page 4 of pdf), I think I can see what the authors are getting at when they claim "Only Ctx-dep.01 engages context-gated reward predictions", because the same reward is available in each context, and the animal must use contextual information to determine which cue will be rewarded. In other words, it has a discriminative purpose. In Ctx-dep.O1/O2, however, although the context doesn't serve a discriminative purpose in the sense that one cue will always earn a unique outcome, regardless of context, the fact that these cues are differentially rewarded in the different context means that animals may well form context-gated cue-outcome associations (e.g. CtxA-(CS1-O1), CtxnoA-(CS2-O2)). Moreover, the context is informative in this group in telling the animal which cue will be rewarded, even prior to outcome delivery, such that I don't think contextual information will fade to the background of the association and attention be lost to it in the way, say Mackintosh (1975) might predict. Therefore, I don't think this statement is correct.
2. I think the results shown in Figure 1 are very interesting, and well supported by the statistics. It's so nice to see a significant interaction, as so many papers try to report these types of effects without it. However, I do wonder how specific the results are to contextual modulation. That is, should a discriminative discrete cue be used instead of each context (e.g. CS1 indicates CS2 earns O1, CS3 indicates CS4 earns O1), would female rats still be as slow to learn the discrimination?
3. Pages 8-9 of pdf, where the biological basis or the delayed acquisition of contextual control in females is considered, I find this to be written from a place of assuming that what is observed in the males is the default behaviour. That is, although the estrous cycle and its effects on synaptic plasticity/physiology may well account for the results, is there not a similar argument to be made for androgens in males? Perhaps the androgens also somehow alter synaptic plasticity/physiology, leading to their faster speed, reduced performance stability, and increased susceptibility to stress.
4. In addition, the OFC - which is the brain region found to have differential expression of c-fos in males and females in Figure 5 - is not explicitly discussed with regard to the biological mechanisms of differences, which seems odd.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The authors presented a new MNase-based proximity ligation method called MChIP-C, allowing for the measurement of protein-mediated chromatin interactions at single-nucleosome resolution on a genome-wide scale. With improved resolution and sensitivity, they explored the spatial connectivity of active promoters and identified the potential candidates for establishing/maintaining E-P interactions. Finally, with published CRISPRi screens, they found that most functionally verified enhancers do physically interact with their cognate promoters, supporting the enhancer-promoter looping model.
The study's experimental approach and findings are interesting. However, several issues need to be addressed.
1. The authors described that "the lack of interaction between experimentally-validated enhancers and their cognate promoters in some studies employing C-methods has raised doubts regarding the classical promoter-enhancer looping model", so it's intriguing to see whether the MChIP-C could indeed detect the E-P interactions which were not identified by C-methods as they mentioned (Benabdallah et al., 2019; Gupta et al., 2017). I agree that they identified more E-P interactions using MChIP-C, but specifically, they should show at least 2-3 cases. It's important since this is the main conclusion the authors want to draw.
2. The authors compared their data to those of Chen et al. (Chen et al., 2022), who used PLAC-seq with anti-H3K4me3 antibodies in K562 cells and standard Micro-C data previously reported for K562, concluding that "MChIP-C achieves superior sensitivity and resolution compared to C-methods based on standard restriction enzymes.". This is not convincing since they only compared their data to one dataset. More datasets from other cell lines should be included.
3. The reasons for choosing Chen's data (Chen et al., 2022) and CRISPRi screens (Fulco et al., 2019; Gasperini et al., 2019) should be provided since there are so many out there.
4. The authors identify EP300 histone acetyltransferase and the SWI/SNF remodeling complex as potential candidates for establishing and/or maintaining enhancer-promoter interactions, but not RNA polymerase II, mediator complex, YY1, and BRD4. More explanation is needed for this point since they're previously suggested to be associated with E-P interactions.
5. The limitations of the method should be discussed.
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arxiv.org arxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The manuscript considers a mechanistic extension of MacArthur's consumer-resource model to include chasing down food and potential encounters between the chasers (consumers) that lead to less efficient feeding in the form of negative feedback. After developing the model, a deterministic solution and two forms of stochastic solutions are presented, in agreement with each other. Finally, the model is applied to explain observed coexistence and rank-abundance data.
Strengths:<br /> - The application of the theory to natural rank-abundance curves is impressive.<br /> - The comparison with the experiments that reject the competitive exclusion principle is promising. It would be fascinating to see if in, e.g. insects, the specific interference dynamics could be observed and quantified and whether they would agree with the model.<br /> - The results are clearly presented; the methods adequately described; the supplement is rich with details.<br /> - There is much scope to build upon this expansion of the theory of consumer-resource models. This work can open up new avenues of research.
Weaknesses:<br /> - I am questioning the use of carrying capacity (Eq. 4) instead of using nutrient limitation directly through Monod consumption (e.g. Posfai et al. who the authors cite). I am curious to see how these results hold or are changed when Monod consumption is used.
- Following on the previous comment, I am confused by the fact that the nutrient consumption term in Eq. 1 and how growth is modeled (Eq. 4) are not obviously compatible and would be hard to match directly to experimentally accessible quantities such as yield (nutrient to biomass conversion ratio). Ultimately, there is a conservation of mass ("flux balance"), and therefore the dynamics must obey it. I don't quite see how conservation of mass is imposed in this work.
- These models could be better constrained by more data, in principle, thereby potential exists for a more compelling case of the relevance of this interference mechanism to natural systems.
- The underlying frameworks, B-D and MacArthur are not properly exposed in the introduction, and as a result, it is not obvious what is the specific contribution in this work as opposed to existing literature. One needs to dig into the literature a bit for that. The specific contribution exists, but it might be more clearly separated and better explained. In the process, the introduction could be expanded a bit to make the paper more accessible, by reviewing key features from the literature that are used in this manuscript.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
This paper explores how minimal active matter simulations can model tissue rheology, with applications to the in vivo situation of zebrafish morphogenesis. The authors explore the idea of active noise, particle softness and size heterogeneity cooperating to give rise to surprising features of experimental tissue rheologies (in particular an increase and then a plateau in viscosity with fluid fraction). In general, the paper is interesting from a theoretical standpoint, by providing a bridge between concepts from jamming of particulate systems and experiments in developmental biology. The idea of exploring a free space picture in this context is also interesting. It will be interesting in the future to see whether and how the findings change when considering 3D tissues with less size heterogeneity or how viscosity is impacted by the time scale of measurements.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Gazula and co-workers presented in this paper a software tool for 3D structural analysis of human brains, using slabs of fixed or fresh brains. This tool will be included in Freesurfer, a well-known neuroimaging processing software. It is possible to reconstruct a 3D surface from photographs of coronal sliced brains, optionally using a surface scan as a model. A high-resolution segmentation of 11 brain regions is produced, independent of the thickness of the slices, interpolating information when needed. Using this method, the researcher can use the sliced brain to segment all regions, without the need for ex vivo MRI scanning.
The software suite is freely available and includes 3 modules. The first accomplishes preprocessing steps, for correction of pixel sizes and perspective. The second module is a registration algorithm that registers a 3D surface scan obtained prior to sectioning (reference) to the multiple 2D slices. It is not mandatory to scan the surface - a probabilistic atlas can also be used as a reference - however, the accuracy is lower. The third module uses machine learning to perform the segmentation of 11 brain structures in the 3D reconstructed volume. This module is robust, dealing with different illumination conditions, cameras, lenses, and camera settings. This algorithm ("Photo-SynthSeg") produces isotropic smooth reconstructions, even in high anisotropic datasets (when the in-plane resolution of the photograph is much higher than the thickness), interpolating the information between slices.
To verify the accuracy and reliability of the toolbox, the authors reconstructed 3 datasets, using real and synthetic data. Real data of 21 postmortem confirmed Alzheimer's disease cases from the Massachusetts Alzheimer's Disease Research Center (MADRC) and 24 cases from the AD Research at the University of Washington (who were MRI scanned prior to processing) were employed for testing. These cases represent a challenging real-world scenario. Additionally, 500 subjects of the Human Connectome project were used for testing error as a continuous function of slice thickness. The segmentations were performed with the proposed deep-learning new algorithm ("Photo-SynthSeg") and compared against MRI segmentations performed to "SAMSEG" (an MRI segmentation algorithm, computing Dice scores for the segmentations. The methods are sound and statistically showed correlations above 0.8, which is good enough to allow volumetric analysis. The main strengths of the methods are the datasets used (real-world challenging and synthetic) and the statistical treatment, which showed that the pipeline is robust and can facilitate volumetric analysis derived from brain sections and conclude which factors can influence the accuracy of the method (such as using or not 3D scan and using constant thickness).
Although very robust and capable of handling several situations, the researcher has to keep in mind that processing has to follow some basic rules in order for this pipeline to work properly. For instance, fiducials and scales need to be included in the photograph, and the slabs must be photographed against a contrasting background. Also, only coronal slices can be used, which can be limiting for certain situations.
The authors achieved their aims, and the statistical analysis confirms that the machine learning algorithm performs segmentations comparable to the state-of-the-art of automated MRI segmentations.
Those methods will be particularly interesting to researchers who deal with post-mortem tissue analysis and do not have access to ex vivo MRI. Quantitative measurements of specific brain areas can be performed in different pathologies and even in the normal aging process. The method is highly reproducible, and cost-effective since it allows the pipeline to be applied by any researcher with small pre-processing steps.
The paper is very interesting and well structured, adding an important tool for fixed and fresh brain analysis. The software tool is robust and demonstrated good and consistent results in the hard task of managing automated segmentation from brain slices. In the future, segmentation of the histological slices could be developed and histological structures added (such as small brainstem nuclei, for instance). Also, dealing with axial and sagittal planes can be useful to some labs.
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Reviewer #1 (Public Review):
Gumaste et al studied if a parameter of odor plumes, the intermittency can be detected by an animal species, such as mice that heavily rely on olfaction to navigate and search for food and mates, among other behaviors. They also ask if the animals can extract information from this to gain knowledge about the odor source. Intermittency is defined as the fraction of time an odorant is present at a sampled point within the odor plume space. Their findings could be summarized as follows: they found that animals are capable of detecting differences in intermittency levels and suggest that this parameter of odor plumes is important for odor-based navigation in mammals, as it has been seen in other animals such as flying insects. The authors used a combination of behavioral training while concomitantly performing calcium imaging of olfactory receptor neurons (input to the olfactory bulb) and also mitral cells (output of the olfactory bulb). They found that mice are able to behaviorally discriminate between odor plumes of high and low intermittency. Interestingly, they found that the response of both input and output neurons of the olfactory bulb is capable to encode the intermittency experienced by the animals. The methods utilized in this work are very well suited for the kind of questions that the authors are asking. The combination of behavior and imaging, as opposed to only anesthetized imaging gives the authors a lot of power to interpret their data. A very relevant point is the generation of the olfactory stimuli that will be used to test the animals. The authors go to great lengths to generate more naturalistic odorant stimulations, as opposed to the typically used square pulses. Although there are some issues that can be addressed, the authors succeeded in answering the questions they set at the beginning of this work, and their conclusions are supported by their experiments. This work would generate interest among a relatively broad audience because the issue presented here (how the temporal structure of the odor plume affects the detection and encoding of an odorant) is novel in mice olfactory research.
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Reviewer #1 (Public Review):
With MERGEseq, the authors sought to develop a scalable and accessible method for getting both projectome and transcriptome information at the single-cell level from multiple projection targets within a single animal. MERGEseq uses a retro rAAV2 to deliver a 15-nucleotide barcode driven by a CAG promoter with co-expression of eGFP to enrich barcoded cells using FACS. Injection of this rAAV2 in distinct regions (with each injection region distinguished by a unique barcode that is specific to the virus used) allows retrograde trafficking and expression of the barcodes in cells that project to the injected region. In this manuscript, rAAVs harboring 5 unique barcodes were stereotactically delivered to 5 targets of the mouse: dorsomedial striatum (DMS), mediodorsal thalamic nucleus (MD), basal amygdala (BLA), lateral hypothalamus (LH), and agranular insular cortex (AI). After a 6-week period to allow for viral transduction and expression, the ventromedial prefrontal cortex (vmPFC) was harvested for scRNAseq. vmPFC scRNAseq data were validated against previously published PFC datasets, demonstrating that MERGEseq does not disrupt transcript expression and identifies the same principal cell types as annotated in previous studies. Importantly, MERGEseq enabled the identification of cell types in the vmPFC that project to distinct areas, with separation occurring largely based on cell type and cortical layer. The application of stringent criteria for barcode index determination is rigorous and improves confidence that barcoded cells are correctly identified. The observation that all barcoded cells were excitatory is consistent with prior work, although it is not clear if viral tropism contributes to this in some way. In a parallel experiment, FAC-sorted cells (vmPFC cells expressing EGFP) were isolated as a comparison. Notably, EGFP+ cells were exclusively excitatory neurons, consistent with literature showing PFC projection neurons are excitatory. Next, barcode analysis was combined with transcriptional identification of neuronal subtypes to define general projection patterns and single-cell projection patterns, which were validated by the DMS and MD in situ using retrograde tracing in combination with RNA FISH. MERGEseq data were also used to identify transcriptional differences between neurons with dedicated and bifurcated projections. DMS+LH and DMS+MD projecting neurons had distinct transcriptional profiles, unlike cells with other targets. RNA FISH for marker gene Pou3f and retrograde tracing from DMS+LH projecting cells demonstrate enrichment of this gene in this projection population. Finally, machine-learning was used to predict projection targets based on transcriptional profiles. In this dataset, 50 highly variable genes (HVGs) were optimal for predicting projection patterns, though this might vary in different circuits. Overall, the results of this manuscript are well presented and include rigorous validation for select vmPFC targets with in situ techniques. The application of unique barcodes for retro-AAV delivery is an accessible tool that other labs can implement to study other brain circuits.
Ultimately, MERGEseq is a subtle conceptual advancement over VECTORseq (retro-AAV delivered transgenes rather than barcodes, in combination with scRNAseq) that offers higher confidence in the described projectome diversity in comparison. The use of a retrograde AAV inherently limits the number of projection areas that can be assessed, a weakness compared to anterograde approaches such as MAPseq/BARseq. However, BARseq demands more time and resources; further, the use of the highly toxic Sindbis virus limits the application of this technique. This manuscript builds upon previous work by utilizing machine learning to predict projection targets. BARseq2 could be used to rigorously validate predicted projectomes and gain single-cell information regarding target neurons. Overall, MERGEseq is an accessible technique that can be used across many animal models and serve as an important starting point to define circuits at the single-cell level.
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Reviewer #1 (Public Review):
This study characterized the role of the Drosophila odorant-binding protein Obp56g in mediating post-mating responses in females. The authors show that this Obp56g is expressed in the male ejaculatory bulb, use genetic approaches to disrupt Obp56g, and show that males with disrupted Obp56g fail to form mating plugs in their mates.
Despite the fact that Obp56g deficient males generate and transfer sperm normally, the lack of formation of a functional mating plug leads to sperm loss in females mated to these males and thus a failure to induce normal post-mating responses.
This study identifies an unexpected role for an Obp and raises new questions about the function of this class of protein and the variety of roles that they may play in sensory function and reproduction.
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Reviewer #1 (Public Review):
In this paper, the authors develop new models of sequential effects in a simple Bernoulli learning task. In particular, the authors show evidence for both a "precision-cost" model (precise posteriors are costly) and an "unpredictability-cost" model (expectations of unpredictable outcomes are costly). Detailed analyses of experimental data partially support the model predictions.
Strengths:<br /> - Well-written and clear.<br /> - Addresses a long-standing empirical puzzle.<br /> - Rigorous modeling.
Weaknesses:<br /> - No model adequately explains all of the data.<br /> - New empirical dataset is somewhat incremental.<br /> - Aspects of the modeling appear weakly motivated (particularly the unpredictability model).<br /> - Missing discussion of some relevant literature.
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Reviewer #1 (Public Review)
This work challenges previously published results regarding the presence and abundance of 6mA in Drosophila genome, as well as the claim that the TET or DMAD enzyme serves as the "eraser" of this DNA methylation mark and its roles in development. This information is needed to clarify these questions in the field. Generally speaking, the methods for fly husbandry and treatment seem to be in accordance with those established ones in the field.
Here are a couple of suggestions that could be discussed with the current work and addressed in the future, in order to better understand the roles of 6mA and TET.
1. Regarding the estimated "200 to 400 methylated adenines per haplogenome", some insights regarding where they are enriched in the genome could inform the potential target sites regulated by 6mA.
2. The TET-GFP and TET-CD-GFP knock-in lines give proper nuclear localization and could be used to identify genomic regions bound with full-length TET and TET-CD using anti-GFP for ChIP-seq or CUT&RUN (or CUT&TAG).
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Reviewer #1 (Public Review):
Summary:
Using chromaffin cells as powerful model systems for studying secretion, the authors study the regulatory role of complexin in secretion. Complexin is still enigmatic in its regulatory role, as it both provides inhibitory and facilitatory functions in release. The authors perform an extensive structure-function analysis of both the C- and N-terminal regions of complexin. There are several interesting findings that significantly advance our understanding of cpx/SNARe interactions in regulating release. C-terminal amphipathic helix interferes with SNARE complex assembly and thus clamps fusion. There are acidic residues in the C-term that may be seen as putative interaction partners for Synaptotagmin. The N-terminus of Complexin promoting role may be associated with an interaction with Syt1. In particular, the putative interaction with Syt1 is of high interest and supported by quite strong functional and biochemical evidence. The experimental approaches are state-of-the-art, and the results are of the highest quality and convincing throughout. They are adequately and intelligently discussed in the rich context of the standing literature. Whilst there are some concerns about whether the facilitatory actions of complexion have to be tightly linked to Syt1 interactions, the proposed model will significantly advance the field by providing new directions in future research.
I have only minor comments related to the interpretation of the data:
Fig 5 While the data very nicely show that CPX and Syt1 have interdependent interactions in the chromaffin neurons, this seems to be not the case in neurons, where the loss of complexins and synaptotagmins have additive effects, suggesting independent mechanisms (eg Xue et al., 2010). This would be a good opportunity to discuss some possible differences between secretion in endocrine cells vs neurons.
Fig 8 Shows an apparent shift in Ca sensitivity in N-terminal mutants suggesting a modification of Ca sensitivity of Syt1. Could there be also an alternative mechanism, that explains this phenotype which is based on a role of the n-term lowering the energy barrier for fusion, that in turn shifts corresponding fusion rates to take place at lower Ca saturation levels?
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Reviewer #1 (Public Review):
Summary:<br /> Yang et al combine high-speed video tracking of the limbs of freely moving mice with in vivo electrophysiology to demonstrate how striatal neurons encode single-limb gait. They also examine encoding other well-known aspects of locomotion, such as movement velocity and the initiation/termination of movement. The authors show that striatal neurons exhibit rhythmic firing phase-locked with mouse gait, while mice engage in spontaneous locomotion in an open field arena. Moreover, they describe gait deficits induced by severe unilateral dopamine neuron degeneration and associate these deficits with a relative strengthening of gait-modulation in the firing of D2-expressing MSNs. Although the source and function of this gait-modulation remain unclear, this manuscript uncovers an important physiological correlate of striatal activity with gait, which may have implications for gait deficits in Parkinson's Disease.
Strengths:<br /> While some previous work has looked at the encoding of gait variables in the striatum and other basal ganglia nuclei, this paper uses more careful quantification of gait with video tracking. In addition, few if any papers do this in combination with optically-labeled recordings as were performed here.
Weaknesses:<br /> The data collected has a great richness at the physiological and behavioral levels, and this is not fully described or explored in the manuscript. Additional analysis and display of data would greatly expand the interest and interpretability of the findings.
There are also some caveats to the interpretation of the analyses presented here, including how to compare encoding of gait variables when animals have markedly different behaviors (eg comparing sham and unilaterally 6-OHDA treated mice), or how to interpret the loss of gait modulation when single unit activity is overall very low.
1. The authors use circular analysis to quantify the degree to which striatal neurons are phase-locked to individual limbs during gait. The result of this analysis is shown as the proportion of units phase-locked to each limb, vector length, and vector angle (Fig 2H-K; Fig 4E-F; Fig 6E-F). Given that gait is a cyclic oscillation of the trajectories of all four limbs, one could expect that if one unit is phase-locked to one limb, it will also be phase-locked to the other three limbs but at a different phase. Therefore, it is not clear in the manuscript how the authors determine to which limb each unit is locked, and how some units are locked to more than one limb (Fig 2H). More methodological/analytical detail would be especially helpful.
2. In Figures 2 and 3, the authors describe the modulation of striatal neurons by gait, velocity, and movement transitions (start/end), with most of their examples showing firing rates compatible with rates typical of striatal interneurons, not MSNs. In order to have a complete picture of the relationship between striatal activity and gait, a cell type-specific analysis should be performed. This could be achieved by classifying units into putative MSN, FS interneurons, and TANs using a spike waveform-based unit classification, as has been done in other papers using striatal single-unit electrophysiology. An example of each cell type's modulation with gait, as well as summary data on the % modulation, would be especially helpful.
3. By normalizing limb trajectories to the nose-tail axis, the analysis ignores whether the mouse is walking straight, or making left/right turns. Is the gait-modulation of striatal activity shaped by ipsi- and contralateral turning? This would be especially important to understand changes in the unilateral disease model, given the imbalance in turning of 6-OHDA mice.
4. It looks like the data presented in Figure 4 D-F comes from all opto-identified D1- and D2-MSNs. How many of these are gait-modulated? This information is missing (line 110). Pooling all units may dilute differences specific to gait-modulated units, therefore a similar analysis only on gait-modulated units should be performed.
5. Since 6-OHDA lesions are on the right hemisphere, we would expect left limbs to be more affected than right limbs (although right limbs may also compensate). It is therefore surprising that RF and RR strides seem slightly shorter than LF and LR (Fig 5G), and no differences in other stride parameters (Fig 5H-J). Could the authors comment on that? It may be that this is due to rotational behavior. One interesting analysis would be to compare activity during similar movements in healthy and 6-OHDA mice, eg epochs in which mice are turning right (which should be present in both groups) or walking a few steps straight ahead (which are probably also present in both groups).
6. Multiple publications have shown that firing rates of D1-MSN and D2-MSN are dramatically changed after dopamine neuron loss. Is it possible that changes observed in gait-modulation might be biased by changes in firing rates? For example, dMSNs have exceptionally low overall activity levels after dopamine depletion (eg Parker...Schnitzer, 2018; Ryan...Nelson, 2018; Maltese...Tritsch, 2021); this might reduce the ability to detect modulation in the firing of dMSNs as compared to iMSNs, which have similar or increased levels of activity in dopamine depleted mice. Does vector length correlate with firing rate? In addition, the normalization method used (dividing firing rate by minimum) may amplify very small changes in absolute rates, given that the firing rates for MSN are very low. The authors could show absolute values or Z-score firing rates (Figure 6 A, D).
7. The analysis shown in Fig 3C should also be done for opto-identified D1- and D2-MSNs (and for waveform-based classified units as noted above).
8. Discussion: the origin of the gait-modulation as well as the possible mechanisms driving the alterations observed in 6-OHDA mice should be discussed in more detail.
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Reviewer #1 (Public Review):
The brain's code is not static. Neuronal activity patterns change as a result of learning, aging, and disease. Reliable tracking of activity from individual neurons across long time periods would enable detailed studies of these important dynamics. For this reason, the authors' efforts to track electrophysiological activity across days without relying on matching neural receptive fields (which can change due to learning, aging, and disease) are very important.
By utilizing the tightly-spaced electrodes on Neuropixels probes, they are able to measure the physical distance and the waveform shape 'distance' between sorted units recorded on different days. To tune the matching algorithm and validate the results, they used the visual receptive fields of neurons in the mouse visual cortex (which tend to change little over time) as ground truth. Their approach performs quite well, with a high proportion of neurons accurately matched across multiple weeks. This suggests that the method may be useable in other cases where the receptive fields can't be used as ground truth to validate the tracking. This potential extendibility to tougher applications is where this approach holds the most promise.
The main caveat (and disappointment) is that this paper does not address generalizability to other experimental conditions. Because it only looks at one brain area (visual cortex), in one species (mouse), using one type of spike sorter (Kilosort), and one type of behavioral prep (head-fixed), it is not clear if this approach is overfit to those conditions or if it will perform equally well in other conditions. Most importantly, in brain areas where neuronal receptive fields are more dynamic and can't be used as a ground truth diagnostic, it isn't clear how to apply the technique outlined in this study, since many of the parameters are tuned to a very specific set of conditions using visual receptive fields as ground truth.
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Reviewer #1 (Public Review):
Summary:<br /> The authors have nicely demonstrated the efficiency of the HCR v.3.0 using hr38 mRNA expression as a marker of neuronal activity. This is very important in the Drosophila neuroscience field as in situ hybridization in adult Drosophila brains has been so far very challenging to do and replicate. However, this method has been described before [Choi et al., (2018)] and, if I understand correctly, is now the property of the non-profit organization molecular Technologies, who are the ones responsible for designing the probes (the sequences are not provided). Here the authors present their work as a description of a new method, called HI-FISH. However, I do not consider this as a new method but rather an application, a "proof of principle" that HCR v.3.0 can be done even on challenging tissues such as the adult Drosophila brain. Hence, if HCR v3.0 is sensitive enough and powerful enough to be used as a marker of neuronal activity, we can use it, for other neurobiological purposes, using other gene probes.<br /> To demonstrate the efficiency of HI-FISH, the authors have addressed two biological questions. The first one addressed whether specific groups of neurons, known to trigger specific behaviours (here courtship and/or aggression) are indeed activated by the behavioural context they can promote. In other words: is the behavioural output of these neurons also a trigger for their activation? The second question addressed whether this method is powerful enough to distinguish two subgroups of a class of neurons called P1 known to be involved in the two behaviours considered. In other words, are the same P1 neurons that promote aggression and courtship?
Strengths: The demonstration of the efficiency of the method is very convincing and well-performed. It gives the will for the reader to apply the method to their own subject.
Weakness: The pictures provided for HI-FISH and catFISH do not corroborate with the quantification and therefore I am not convinced about the author's biological interpretation of their data. See below for details.
Previously, using a split-gal4 line to restrict the Gal4 expression to a subset of P1 neurons, the authors have shown that these particular neurons when activated can trigger both aggressivity and courtship behaviour [Hoopfer et al., 2015]. The P1 neurons are composed of about 20 FruM neurons/hemibrain but are part of a bigger group that comprises about the same number of Fru- neurons that seem to be exclusively aggression-promoting neurons [Koganezawa et al., 2016]. Hence, this group of 40 neurons (pC1 neurons) contains aggressive-promoting neurons, courtship-promoting neurons, and perhaps neurons that can do both. Therefore, to address the first question, the authors compared hr38 expression in different groups of neurons, with a focus on subgroups, under different social contexts. While there is no ambiguity concerning the function of the Tk neurons as being exclusively aggressive-promoting neurons [Asahina et al., 2014], it is less clear when we look at the pC1 neurons. This is particularly evident for the P1a neurons which have been shown to be ambiguous as they can promote both aggression and courtship. For example, while optogenetic activation of these neurons promotes hr38 expression (Fig. 3D and fig sup. 4), it is less clear in the pictures to determine whether these specific P1a neurons labeled by the split-gal4 line are specifically activated by an aggressive behavioural context or a courtship behavioural context (Fig1, supp. 2 and fig. 4). Furthermore, the pictures chosen do not reflect the reality of the quantification (Fig. 2 B-D compared to sup. 2 or fig. 4C compared to fig. 4D) and therefore the authors conclusion. Because the drivers used are only expressed by a small subset of a larger population, I believe it would be more informative to separate in the quantification between the Gal4-expressing neurons and the non-expressing ones. Notably, based on the pictures provided, it looks like more P1 neurons (or rather pC1 neurons) are activated by a male-male encounter than by a male-female encounter. On the other hand, the splitGal4+ P1a seem to be more responsive to a courtship behavioural context (2/6 P1a neurons express hr38 in a courtship behavioural context while 0/9 _if we mentally abstract the increase of the background signal compared to the control picture_ seem to express hr38 in an aggression behavioural context). Hence, while activation of this P1asplit-Gal4 can promote both aggressive behaviour and courtship behaviour [Hoopfer et al., 2015], the authors didn't provide clear evidence (pictures not corroborating the quantification) that these specific small subpopulation of neurons are activated by one or the other or both behavioural conditions. Therefore my suggestion of differentiating in the quantification between the Gal4+ neurons from the others in the same local area.
Fig. 3, suppl. 3: In this section the authors addressed the question of whether the HI-FISH can be used to identify the downstream targets of this subpopulation. As positive controls of known downstream targets, the authors looked at the pCd population which they recently published as being an indirect downstream target of the P1a neurons [Jung et al., Neuron 2020]. They identified the Kenyon cells and a group of dopaminergic neurons, the PAM neurons as being activated by the P1a neurons. To confirm the increase of hr38 expression is indeed the result of a neuronal response of these neurons to the P1a activation, the authors used a different strategy used by them and others before. Using Gcamp signal to monitor the neuronal response of the presumably downstream targets the authors activated the P1a neurons using optogenetic (chrimson). It is important to note that they have previously shown that depending on the frequency of the light pulses activation of the P1a neurons can trigger only aggression, both aggression and wing extension or only wing extension [Hoopfer et al., eLife 2015]. Here the authors use 50Hz which is a frequency that leads to wing extension during the stimulation and aggressive behaviour at the offset of the stimulus [Hoopfer et al., eLife 2015]. Interestingly, the Gcamp experiment shows activation of the Kenyon cells and the PAM neurons but this activity is not maintained when the stimulus is turned off, suggesting that these neurons are activated during a courtship context rather than an aggressive behavioural context. I think it would be nice to see in which behavioural context the Kenyon cells and PAM neurons are activated (hr38 expression in the different behavioural context using the corresponding Gal4).<br /> Fig.4 and supp.4: The demonstration that the catFISH can now be done in Drosophila brain with a new in situ method was nicely performed. Notably, the intronic Hr38 probe appears to be an excellent marker for recent neuronal activation. However, while the optogenetic activation of the P1a neurons used to quantify the time lapse for both probes nicely distinguishes between nuclear and cytoplasmic exonic hr38, it is very difficult to use the localization of this probe in the experimental setup the authors used. Also, With their setup, I would simply use the frequency of intronic hr38 as a marker of recent activation correlating or not with the frequency of exonic hr38 marker (present in both conditions first and second encounter). This is important as this experiment addresses the second biological question. Once again, the pictures chosen absolutely do not corroborate the quantification. For example, the picture of the double encounter with the same gender male-male context clearly shows a higher number of cells that are hr38INT positive (and therefore nuclear) than the picture of the female-female context (Fig. 4C), and thus even if we only considered the P1asplit-Gal4 positive cells. In the male-male picture, 5/6 P1a cells have the Hr38INT marker while the presence of this marker is debatable in the female-female context. Especially, in some of the cells these magenta dots appear to be localized in the cytoplasm, suggesting a non-specific signal. Therefore, I would suggest to quantify the percentage of Hr38INT positive cells as the only marker for recent activation and the relative level of Hr38EXN immunostaining, and this among the P1asplit-Gal4 positive cells and the gal4- ones. A high Hr38EXN level associated with the presence of hr38INT would indicate that the cell has been activated during both encounters, while a lower hr38EXN with no hr38INT would suggest only an activation during the 1st behavioural context. Finally, a lower hr38EXN associated with the presence of hr38INT would suggest the opposite, an activation only during the 2nd behaviour.<br /> Overall, by only looking at the pictures provided, I would conclude that the HCR applied with the hr38 probes seems to efficiently work and is usable to address the question of whether a specific group of neurons are indeed activated by a specific social behavioural context. However, I would also conclude that this technique nicely demonstrated that flies are not robots and that even in a "simple" organism model such as Drosophila melanogaster individual variability is present among a group of neurons. Hence, only the quantification of the gal4-expressing neurons in comparison with their neighbor neurons known to belong to the same functional group, would allow a conclusion toward a specificity of contextual response. Therefore, although activation of a small group of neurons can be enough to trigger a specific behaviour that shouldn't happen under a certain environmental context [Hoopfer et al., eLife 2015], the results presented here suggest that we should, using this method, considering the response of the neighbour cells of the Gal4+ ones. Although currently, the quantification of the author's data does not allow such analysis, to strengthen the author's argumentation, I would distinguish in their quantification between gal4+ from the others (Fig. 2 and 4). Furthermore, I am not certain that the distinction between cytoplasmic and nuclear hr38EXN is 100% feasible (based on the pictures provided). I would instead for the hr38EXN marker only use the relative intensity (Fig. 4D).
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Reviewer #1 (Public Review):
Bischoff et al present a carefully prepared study on a very interesting and relevant topic: the role of ion channels (here a Ca2+-activated K+ channel BK) in regulating mitochondrial metabolism in breast cancer cells. The potential impact of these and similar observations made in other tumor entities has only begun to be appreciated. That being said, the authors pursue in my view an innovative approach to understanding breast cancer cell metabolism.
Considering the following points would further strengthen the manuscript:
Methods:
1. The authors use an extracellular Ca2+ concentration (2 mM) in their Ringer's solutions that is almost twice as high as the physiologically free Ca2+ concentration (ln 473). Moreover, the free Ca2+ concentration of their pipette solution is not indicated (ln 487).
2. Ca2+I measurements: The authors use ATP to elicit intracellular Ca2+ signals. Is this then a physiological stimulus for Ca2+ signaling in breast cancer? What is the rationale for using ATP? Moreover, it would be nice to see calibrated baseline values of Ca2+i.
3. Membrane potential measurements: It would be nice to see a calibration of the potential measurements; this would allow us to correlate the IV relationship with membrane potential. Without calibration, it is hard to compare unless the identical uptake of the dye is shown.
Does paxilline or IbTx also induce depolarization?
4. Mito-potential measurements: Why did the authors use such a long time course and preincubate cells with channel blockers overnight? Why did they not perform paired experiments and record the immediate effect of the BK channel blockers in the mito potential?
5. MTT assays are also based on mitochondrial function - since modulation of mito function is at the core of this manuscript, an alternative method should be used.
Results:
1. Fig. 5G: The number of BK "positive" mitoplasts is surprisingly low - how does this affect the interpretation? Did the authors attempt to record mitoBK current in the "whole-mitoplast" mode? How does the mitoBK current density compare with that of the plasma membrane? Is it possible to theoretically predict the number of mitoBK channels per mitochondrion to elicit the observed effects? Can these results be correlated with the immuno-localization of mitoBK channels?
2. There are also reports about other mitoK channels (e.g. Kv1.3, KCa3.1, KATP) playing an important role in mitochondrial function. Did the authors observe them, too? Can the authors speculate on the relative importance of the different channels? Is it known whether they are expressed organ-/tumor-specifically?
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Reviewer #1 (Public Review):
Summary:
In this study, the authors showed that activation of RelA and Stat3 in hepatocytes of DSS-treated mice induced CYPs and thereby produced primary bile acids, particularly CDCA, which exacerbated intestinal inflammation.
Strengths:
This study reveals the RelA/Stat3-dependent gene program in the liver influences intestinal homeostasis.
Weaknesses:
Additional evidence will strengthen the conclusion.
1. In Fig. 1C, photos show that phosphorylation of RelA and Stat3 was induced in only a few hepatocytes. The authors conclude that activation of both RelA and Stat3 induces inflammatory pathways. Therefore, the authors should show that phosphorylation of RelA and Stat3 is induced in the same hepatocytes during DSS treatment.
2. In Fig. 5, the authors treated mice with CDCA intraperitoneally. In this experiment, the concentration of CDCA in the colon of CDCA-treated mice should be shown.
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Reviewer #1 (Public Review):
Summary:
Ravichandran et al investigate the regulatory panels that determine the polarization state of macrophages. They identify regulatory factors involved in M1 and M2 polarization states by using their network analysis pipeline. They demonstrate that a set of three regulatory factors (RFs) i.e., CEBPB, NFE2L2, and BCL3 can change macrophage polarization from the M1 state to the M2 state. They also show that siRNA-mediated knockdown of those 3-RF in THP1-derived M0 cells, in the presence of M1 stimulant increases the expression of M2 markers and showed decreased bactericidal effect. This study provides an elegant computational framework to explore the macrophage heterogeneity upon different external stimuli and adds an interesting approach to understanding the dynamics of macrophage phenotypes after pathogen challenge.
Strengths:
This study identified new regulatory factors involved in M1 to M2 macrophage polarization. The authors used their own network analysis pipeline to analyze the available datasets. The authors showed 13 different clusters of macrophages that encounter different external stimuli, which is interesting and could be translationally relevant as in physiological conditions after pathogen challenge, the body shows dynamic changes in different cytokines/chemokines that could lead to different polarization states of macrophages. The authors validated their primary computational findings with in vitro assays by knocking down the three regulatory factors-NCB.
Weaknesses:
One weakness of the paper is the insufficient analysis performed on all the clusters. They used macrophages treated with 28 distinct stimuli, which included a very interesting combination of pro- and anti-inflammatory cytokines/factors that can be very important in the context of in vivo pathogen challenge, but they did not characterize the full spectrum of clusters. Although they mentioned that their identified regulatory panels could determine the precise polarization state, they restricted their analysis to only the two well-established macrophage polarization states, M1 and M2. Analyzing the other states beyond M1 and M2 could substantially advance the field. They mentioned the regulatory factors involved in individual clusters but did not study the potential pathway involving the target genes of these regulatory factors, which can show the importance of different macrophage polarization states. Importantly, these findings were not validated in primary cells or using in vivo models.
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Reviewer #1 (Public Review):
Summary:<br /> In this paper, the authors provide a characterisation of auditory responses (tones, noise, and amplitude-modulated sounds) and bimodal (somatosensory-auditory) responses and interactions in the higher-order lateral cortex (LC) of the inferior colliculus (IC) and compare these characteristics with the higher order dorsal cortex (DC) of the IC - in awake and anaesthetised mice. Dan Llano's group has previously identified gaba'ergic patches (modules) in the LC distinctly receiving inputs from somatosensory structures, surrounded by matrix regions receiving inputs from the auditory cortex. They here use 2P calcium imaging combined with an implanted prism to - for the first time - get functional optical access to these subregions (modules and matrix) in the lateral cortex of IC in vivo, in order to also characterise the functional difference in these subparts of LC. They find that both DC and LC of both awake and anaesthetised mice appear to be more responsive to more complex sounds (amplitude-modulated noise) compared to pure tones and that under anesthesia the matrix of LC is more modulated by specific frequency and temporal content compared to the gabaergic modules in LC. However, while both LC and DC appear to have low-frequency preferences, this preference for low frequencies is more pronounced in DC. Furthermore, in both awake and anesthetized mice, somatosensory inputs are capable of driving responses on their own in the modules of LC, but very little (possibly not at all) in the matrix. However, bimodal interactions may be different under awake and anesthesia in LC, which warrants deeper investigation by the authors: They find, under anesthesia, more bimodal enhancement in modules of LC compared to the matrix of LC and bimodal suppression dominating the matrix of LC. In contrast, under awake conditions bimodal enhancement is almost exclusively found in the matrix of LC, and bimodal suppression dominates both matrix and modules of LC.
The paper provides new information about how subregions with different inputs and neurochemical profiles in the higher-order auditory midbrain process auditory and multisensory information, and is useful for the auditory and multisensory circuits neuroscience community.
Strengths:<br /> The major strength of this study is undoubtedly the fact that the authors for the first time provide optical access to a subcortical region (the lateral cortex of the inferior colliculus (i.e. higher order auditory midbrain)) which we know (from previous work by the same group) have optically identifiable subdivisions with unique inputs and neurotransmitter release, and plays a central role in auditory and multisensory processing. A description of basic auditory and multisensory properties of this structure is therefore very useful for understanding auditory processing and multisensory interactions in subcortical circuits.
Weaknesses:<br /> I have divided my comments about weaknesses and improvements into major and minor comments. All of which I believe are addressable by the reviewers to provide a more clear picture of their characterisation of the higher-order auditory midbrain.
Major comment:<br /> 1. The differences between multisensory interactions in LC in anaesthetised and awake preparations appear to be qualitatively different, though the authors claim they are similar (see also minor comment related to figure 10H for further explanation of what I mean). However, the findings in awake and anaesthetised conditions are summarised differently, and plotting of similar findings in the awake figures and anaesthetised figures are different - and different statistics are used for the same comparisons. This makes it very difficult to assess how multisensory integration in LC is different under awake and anaesthetised conditions. I suggest that the authors plot (and test with similar statistics) the summary plots in Figure 8 (i.e. Figure 8H-K) for awake data in Figure 10, and also make similar plots to Figures 10G-H for anaesthetised data. This will help the readers understand the differences between bimodal stimulation effects on awake and anaesthetised preparations - which in its current form, looks very distinct. In general, it is unclear to me why the awake data related to Figures 9 and 10 is presented in a different way for similar comparisons. Please streamline the presentation of results for anaesthetised and awake results to aid the comparison of results in different states, and explicitly state and discuss differences under awake and anaesthetised conditions.
2. The claim about the degree of tonotopy in LC and DC should be aided by summary statistics to understand the degree to which tonotopy is actually present. For example, the authors could demonstrate that it is not possible/or is possible to predict above chance a cell's BF based on the group of other cells in the area. This will help understand to what degree the tonotopy is topographic vs salt and pepper. Also, it would be good to know if the gaba'ergic modules have a higher propensity of particular BFs or tonotopic structure compared to matrix regions in LC, and also if general tuning properties (e.g. tuning width) are different from the matrix cells and the ones in DC.
3. Throughout the paper more information needs to be given about the number of cells, sessions, and animals used in each panel, and what level was used as n in the statistical tests. For example, in Figure 4 I can't tell if the 4 mice shown for LC imaging are the only 4 mice imaged, and used in the Figure 4E summary or if these are just examples. In general, throughout the paper, it is currently not possible to assess how many cells, sessions, and animals the data shown comes from.
4. Throughout the paper, to better understand the summary maps and plots, it would be helpful to see example responses of the different components investigated. For example, given that module cells appear to have more auditory offset responses, it would be helpful to see what the bimodal, sound-only, and somatosensory responses look like in example cells in LC modules. This also goes for just general examples of what the responses to auditory and somatosensory inputs look like in DC vs LC. In general example plots of what the responses actually look like are needed to better understand what is being summarised.
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Joint Public Review:
This study sought to characterize the influence of acute stress on prosocial behavior, combining an effort-based task with neuroimaging, neuroendocrinological measures, and computational cognitive modeling. Two major results are reported: 1) Compared to controls, participants who experienced acute stress were less willing to exert effort for others, with more prominent effects for those who were more selfish; 2) More stressed participants exhibited an increase in activation in the dorsal anterior cingulate cortex and anterior insula, which are implicated in self-benefiting behavior. The approach is sophisticated and the findings are informative. Concerns regarding potential confounds and data reporting were addressed in a revised submission.
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Reviewer #1 (Public Review):
This paper studies the effects of tACS on detection of silence gaps in an FM modulated noise stimulus. Both FM modulation of the sound and the tACS are at 2Hz, and the phase of the two is varied to determine possible interactions between the auditory and electric stimulation. Additionally, two different electrode montages are used to determine if variation in electric field distribution across the brain may be related to the effects of tACS on behavioral performance in individual subjects.
Major strengths and weaknesses of the methods and results.
The study appears to be well powered to detect modulation of behavioral performance with N=42 subjects. There is a clear and reproducible modulation of behavioral effects with the phase of the FM sound modulation. The study was also well designed and executed in terms of fMRI, current flow modeling, montage optimization targeting, and behavioral analysis. A particular merit of this study is to have repeated the sessions for most subjects in order to test repeat-reliability, which is so often missing in human experiments. The results and methods are generally well described and well conceived. The portion of the analysis related to behavior alone is excellent. The analysis of the tACS results are also generally well described, candidly highlighting how variable results are across subjects and sessions. The figures are all of high quality and clear. One weakness of the experimental design is that no effort was made to control for sensation effects. tACS at 2Hz causes prominent skin sensations which could have interacted with auditory perception and thus, detection performance.
The central claim is that tACS modulates behavioral detection performance across the 0.5s cycle of stimulation. Statistical analysis with randomize relative phase (between audio and tACS) show that detection performance is modulated by tACS. Neither the relative phase or the strength of this effect reproduces across subjects or sessions, which makes the interpretation of these results difficult. These result could be of interest to investigators in the field of tACS.
The claim that the variation in the strength of the effect can be explained by variation of electric fields is not compelling.
The following are more detailed comments to specific sections of the paper, including details on the concerns with the statistical analysis of the tACS effects.<br /> The introduction is well balanced, discussing the promise and limitations of previous results with tACS. The objectives are well defined.
The analysis surrounding behavioral performance and its dependence on phase of the FM modulation (Figure 3) is masterfully executed and explained. It appears that it reproduces previous studies and points to a very robust behavioral task that may be of use in other studies.
The definition of tACS(+) vs tACS(-) phase is adjusted to each subject/session, which seems unconventional. For argument sake, let's assume the curves in Fig. 3E are random fluctuations. Then aligning them to best-fitting cosine will trivially generate a FM-amplitude fluctuation with cosine shape as shown in Fig. 4a. Selecting the positive and negative phase of that will trivially be larger and smaller than sham, respectively, as shown in Fig 4b.
"Data from the optimal tACS lag and its opposite lag (corresponding trough) were excluded to avoid any artificial bias in estimating tACS effects induced by the alignment procedure (33)." The delay was found by fitting a cosine, so removing just the peaks of that cosine does little to avoid this problem.
To demonstrate that this is not a trivial result of the definition, the analysis compares this to the same analysis but with a randomize alignment to the two stimuli (audio and tACS) in Figure 4d. Assuming this shuffle was done correctly, this shows that the modulation observed in 4b is not just a result of the analysis procedure.
The authors are to be commended for analyzing the robustness of their observation across subjects and across sessions in Fig. 5. The lack of consistency in the optimal time delay between the two stimuli is hard to reconcile with the common theory that tACS entrains brain function.
"To better understand what factors might be influencing inter-session variability in tACS effects, we estimated multiple linear models ..." "Inter-individual variability in the simulated E-field predicts tACS effects" Authors here are attempting to predict a property of the subjects that was just shown to not be a reliable property of the subject. Authors are picking 9 possible features for this, testing 33 possible models with N=34 data points. With these circumstances it is not hard to find something that correlates by chance. And some of the models tested had interaction terms, possibly further increasing the number of comparisons. In the absence of multiple comparison correction, what is happening here is that multiple models are fit to the data, and a statistical test is performed for the best model on the same (training) data. The corresponding claim that variations are explained by variations in electric field is not persuasive.
"Can we reduce inter-individual variability in tACS effects ..." This section seems even more speculative and with mixed results.
Given the concerns with the statistical analysis above, there are concerns about the following statements in the summary of the Discussion:
"4) individual variability in tACS effect size was partially explained by two interactions: between the normal component of the E-field and the field focality, and between the normal component of the E-field and the distance between the peak of the electric field and the functional target ROIs."
The complexity of this statement alone may be a good indication that this could be the result of false discovery due to multiple comparisons.
For the same reason as stated above, the following statements in the Abstract do not appear to have adequate support in the data:
"Inter-individual variability of tACS effects was best explained by the strength of the inward electric field, depending on the field focality and proximity to the target brain region. Although additional evidence is necessary, our results<br /> 42 also provided suggestive insights that spatially optimizing the electrode montage could be a promising tool to reduce inter-individual variability of tACS effects."
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Joint Public Review:
This manuscript tackles an important question, namely how K+ affects substrate transport in the SLC6 family. K+ effects have previously been reported for DAT and SERT, but the prototypical SLC6-fold transporter LeuT was not known to be sensitive to the K+ concentration. In this manuscript, the authors demonstrate convincingly that K+ inhibits Na+ binding, and Na+-dependent amino acid binding at high concentrations, and that K+ inside of vesicles containing LeuT increases the transport rate. However, outside K+ apparently had very little effect. Uptake data are supplemented with binding data, using the scintillation proximity assay, and transition metal FRET, allowing the observation of the distribution of distinct conformational states of the transporter.
Overall, the data are of high quality. I was initially concerned about the use of solutions of very high ionic strength (the Km for K+ is in the 200 mM range), however, the authors performed good controls with lower ionic strength solutions, suggesting that the K+ effect are specific and not caused by artifacts from the high salt concentrations.
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Reviewer #1 (Public Review):
Summary:<br /> In this study, Nandy and colleagues examine neural and behavioral correlates of perceptual variability in monkeys performing a visual change detection task. They used a laminar probe to record from area V4 while two macaque monkeys detected a small change in stimulus orientation that occurred at a random time in one of two locations, focusing their analysis on stimulus conditions where the animal was equally likely to detect (hit) or not-detect (miss) a briefly presented orientation change (target). They discovered two behavioral measures that are significantly different between hit and miss trials - pupil size tends to be slightly larger on hits vs. misses, and monkeys are more likely to miss the target on trials in which they made a microsaccade shortly before target onset. They also examined multiple measures of neural activity across the cortical layers and found some measures that are significantly different between hits and misses.
Strengths:<br /> Overall the study is well executed and the analyses are appropriate (though multiple issues do need to be addressed).
Weaknesses:<br /> My main concern with this study is that with the exception of the pre-target microsaccades, the physiological and behavioral correlates of perceptual variability (differences between hits and misses) appear to be very weak and disconnected. Some of these measures rely on complex analyses that are not hypothesis-driven and where statistical significance is difficult to assess. The more intuitive analysis of the predictive power of trial outcomes based on the behavioral and neural measures is only discussed at the end of the paper. This analysis shows that some of the significant measures have no predictive power, while others cannot be examined using the predictive power analysis because these measures cannot be estimated in single trials. Given these weak and disconnected effects, my overall sense is that the current results do not significantly advance our understanding of the neural basis of perceptual variability.
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Reviewer #1 (Public Review):
Zhu, et al present a genome-wide histone modification analysis comparing patients with schizophrenia (on or off antipsychotics) to non-psychiatric controls. The authors performed analyses across the dorsolateral prefrontal cortex and tested for enrichment of nearby genes and pathways. The authors performed an analysis measuring the effect of age on the epigenomic landscape as well. While this paper provides a unique resource around SCZ and its epigenetic correlates, and some potentially intriguing findings in the antipsychotic response dataset there were some potential missed opportunities - related to the integration of outside datasets and genotypes that could have strengthened the results and novelty of the paper.
Major Comments
1. Is there genotype data available for this cohort of donors or can it be generated? This would open several novel avenues of investigation for the authors. First the authors can test for enrichment of heritability for SCZ or even highly comorbid disorders such as bipolar. Second, it would allow the authors to directly measure the genetic regulation of histone markers by calculating QTLs (in this case histone hQTLs). The authors assert that although interesting, ATAC-seq approach does not provide the same chromatin state information as histone mods mapped by ChiP. Why do the authors not test this? There are several ATAC-seq datasets available for SCZ [https://pubmed.ncbi.nlm.nih.gov/30087329/]and an additional genomic overlap could help tease apart genetic regulation of the changes observed.
2. Can the authors theorize why their analysis found significant effects for H3K27Ac for antipsychotic use when a recent epigenomic study of SCZ using a larger cohort of samples and including the same histone modifications did not [https://pubmed.ncbi.nlm.nih.gov/30038276/]? Given the lower n and lower number of cells in this group, it would be helpful if the authors could speculate on why they see this. Do the authors know if there is any overlap with the Girdhar study donors or if there are other phenotypic differences that could account for this?
3. The reviewer is concerned about the low concordance between bulk nuclei RNA-seq and single-cell RNA-seq for SCZ (236 of 802 DEGs in NeuN+ and 63 of 1043 NEuN-). While it is not surprising for different cohorts to have different sets of DEGs these seem to be vastly different. Was there a particular cell type(s) that enriched for the authors' DEGs in the single-cell dataset? Do the authors know if any donors overlapped between these cohorts?
4. Functional enrichment analyses: details are not provided by the authors and should be added. The authors need to consider a) providing a gene universe, ie only considering the sets of genes with nearby H3K4me3/ H3K27ac levels, to such pathway tools, and b) should take into account the fact that some genes have many more peaks with data. There are known biases in seemingly just using the best p-value per gene in other epigenetic analysis (ie. DNA methylation data) and software is available to run correct analyses: https://pubmed.ncbi.nlm.nih.gov/23732277.
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Reviewer #1 (Public Review):
Summary:<br /> The circuit mechanism underlying the formation of grid cell activity and the organization of grid cells in the medial entorhinal cortex (MEC) is still unclear. To understand the mechanism, the current study investigated synaptic interactions between stellate cells (SC) and PV+ interneurons (IN) in layer 2 of the MEC by combing optogenetic activations and paired patch-clamp recordings. The results convincingly demonstrated highly structured interactions between these neurons: specific and direct excitatory-inhibitory interactions existed at the scale of grid cell phase clusters, and indirect interactions occurred at the scale of grid modules.
Strengths:<br /> Overall, the manuscript is very well written, the approaches used are clever, and the data were thoroughly analyzed. The study conveyed important information for understanding the circuit mechanism that shapes grid cell activity. It is important not only for the field of MEC and grid cells, but also for broader fields of continuous attractor networks and neural circuits.
Weaknesses:<br /> (1) The study largely relies on the fact that ramp-like wide-field optogenetic stimulation and focal optogenetic activation both drove asynchronous action potentials in SCs, and therefore, if a pair of PV+ INs exhibited correlated activity, they should receive common inputs. However, it is unclear what criteria/thresholds were used to determine the level of activity asynchronization, and under these criteria, what percentage of cells actually showed synchronized or less asynchronized activity. A notable percentage of synchronized or less asynchronized SCs could complicate the results, i.e., PV+ INs with correlated activity could receive inputs from different SCs (different inputs), which had synchronized activity. More detailed information/statistics about the asynchronization of SC activity is necessary for interpreting the results.
(2) The hypothesis about the "direct excitatory-inhibitory" synaptic interactions is made based on the GABAzine experiments in Figure 4. In the Figure 8 diagram, the direct interaction is illustrated between PV+ INs and SCs. However, the evidence supporting this "direct interaction" between these two cell types is missing. Is it possible that pyramidal cells are also involved in this interaction? Some pieces of evidence or discussions are necessary to further support the "direction interaction".
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Reviewer #1 (Public Review):
The authors used mathematical models to explore the mechanism(s) underlying the process of polar tube extrusion and the transport of the sporoplasm and nucleus through this structure. They combined this with experimental observations of the structure of the tube during extrusion using serial block face EM providing 3 dimensional data on this process. They also examined the effect of hyperosmolar media on this process to evaluate which model fit the predicted observed behavior of the polar tube in these various media solutions. Overall, this work resulted in the authors arriving at a model of this process that fit the data (model 5, E-OE-PTPV-ExP). This model is consistent with other data in the literature and provides support for the concept that the polar tube functions by eversion (unfolding like a finger of a glove) and that the expanding polar vacuole is part of this process. Finally, the authors provide important new insights into the bucking of the spore wall (and possible cavitation) as providing force for the nucleus to be transported via the polar tube. This is an important observation that has not been in previous models of this process.
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Reviewer #1 (Public Review):
In this study, the authors investigated the role of MAM and the Notch signalling pathway in the onset of the atrophic phenotype in both in vivo and in vitro models. The rationale used to obtain the data is one of the main strengths of the study. Already from the reading, the reasoning scheme used by the authors in setting up the study and evaluating the data obtained is clear. Using both cellular and mouse models in vivo consolidates the data obtained. The authors also methodologically described all the choices made in the supplementary section.
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Joint Public Review:
The biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria is not fully understood, particularly client recognition and insertion by the conserved beta-assembly machinery (BAM), which is itself integrated in the outer membranes. So far, the last strand of an OMP, referred to as the beta-signal, has been known as a primary recognition motif by BAM. Here, authors have identified additional sequence motifs that are located in the upstream of the last strand.
Here, authors carried out rigorous biochemical, biophysical, and genetic approaches to prove that the newly identified internal motifs are critical to the assembly of outer membrane proteins as well as to the interaction with the BAM complex. The identification of important regions on the substrates and Bam proteins during biogenesis is an important contribution that gives clues to the path substrates take en route to the membrane. Assessing the effect of the internal motifs in the assembly of model OMPs in the absence (in vitro) and presence (in vitro and in vivo) of BAM machinery aids a precise definition of the role of the motifs, solidifying the conclusions.
The initial reviews raised several concerns:
1. Strengthening the claim that the recognition of the internal signal by BAM is mediated by BamA and BamD via specific interactions.
2. Justification of the rationale of the peptide inhibition assays as a primary tool to identify significant recognition motifs.
3. More careful interpretation of the mutational effects on OMP assembly - that is, discerning the impairment of BAM-nascent polypeptide chain interaction from the impairment of intrinsic folding.
4. Providing further clarification of the discrepancy between in vitro assay and in vivo assay regarding the effect of the mutation Y286A on OMP assembly.
5. More elaboration on the rationale, interpretation, and representation of neutron refractory data.
6. An explanation is lacking why the strain with BamD R197A does not display VCN sensitivity in contrast to the strain with BamD Y62A.
Those concerns were well addressed in the revised manuscript in a rigorous manner.
Overall, this study comprehensively addresses an important question in the field. The notion that additional signals assist in biogenesis is a novel concept that has been tested and verified at least for a subset of model OMPs in this study. The generalization of the conclusion awaits a further proof of the concept.
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Reviewer #1 (Public Review):
Summary:<br /> This manuscript examined the impact of prenatal alcohol exposure on genome-wide DNA methylation in the brain and liver, comparing ethanol-exposed mice to unexposed controls. They also investigated whether a high-methyl diet (HMD) could prevent the DNA methylation alterations caused by alcohol. Using bisulfite sequencing (n=4 per group), they identified 78 alcohol-associated differentially methylated regions (DMRs) in the brain and 759 DMRs in the liver, of which 85% and 84% were mitigated by the HMD group, respectively. The authors further validated 7 DMRs in humans using previously published data from a Canadian cohort of children with FASD.
Overall, the findings from this study provide new insight into the impact of prenatal alcohol exposure, while also showing evidence for methyl-rich diets as an intervention to prevent the effects of alcohol on the epigenome. However, several methodological concerns limit the robustness of these results and should be addressed to further strengthen the conclusions of this study and its applicability to broader settings.
Strengths:<br /> - The use of whole genome bisulfite sequencing allowed for the interrogation of the entire DNA methylome and DMR analysis, rather than a subset of CpGs.<br /> - The combination of data from animal models and humans allowed the authors to make stronger inferences regarding their findings.<br /> - The authors investigated a potential mechanism (high methyl diet) to buffer against the effects of prenatal alcohol exposure, which increases the relevance and applicability of this research.
Weaknesses:<br /> - Mistakes and discontinuities in the reporting of results and methods made the manuscript difficult to follow. There was also some overuse of causal language and overinterpretation of differences.<br /> - The authors provide insufficient details to replicate their analyses, particularly for data quality control steps and statistical analyses.<br /> - The sample size was very small for the epigenetic analyses, which limits the robustness of the findings. This limitation is further emphasized by the cutoffs used to identify DMRs, which did not include multiple test corrections and used a delta cutoff that was not supported by the sequencing depth.<br /> - The authors do not account for potential confounders in their analyses, including birthweight, alcohol levels, and sex. This is a particular problem for the high-methyl diet analyses, in which the alcohol-exposed mice seemed to consume less alcohol than their non-diet counterparts.
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Reviewer #1 (Public Review):
Summary:<br /> In their manuscript, Massa and colleagues provide a map of the epigenetic landscape in podocytes and analyze the role of the transcription factor MafB in podocyte gene expression. They initially map the histone profile in adult podocytes of the mouse by assaying three different histone methylation marks, namely H3K4me3, H3K4me1, and H3K27me3 for active, primed, and repressed states. They then perform Wt1- and MafB-ChIP-Seq analysis to identify respective direct targets of those transcription factors. Subsequently, they employ an inducible MafB knockout model and show that homozygous knockout mice show proteinuria and FSGS, suggesting an important role for MafB in podocyte homeostasis. RNA-Seq analysis in mice two days after tamoxifen application identified direct and indirect MafB target genes. Finally, the authors turn to a constitutive MafB knockout model, carry out anti-H3K4me3 and anti-Wt1 ChIP experiments, and examine selected promoters. One main conclusion from this work is that MafB opens chromatin and thus facilitates the binding of other transcription factors like Wt1 to podocyte-specific genes.
Strengths and weaknesses:<br /> The authors have performed an impressive number of experiments and generated very valuable data. They use state-of the-art technology and the data are presented well and are sound. This being said the manuscript contains significant novel data, but also experiments that are already available in some sort. The histone profile in adult mouse podocytes is novel and provides an interesting map of epigenetic marks in this particular cell type. It is maybe not too surprising that podocyte-differentiation genes have different chromatin accessibility than genes associated with general development. The Wt1-ChIP has been done before by several labs but is certainly an important control in this work. The MafB-ChIP is new. The inducible MafB knockout model including the identification of Tcf21 as a target gene has been published by others in 2020 (and is acknowledged by the authors). The experiments addressing the potential role of MafB in chromatin opening are new. I find that the data are certainly compatible with the model put forward by the authors, but they are not compelling.
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Reviewer #1 (Public Review):
Summary:<br /> The manuscript titled "Coevolution due to physical interactions is not a major driving force behind evolutionary rate covariation" by Little et al., explores the potential contribution of physical interaction between correlated evolutionary rates among gene pairs. The authors find that physical interaction is not the main driving of evolutionary rate covariation (ECR). This finding is similar to a previous report by Clark et al. (2012), Genome Research, wherein the authors stated that "direct physical interaction is not required to produce ERC." The previous study used 18 Saccharomycotina yeast species, whereas the present study used 332 Saccharomycotina yeast species and 11 outgroup taxa. As a result, the present study is better positioned to evaluate the interplay between physical interaction and ECR more robustly.
Strengths & Weaknesses:<br /> Various analyses nicely support the authors' claims. Accordingly, I have only one significant comment and several minor comments that focus on wordsmithing - e.g., clarifying the interpretation of statistical results and requesting additional citations to support claims in the introduction.
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Reviewer #1 (Public Review):
The study provides a complete comparative interactome analysis of α-arrestin in both humans and drosophila. The authors have presented interactomes of six humans and twelve Drosophila α-arrestins using affinity purification/mass spectrometry (AP/MS). The constructed interactomes helped to find α-arrestins binding partners through common protein motifs. The authors have used bioinformatic tools and experimental data in human cells to identify the roles of TXNIP and ARRDC5: TXNIP-HADC2 interaction and ARRDC5-V-type ATPase interaction. The study reveals the PPI network for α-arrestins and examines the functions of α-arrestins in both humans and Drosophila. The authors have carried out the necessary changes that were suggested.
I would like to congratulate the authors and the corresponding authors of this manuscript for bringing together such an elaborate study on α-arrestin and conducting a comparative study in drosophila and humans.
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Reviewer #1 (Public Review):
Zhou et al. have slightly expanded and improved their web tool from the previous submission, fixing some small issues and adding in additional sets of data from HMDP mice. Essentially, the authors have created a tool that facilitates the integrated analysis of omics datasets (particularly transcriptomics, but could be easily adapted to include proteomics) across tissues.
The strength is that this is new; as far as I know, any other multi-tissue analysis software is relatively ad hoc and it is not easily supported by e.g. SRA/GEO, but rather you'd need to download the multiple datasets and DIY. The authors have now shown some statistically significant (albeit expected from literature) results created using their pipeline. Whether the method will be generally useful for the community depends on its further development and support, but of course whether a project is supported also depends on whether its first publication is accepted - somewhat of a Catch-22 for a reviewer. Right now, the results shown are a convincing proof-of-concept that would likely be of utility mostly to the hosting laboratory and their direct collaborators, but which, with continued development at a similar level of effort, could be more generally useful for the growing number of groups interested in cross-tissue analysis.
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Reviewer #1 (Public Review):
Summary:<br /> The CPC plays multiple essential roles in mitosis such as kinetochore-microtubule attachment regulation, kinetochore assembly, spindle assembly checkpoint activation, anaphase spindle stabilization, cytokinesis, and nuclear envelope formation, as it dynamically changes its mitotic localization: it is enriched at inner centromeres from prophase to metaphase but it is relocalized at the spindle midzone in anaphase. The business end of the CPC is Aurora B and its allosteric activation module IN-box, which is located at the C-terminal part of INCENP. In most well-studied eukaryotic species, Aurora B activity is locally controlled by the localization module of the CPC, Survivin, Borealin, and the N-terminal portion of INCENP. Survivin and Borealin, which bind the N terminus of INCENP, recognize histone residues that are specifically phosphorylated in mitosis, while anaphase spindle midzone localization is supported by the direct microtubule-binding capacity of the SAH (single alpha helix) domain of INCENP and other microtubule-binding proteins that specifically interact with INCENP during anaphase, which are under the regulation of CDK activity. One of these examples includes the kinesin-like protein MKLP2 in vertebrates.
Trypanosoma is an evolutionarily interesting species to study mitosis since its kinetochore and centromere proteins do not show any similarity to other major branches of eukaryotes, while orthologs of Aurora B and INCENP have been identified. Combining molecular genetics, imaging, biochemistry, cross-linking IP-MS (IP-CLMS), and structural modeling, this manuscript reveals that two orphan kinesin-like proteins KIN-A and KIN-B act as localization modules of the CPC in Trypanosoma brucei. The IP-CLMS, AlphaFold2 structural predictions, and domain deletion analysis support the idea that (1) KIN-A and KIN-B form a heterodimer via their coiled-coil domain, (2) Two alpha helices of INCENP interact with the coiled-coil of the KIN-A-KIN-B heterodimer, (3) the conserved KIN-A C-terminal CD1 interacts with the heterodimeric KKT9-KKT11 complex, which is a submodule of the KKT7-KKT8 kinetochore complex unique to Trypanosoma, (4) KIN-A and KIN-B coiled-coil domains and the KKT7-KKT8 complex are required for CPC localization at the centromere, (5) CD1 and CD2 domains of KIN-A support its centromere localization. The authors further show that the ATPase activity of KIN-A is critical for spindle midzone enrichment of the CPC. The imaging data of the KIN-A rigor mutant suggest that dynamic KIN-A-microtubule interaction is required for metaphase alignment of the kinetochores and proliferation. Overall, the study reveals novel pathways of CPC localization regulation via KIN-A and KIN-B by multiple complementary approaches.
Strengths:<br /> The major conclusion is collectively supported by multiple approaches, combining site-specific genome engineering, epistasis analysis of cellular localization, AlphaFold2 structure prediction of protein complexes, IP-CLMS, and biochemical reconstitution (the complex of KKT8, KKT9, KKT11, and KKT12).
Weaknesses:<br /> - The predictions of direct interactions (e.g. INCENP with KIN-A/KIN-B, or KIN-A with KKT9-KKT11) have not yet been confirmed experimentally, e.g. by domain mutagenesis and interaction studies.
- The criteria used to judge a failure of localization are not clearly explained (e.g., Figure 5F, G).
- It remains to be shown that KIN-A has motor activity.
- The authors imply that KIN-A, but not KIN-B, interacts with microtubules based on microtubule pelleting assay (Fig. S6), but the substantial insoluble fractions of 6HIS-KINA and 6HIS-KIN-B make it difficult to conclusively interpret the data. It is possible that these two proteins are not stable unless they form a heterodimer.
- For broader context, some prior findings should be introduced, e.g. on the importance of the microtubule-binding capacity of the INCENP SAH domain and its regulation by mitotic phosphorylation (PMID 8408220, 26175154, 26166576, 28314740, 28314741, 21727193), since KIN-A and KIN-B may substitute for the function of the SAH domain.
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Reviewer #1 (Public Review):
Summary:<br /> In this study, Faniyan and colleagues build on their recent finding that renal Glut2 knockout mice display normal fasting blood glucose levels despite massive glucosuria. Renal Glut2 knockout mice were found to exhibit increased endogenous glucose production along with decreased hepatic metabolites associated with glucose metabolism. Crh mRNA levels were higher in the hypothalamus while circulating ACTH and corticosterone were elevated in this model. While these mice were able to maintain normal fasting glucose levels, ablating afferent renal signals to the brain resulted in substantially lower blood glucose levels compared to wildtype mice. In addition, the higher CRH and higher corticosterone levels of the knockout mice were lost following this denervation. Finally, acute phase proteins were altered, plasma Gpx3 was lower, and major urinary protein MUP18 and its gene expression were higher in renal Glut2 knockout mice. Overall, the main conclusion that afferent signaling from the kidney is required for renal glut2 dependent increases in endogenous glucose production is well supported by these findings.
Strengths:<br /> An important strength of the paper is the novelty of the identification of kidney-to-brain communication as being important for glucose homeostasis. Previous studies had focused on other functions of the kidney modulated by or modulating brain activity. This work is likely to promote interest in CNS pathways that respond to afferent renal signals and the response of the HPA axis to glucosuria. Additional strengths of this paper stem from the use of incisive techniques. Specifically, the authors use isotope-enabled measurement of endogenous glucose production by GC-MS/MS, capsaicin ablation of afferent renal nerves, and multifiber recording from the renal nerve. The authors also paid excellent attention to rigor in the design and performance of these studies. For example, they used appropriate surgical controls, confirmed denervation through renal pelvic CGRP measurement, and avoided the confounding effects of nerve regrowth over time. These factors strengthen confidence in their results. Finally, humans with glucose transporter mutations and those being treated with SGLT2 inhibitors show a compensatory increase in endogenous glucose production. Therefore, this study strengthens the case for using renal Glut2 knockout mice as a model for understanding the physiology of these patients.
Weaknesses:<br /> A few weaknesses exist. Clarification of some aspects of the experimental design would improve the manuscript. However, most concerns relate to the interpretation of this study's findings. The authors state that loss of glucose in urine is sensed as a biological threat based on the HPA axis activation seen in this mouse model. This interpretation is understandable but speculative. Importantly, whether stress hormones mediate the increase in endogenous glucose production in this model and in humans with altered glucose transporter function remains to be demonstrated conclusively. For example, the paper found several other circulating and local factors that could be causal. In addition, the approach used in these studies cannot definitively determine whether renal glucose production or only hepatic glucose production was altered. This model is also unable to shed light on how elevated stress hormones might interact with insulin resistance, which is known to increase endogenous glucose production. That issue is of substantial clinical relevance for patients with T2D and metabolic disease. Finally, while findings from the Glut2 knockout mice are of scientific interest, it should be noted that the Glut2 receptor is critical to the function of pancreatic islets and as such is not a good candidate for pharmacological targeting.
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Reviewer #1 (Public Review):
Summary:<br /> In their study, Petersen et al. investigated the relationship between parameters of metabolic syndrome (MetS) and cortical thickness using partial least-squares correlation analysis (PLS) and performed subsequently a group comparison (sensitivity analysis). To do this, they utilized data from two large-scale population-based cohorts: the UK BioBank (UKB) and the Hamburg City Health Study (HCHS). They identified a latent variable that explained 77% of the shared variance, driven by several measures related to MetS, with obesity-related measures having the strongest contribution. Their results highlighted that higher cortical thickness in the orbitofrontal, lateral prefrontal, insular, anterior cingulate, and temporal areas is associated with lower MetS metric severity. Conversely, the opposite pattern was observed in the superior frontal, parietal, and occipital regions. A similar pattern was then observed in the sensitivity analysis when comparing two groups (MetS vs. matched controls) separately. They then mapped local cellular and network topological attributes to the observed cortical changes associated with MetS. This was achieved using cell-type-specific gene expressions from the Allen Human Brain Atlas and the group consensus functional and structural connectomes of the Human Connectome Project (HCP), respectively. This contextualization analysis allowed them to identify potential cellular contributions in these structures driven by endothelial cells, microglial cells, and excitatory neurons. It also indicated functional and structural interconnectedness of areas experiencing similar MetS effects.
Strengths:<br /> The effects of metabolic syndrome on the brain are still incompletely understood, and such multi-scale analyses are important for the field. Despite the study's sole 'correlation-based' nature, it yields valuable results, including several scales of brain parameters (cortical thickness, cellular, and network-based). The results are robust and benefit from two 'large-scale' datasets, resulting in highly powered statistics.
Weaknesses:<br /> However, some concerns arise regarding certain interpretations and claims made by the authors. In particular, it is not entirely convincing that the authors' results are relevant for studying insulin resistance as a clinical measure of MetS. This is due to the use of non-fasting glycemia as a metric, which the authors claim represents insulin resistance. While non-fasting blood glucose is a potential, albeit poor, indicator of insulin resistance, claiming a direct correlation between insulin resistance and cortical thickness does not seem entirely convincing. By doing so, the authors suggest that insulin resistance might have a weak contribution to cortical thickness abnormalities, with a rather low 'loading' of glycemia compared to the other MetS metrics, although this cannot be conclusively determined from these results.
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Reviewer #1 (Public Review):
Smirnova et al. present a cryo-EM structure of a nucleosome-SIRT6 complex to understand how the histone deacetylase SIRT6 deacetylates the N-terminal tail of histone H3. The authors obtained the structure at sub-4 Å resolution and can visualize how interactions between the nucleosome and SIRT6 position SIRT6 to allow for H3 tail deacetylation. Through additional conformational analysis of their cryo-EM data, they reveal that SIRT6 positioning is flexible on the nucleosome surface, and this could accommodate the targeting of certain H3 tail residues. This work is significant as it represents the visualization of a histone deacetylase on its native nucleosomal target and reveals how substrate specificity is achieved. Importantly, it should be noted that recently two additional structures of the nucleosome-SIRT6 complex were already published. Therefore, Smirnova et al. confirm and complement these previous findings. Additionally, Smirnova et al. expand our understanding of the structural flexibility of SIRT6 on the nucleosome and clarify that SIRT6 also shows histone deacetylase activity on H3K27Ac.
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Reviewer #1 (Public Review):
Summary:<br /> This manuscript explores the impact of serotonin on olfactory coding in the antennal lobe of locusts and odor-evoked behavior. The authors use serotonin injections paired with an odor-evoked palp-opening response assay and bath application of serotonin with intracellular recordings of odor-evoked responses from projection neurons (PNs).
Strengths:<br /> The authors make several interesting observations, including that serotonin enhances behavioral responses to appetitive odors in starved and fed animals, induces spontaneous bursting in PNs, and uniformly enhances PN responses to odors. Overall, I had no technical concerns.
Weaknesses:<br /> While there are several interesting observations, the conclusions that serotonin enhanced sensitivity specifically and that serotonin had feeding-state-specific effects, were not supported by the evidence provided. Furthermore, there were other instances in which much more clarification was needed for me to follow the assumptions being made and inadequate statistical testing was reported.
Major concerns.<br /> -To enhance olfactory sensitivity, the expected results would be that serotonin causes locusts to perceive each odor as being at a relatively higher concentration. The authors recapitulate a classic olfactory behavioral phenomenon where higher odor concentrations evoke weaker responses which is indicative of the odors becoming aversive. If serotonin enhanced the sensitivity to odors, then the dose-response curve should have shifted to the left, resulting in a more pronounced aversion to high odor concentrations. However, the authors show an increase in response magnitude across all odor concentrations. I don't think the authors can claim that serotonin enhances the behavioral sensitivity to odors because the locusts no longer show concentration-dependent aversion. Instead, I think the authors can claim that serotonin induces increased olfactory arousal.
-The authors report that 5-HT causes PNs to change from tonic to bursting and conclude that this stems from a change in excitability. However, excitability tests (such as I/V plots) were not included, so it's difficult to disambiguate excitability changes from changes in synaptic input from other network components.
-There is another explanation for the theoretical discrepancy between physiology and behavior, which is that odor coding is further processing in higher brain regions (ie. Other than the antennal lobe) not studied in the physiological component of this study. This should at least be discussed.
-The authors cannot claim that serotonin underlies a hunger state-dependent modulation, only that serotonin impacts responses to appetitive odors. Serotonin enhanced PORs for starved and fed locusts, so the conclusion would be that serotonin enhances responses regardless of the hunger state. If the authors had antagonized 5-HT receptors and shown that feeding no longer impacts POR, then they could make the claim that serotonin underlies this effect. As it stands, these appear to be two independent phenomena.
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Reviewer #1 (Public Review):
Summary:<br /> 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.
Major Concerns:<br /> 1. An important point that the authors should clarify in this study is whether mice are detecting qualitative or quantitative differences between fresh and old cat saliva. Do the environmental conditions in which the old saliva was maintained cause degradation of Fel d 4, the main protein known for inducing a defensive response in rodents? (see Papes et al, 2010 again). If that is the case, one would expect that a lower concentration of Fel d 4 in the old saliva after protein degradation would result in reduced antipredator responses. Alternatively, if the authors believe that different proteins that are absent in the old saliva are contributing to the increased defensive responses observed with the fresh saliva, further protein quantification experiments should be performed. An important experiment to differentiate qualitative versus quantitative differences between the two types of saliva would be diluting the fresh saliva to verify if the amount of protein, rather than the type of protein, is the main factor regulating the behavioral differences.
2. The authors claim that fresh saliva is recognized as an immediate danger by rodents, whereas old saliva is recognized as a trace of danger. However, the study lacks empirical tests to support this interpretation. With the current experimental tests, the behavioral differences between animals exposed to fresh vs. old saliva could be uniquely due to the reduced amount of the exact same protein (e.g., Fel d 4) in the two samples of saliva.
3. In Figure 4H, the authors state that there were no significant differences in the number of cFos-positive cells between the two saliva-exposed groups. However, this result disagrees with the next result section showing that fresh and old saliva differentially activate the VMH. It is unclear why cFos quantification and behavioral correlations were not performed in other upstream areas that connect the VNO to the VMH (e.g., BNST, MeA, and PMCo). That would provide a better understanding of how brain activity correlates with the different types of behaviors reported with the fresh vs. old saliva.
4. The interpretation that fresh and old saliva activates different subpopulations of neurons in the VMH based on the observation that cFos positively correlates with freezing responses only with the fresh saliva lacks empirical evidence. To address this question, the authors should use two neuronal activity markers to track the response of the same population of VHM cells within the same animals during exposure to fresh vs. old saliva. Alternatively, they could use single-cell electrophysiology or imaging tools to demonstrate that cat saliva of distinct freshness activates different subpopulations of cells in the VMH. Any interpretation without a direct within-subject comparison or the use of cell-type markers would become merely speculative. Furthermore, the authors assume that differential activations of mitral cells between fresh and old saliva result in the differential activation of VMH subpopulations (page 13, line 3). However, there are intermediate structures between the mitral cells and the VMH, which are completely ignored in this study (e.g., BNST, medial amygdala).
5. The authors incorrectly cited the Papes et al., 2010 article on several occasions across the manuscript. In the introduction, the authors cited the Papes et al 2010 study to make reference to the response of rodents to chemical cues, but the Papes et al. study did not use any of the chemical cues listed by the authors (e.g., fox feces, snake skin, cat fur, and cat collars). Instead, the Papes et al. 2010 article used the same chemical cue as the present study: cat saliva. The Papes et al. 2010 article was miscited again in the results section where the authors cited the study to make reference to other sources of cat odor that differ from the cat saliva such as cat fur and cat collars. Because the Papes et al. 2010 article has previously shown the involvement of Trpc2 receptors in the VNO for the detection of cat saliva and the subsequent expression of defensive behaviors by using Trpc2-KO mice, the authors should properly cite this study in the introduction and across the manuscript when making reference to their findings.
6. In the introduction, the authors hypothesized that the VNO detects predator cues and sends sensory signals to the VMH to trigger defensive behavioral decisions and stated that direct evidence to support this hypothesis is still missing. However, the evidence that cat saliva activates the VMH and that activity in the VMH is necessary for the expression of antipredator defensive response in rodents has been previously demonstrated in a study by Engelke et al., 2021 (PMID: 33947849), which was entirely omitted by the authors.
7. In the discussion, the authors stated that their findings suggest that the induction of robust freezing behavior is mediated by a distinct subpopulation of VMH neurons. The authors should cite the study by Kennedy et al., 2020 (PMID: 32939094) that shows the involvement of VMH in the regulation of persistent internal states of fear, which may provide an alternative explanation for why distinct concentrations of saliva could result in different behavioral outcomes.
8. The anatomical connectivity between the olfactory system and the ventromedial hypothalamus (VMH) in the abstract is unclear. The authors should clarify that the VMH does not receive direct inputs from the vomeronasal organ (VNO) nor the accessory olfactory bulb (AOB) as it seems in the current text.
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Reviewer #1 (Public Review):
Summary:<br /> In this study, the authors investigated the mechanisms to repair DSBs induced in euchromatic (Eu) or heterochromatic (Het) contexts in Drosophila. They used a previously described reporter construct that can be used to differentiate between HR, SSA, and mutagenic end joining in response to an I-SceI-induced DSB. Different sub-pathways of end joining (NHEJ, MMEJ, and SD-MMEJ) could be further distinguished by DNA sequence analysis. The main findings of the study are: (1) HR repair is more frequent in Het than in the Eu context; (2) mutagenic EJ repair is more frequent than HR in both contexts; (3) sub-pathways of mutagenic EJ are variable even within the same chromatin domain; and (4) SD-MMEJ repair is associated with larger deletions in the Eu than within the Het compartment.
Strengths:<br /> Overall, the study is well designed and the use of the Bam promoter to drive I-SceI removes some of the variability observed in previous studies. Importantly, the observation of different repair outcomes using the same reporter integrated at different genomic sites suggests that repair is influenced by chromatin state in addition to local DNA sequence context.
Weaknesses:<br /> The main concern I have is the use of only one Eu site versus four for the Het insertions. Given the variability observed between the Het insertions, analysis of a second Eu insertion would give more confidence that the differences observed are significant. One puzzling finding is that HR is increased when the reporter is inserted within the Het domain relative to the Eu domain, suggesting more end resection, yet deletions are smaller for the Het insertions. Bright Ddc2/ATRIP focus formation at DSBs induced in the Het domain is consistent with extensive end resection in this compartment. The authors speculate that this finding could indicate differences in the density of RPA loading or recruitment of Pol theta near ends. I recognize that measuring RPA density on single-stranded DNA would be extremely challenging, but is it known if Pol theta is recruited to DSBs within the Het domain before they move to the periphery?
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Reviewer #1 (Public Review):
The authors sought to resolve the coordinated functions of the two muscles that primarily power flight in birds (supracoracoideus and pectoralis), with particular focus on the pectoralis. Technology has limited the ability to resolve some details of pectoralis function, so the authors developed a model that can make accurate predictions about this muscle's function during flight. The authors first measured aerodynamic forces, wing shape changes, and pectoralis muscle activity in flying doves. They used cutting-edge techniques for the aerodynamic and wing shape measurements and they used well-established methods to measure activity and length of the pectoralis muscle. The authors then developed two mathematical models to estimate the instantaneous force vector produced by the pectoralis throughout the wing stroke. Finally, the authors applied their mathematical models to other-sized birds in order to compare muscle physiology across species.
The strength of the methods is that they smoothly incorporate techniques from many complementary fields to generate a comprehensive model of pectoralis muscle function during flight. The high-speed structured-light technique for quantifying surface area during flight is novel and cutting-edge, as is the aerodynamic force platform used. These methods push the boundaries of what has historically been used to quantify their respective aspects of bird flight and their use here is exciting. The methods used for measuring muscle activation and length are standard in the field. Together, these provide both a strong conceptual foundation for the model and highlight its novelty. This model allows for estimations of muscle function that are not feasible to measure in live birds during flight at present. The weakness of this approach is that it relies heavily on a series of assumptions. While the research presented in this paper makes use of powerful methods from multiple fields, those methods each have assumptions inherent to them that simplify the biological system of study. This reduction in the complexity of phenomena allows specific measurements to be made. In joining the techniques of multiple fields to study greater complexity of the phenomenon of interest, the assumptions are all incorporated also. Furthermore, assumptions are inherent to mathematical modelling of biological phenomena. That being said, the authors acknowledge and justify their assumptions at each step and their model seems to be quite good at predicting muscle function.
Indeed, the authors achieve their aims. They effectively integrate methods from multiple disciplines to explore the coordination and function of the pectoralis and supracoracoideus muscles during flight. The conclusions that the authors derive from their model address the intended research aim.
The authors demonstrate the value of such interdisciplinary research, especially in studying complex behaviors that are difficult or infeasible to measure in living animals. Additionally, this work provides predictions for muscle function that can be tested empirically. These methods are certainly valuable for understanding flight, but also have implications for biologists studying movement and muscle function more generally.
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Reviewer #1 (Public Review):
Summary:<br /> This manuscript builds upon the authors' previous work on the cross-talk between transcription initiation and post-transcriptional events in yeast gene expression. These prior studies identified an mRNA 'imprinting' phenomenon linked to genes activated by the Rap1 transcription factor (TF), a surprising role for the Sfp1 TF in promoting RNA polymerase II (RNAPII) backtracking, and a role for the non-essential RNAPII subunits Rpb4/7 in the regulation of mRNA decay and translation. Here the authors aimed to extend these observations to provide a more coherent picture of the role of Sfp1 in transcription initiation and subsequent steps in gene expression. They provide evidence for (1) a physical interaction between Sfp1 and Rpb4, (2) Sfp1 binding and stabilization of mRNAs derived from genes whose promoters are bound by both Rap1 and Sfp1 and (3) an effect of Sfp1 on Rpb4 binding or conformation during transcription elongation.
Strengths:<br /> This study provides evidence that a TF (yeast Sfp1), in addition to stimulating transcription initiation, can at some target genes interact with their mRNA transcripts and promote their stability. Sfp1 thus has a positive effect on two distinct regulatory steps. Furthermore, evidence is presented indicating that strong Sfp1 mRNA association requires both Rap1 and Sfp1 promoter binding and is increased at a sequence motif near the polyA track of many target mRNAs. Finally, they provide compelling evidence that Sfp1-bound mRNAs have higher levels of RNAPII backtracking and altered Rpb4 association or conformation compared to those not bound by Sfp1.
Weaknesses:<br /> The Sfp1-Rpb4 association is supported only by a two-hybrid assay that is poorly described and lacks an important control. Furthermore, there is no evidence that this interaction is direct, nor are the interaction domains on either protein identified (or mutated to address function).
The contention that Sfp1 nuclear export to the cytoplasm is transcription-dependent is not well supported by the experiments shown, which are not properly described in the text and are not accompanied by any primary data.<br /> The presence of Sfp1 in P-bodies is of unclear relevance and the authors do not ask whether Sfp1-bound mRNAs are also present in these condensates.
Further analysis of Sfp1-bound mRNAs would be of interest, particularly to address the question of whether those from ribosomal protein genes and other growth-related genes that are known to display Sfp1 binding in their promoters are regulated (either stabilized or destabilized) by Sfp1.
The authors need to discuss, and ideally address, the apparent paradox that their previous findings showed that Rap1 acts to destabilize its downstream transcripts, i.e. that it has the opposite effect of Sfp1 shown here.
Finally, recent studies indicate that the drugs used here to measure mRNA stability induce a strong stress response accompanied by rapid and complex effects on transcription. Their relevance to mRNA stability in unstressed cells is questionable.
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Reviewer #1 (Public Review):
Li et al. report here on the expression of a G-protein subunit Gng13 in ectopic tuft cells that develop after severe pulmonary injury in mice. By deleting this gene in ectopic tuft cells as they arise, the authors observed worsened lung injury and greater inflammation after influenza infection, as well as a decrease in the overall number of ectopic tuft cells. This was in stark contrast to the deletion of Trpm5, a cation channel generally thought to be required for all functional gustatory signaling in tuft cells, where no phenotype is observed. Strengths here include a thorough assessment of lung injury via a number of different techniques. Weaknesses are notable: confusingly, these findings are at odds with reports from other groups demonstrating no obvious phenotype upon influenza infection in mice lacking the transcription factor Pou2f3, which is essential for all tuft cell specification and development. The authors speculate that heterogeneity within nascent tuft cell populations, specifically the presence of pro- and anti-inflammatory tuft cells, may explain this difference, but they do not provide any data to support this idea.
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Reviewer #1 (Public Review):
Summary:<br /> In this paper, Song, Shi, and Lin use an existing deep learning-based sequence model to derive a score for each haplotype within a genomic region, and then perform association tests between these scores and phenotypes of interest. The authors then perform some downstream analyses (fine-mapping, various enrichment analyses, and building polygenic scores) to ensure that these associations are meaningful. The authors find that their approach allows them to find additional associations, the associations have biologically interpretable enrichments in terms of tissues and pathways, and can slightly improve polygenic scores when combined with standard SNP-based PRS.
Strengths:<br /> - I found the central idea of the paper to be conceptually straightforward and an appealing way to use the power of sequence models in an association testing framework.<br /> - The findings are largely biologically interpretable, and it seems like this could be a promising approach to boost power for some downstream applications.
Weaknesses:<br /> - The methods used to generate polygenic scores were difficult to follow. In particular, a fully connected neural network with linear activations predicting a single output should be equivalent to linear regression (all intermediate layers of the network can be collapsed using matrix-multiplication, so the output is just the inner product of the input with some vector). Using the last hidden layer of such a network for downstream tasks should also be equivalent to projecting the input down to a lower dimensional space with some essentially randomly chosen projection. As such, I am surprised that the neural network approach performs so well, and it would be nice if the authors could compare it to other linear approaches (e.g., LASSO or ridge regression for prediction; PCA or an auto-encoder for converting the input to a lower dimensional representation).
- A very interesting point of the paper was the low R^2 between the HFS scores in adjacent windows, but the explanation of this was unclear to me. Since the HFS scores are just deterministic functions of the SNPs, it feels like if the SNPs are in LD then the HFS scores should be and vice versa. It would be nice to compare the LD between adjacent windows to the average LD of pairs of SNPs from the two windows to see if this is driven by the fact that SNPs are being separated into windows, or if sei is somehow upweighting the importance of SNPs that are less linked to other SNPs (e.g., rare variants).
- There were also a number of robustness checks that would have been good to include in the paper. For instance, do the findings change if the windows are shifted? Do the findings change if the sequence is reverse-complemented?
- It was also difficult to contextualize the present work in terms of recent results showing that sequence models tend to not do very well at predicting cross-individual expression changes (and such results presumably hold for predicting cross-individual chromatin changes). In particular, it would be good for the authors to contrast their findings with the work of Alex Sasse and colleagues (https://www.biorxiv.org/content/10.1101/2023.03.16.532969.abstract) and Connie Huang and colleagues (https://www.biorxiv.org/content/10.1101/2023.06.30.547100.abstract).
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Reviewer #1 (Public Review):
Summary:<br /> Shakhawat et al., investigated how enhancement of plasticity and impairment could result in the same behavioral phenotype. The authors tested the hypothesis that learning impairments result from saturation of plasticity mechanisms and had previously tested this hypothesis using mice lacking two class I major histocompatibility molecules. The current study extends this work by testing the saturation hypothesis in a Purkinje-cell (L7) specific Fmr1 knockout mouse mice, which have enhanced parallel fiber-Purkinje cell LTD. The authors found that L7-Fmr1 knockout mice are impaired on an oculomotor learning task and both pre-training, to reverse LTD, and diazepam, to suppress neural activity, eliminated the deficit when compared to controls.
Strengths:
This study tests the "saturation hypothesis" to understand plasticity in learning using a well-known behavior task, VOR, and an additional genetic mouse line with a cerebellar cell-specific target, L7-Fmr1 KO. This hypothesis is of interest to the community as it evokes a novel inquisition into LTD that has not been examined previously.
Utilizing a cell-specific mouse line that has been previously used as a genetic model to study Fragile X syndrome is a unique way to study the role of Purkinje cells and the Fmr1 gene. This increases the understanding in the field in regards to Fragile X syndrome and LTD.
The VOR task is a classic behavior task that is well understood, therefore using this metric is very reliable for testing new animal models and treatment strategies. The effects of pretraining are clearly robust and this analysis technique could be applied across different behavior data sets.
The rescue shown using diazepam is very interesting as this is a therapeutic that could be used in clinical populations as it is already approved.
There was a proper use of controls and all animal information was described. The statistical analysis and figures are clear and well describe the results.
Weaknesses:<br /> While the proposed hypothesis is tested using genetic animal models and the VOR task, LTD itself is not measured. This study would have benefited from a direct analysis of LTD in the cerebellar cortex in the proposed circuits.
Diazepam was shown to rescue learning in L7-Fmr1 KO mice, but this drug is a benzodiazepine and can cause a physical dependence. While the concentrations used in this study were quite low and animals were dosed acutely, potential side-effects of the drug were not examined, including any possible withdrawal. This drug is not specific to Purkinje cells or cerebellar circuits, so the action of the drug on cerebellar circuitry is not well understood for the study presented.
It was not mentioned if L7-Fmr1 KO mice have behavior impairments that worsen with age or if Purkinje cells and the cerebellar microcircuit are intact throughout the lifespan. Connections between Purkinje cells and interneurons could also influence the behavior results found.
While males and females were both used for the current study, only 7 of each sex were analyzed, which could be underpowered. While it might be justified to combine sexes for this particular study, it would be worth understanding this model in more detail.
Training was only shown up to 30 minutes and learning did not seem to plateau in most cases. What would happen if training continued beyond the 30 minutes? Would L7-Fmr1 KO mice catch-up to WT littermates?
The pathway discussed as the main focus for VOR in this learning paradigm was connections between parallel fibers (PF) and Purkinje cells, but the possibility of other local or downstream circuitry being involved was not discussed. PF-Purkinje cell circuits were not directly analyzed, which makes this claim difficult to assess.
The authors mostly achieved their aim and the results support their conclusion and proposed hypothesis. This work will be impactful on the field as it uses a new Purkinje-cell specific mouse model to study a classic cerebellar task. The use of diazepam could be further analyzed in other genetic models of neurodevelopmental disorders to understand if effects on LTD can rescue other pathways and behavior outcomes.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Direction selectivity (DS) in the visual system is first observed in the radiating dendrites of starburst amacrine cells (SACs). Studies over the last two decades have aimed to understand the mechanisms that underlie these unique properties. Most recently, a 'space-time' model has garnered special attention. This model is based on two fundamental features of the circuit. First, distinct anatomical types of bipolar cells (BCs) are connected to proximal/distal regions of each of the SAC dendritic sectors (Kim et al., 2014). Second, that input across the length of the starburst is kinetically diverse, a hypothesis that has only recently gained some experimental support using iGluSnFR imaging (Srivastava et al., 2022). However, in these prior studies, the sustained/transient distinctions in BC input that are proposed to underlie direction selectivity were shown to be present mainly in responses to stationary stimuli. When BC receptive field properties are probed using white noise stimuli, the kinetic differences between proximal/distal BC input are relatively subtle or nonexistent (Gaynes et al., 2022; Strauss et al., 2022, Srivastava et al., 2022). Thus, if and how BCs contribute to direction selectivity driven by moving spots that are commonly used to probe the circuit remains to be clarified. To address this issue, Gaynes et al., combine evolutionary computational modeling (Ankri et al., 2020) with two-photon iGluSnFR imaging to address to what degree BCs contribute to the generation of direction selectivity in the starburst dendrites.
Strengths:
Combining theoretical models and iGluSnFR imaging is a powerful approach as it first provides a basic intuition on what is required for the generation of robust DS, and then tests the extent to which the experimentally measured BC output meets these requirements.
The conclusion of this study builds on the previous literature and comprehensively considers the diverse BC receptive field properties that may contribute to DS (e.g. size, lag, rise time, decay time).
By 'evolving' bipolar inputs to produce robust DS in a model network, these authors provide a sound framework for understanding which kinetic properties could potentially be important for driving downstream DS. They suggest that response delay/decay kinetics, rather than the center/surround dynamics are likely to be most relevant (albeit the latter could generate asymmetric responses to radiating/looming stimuli).
Weaknesses:
Finally, these authors report that the experimentally measured BC responses are far from optimal for generating DS. Thus, the BC-based DS mechanism does not appear to explain the robust DS observed experimentally (even with mutual inhibition blocked). Nevertheless, I feel the comprehensive description of BC kinetics and the solid assessment of the extent to which they may shape DS in SAC dendrites, is a significant advancement in the field.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary: The current study examines the necessity of estrogen receptor alpha (ESR1) in GABA neurons located in the anteroventral and preoptic periventricular nuclei and the medial preoptic nucleus of hypothalamus. This brain area is implicated in regulating the pre-ovulatory LH surge in females, but the identity of the estrogen-sensitive neurons that are required remains unknown. The data indicate that approximately 70% knockdown of ESR1 in GABA neurons resulted in variable reproductive phenotypes. However, when the ESR1 knockdown also results in a decrease in kisspeptin expression by these cells, the females had disrupted LH surges, but no alterations in pulsatile LH release. These data support the hypothesis that kisspeptin cells in this region are critical for the pre-ovulatory LH surge in females.
Strengths: The current study examined the efficacy of two guide RNAs to knockdown ESR1 in GABA neurons, resulting in an approximate 70% reduction in ESR1 in GABA neurons. The efficacy of this knockdown was confirmed in the brain via immunohistochemistry and the reproductive outcomes were analyzed several ways to account for differences in guide RNAs or the precise brain region with the ESR1 knockdown. The analysis was taken one step further by grouping mice based on kisspeptin expression following ESR1 knockdown and examining the reproductive phenotypes. Overall, the aims of the study were achieved, the methods were appropriate, and the data were analyzed extensively. This data supports the hypothesis that kisspeptin neurons in the anterior hypothalamus are critical for the preovulatory LH surge.
Weaknesses: One minor weakness in this study is the conclusion that the two different guide RNAs didn't seem to have unique effects on GnRH cFos expression or the reproductive phenotypes. Though the data indicate a 60-70% knockdown for both gRNA2 and gRNA3, 3 of the 4 gRNA2 mice had no cFos expression in GnRH neurons during the time of the LH surge, whereas all mice receiving gRNA3 had at least some cFos/GnRH co-expression. In addition, when mice were re-categorized based on reduction (>75%) in kisspeptin expression, most of the mice in the unilateral or bilateral groups received gRNA2, whereas many of the mice that received gRNA3 were in the "normal" group with no disruption in kisspeptin expression. Whether these results occurred by chance or due to differences in the gRNAs remains unknown. Thus, additional experiments with increased sample sizes would be needed, even if the efficacy of the ESR1 knockdown was comparable, before concluding these 2 gRNAs don't have unique actions.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary: The authors set out to clarify the molecular mechanism of endocytosis (re-uptake) of synaptic vesicle (SV) membrane in the presynaptic terminal following release. They have examined the role of presynaptic actin, and of the actin regulatory proteins diaphanous-related formins ( mDia1/3), and Rho and Rac GTPases in controlling the endocytosis. They successfully show that presynaptic membrane-associated actin is required for normal SV endocytosis in the presynaptic terminal and that the rate of endocytosis is increased by activation of mDia1/3. They show that RhoA activity and Rac1 activity act in a partially redundant and synergistic fashion together with mDia1/3 to regulate the rate of SV endocytosis. The work adds substantially to our understanding of the molecular mechanisms of SV endocytosis in the presynaptic terminal.
Strengths: The authors use state-of-the-art optical recording of presynaptic endocytosis in primary hippocampal neurons, combined with well-executed genetic and pharmacological perturbations to document effects of alteration of actin polymerization on the rate of SV endocytosis. They show that removal of the short amino-terminal portion of mDia1 that associates with the membrane interrupts the association of mDia1 with membrane actin in the presynaptic terminal. They then use a wide variety of controlled perturbations, including genetic modification of the amount of mDia1/3 by knock-down and knockout, combined with inhibition of activity of RhoA and Rac1 by pharmacological agents, to document the quantitative importance of each agent and their synergistic relationship in regulation of endocytosis.<br /> The analysis is augmented by ultrastructural analyses that demonstrate the quantitative changes in numbers of synaptic vesicles and in uncoated membrane invaginations that are predicted by the optical recordings.<br /> The manuscript is well-written and the data are clearly explained. Statistical analysis of the data is strengthened by the very large number of data points analyzed for each experiment.
Weaknesses: There are no major weaknesses. The optical images as first presented are small and it is recommended that the authors provide larger, higher-resolution images.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> O'Leary and colleagues present data identifying several procedures that alter discrimination between novel and familiar objects, including time, environmental enrichment, Rac-1, context reexposure, and brief reminders of the familiar object. This is complimented with an engram approach to quantify cells that are active during learning to examine how their activation is impacted following each of the above procedures at test. With this behavioral data, authors apply a modeling approach to understand the factors that contribute to good and poor object memory recall.
Strengths:<br /> • Authors systematically test several factors that contribute to poor discrimination between novel and familiar objects. These results are extremely interesting and outline essential boundaries of incidental, nonaversive memory.<br /> • These results are further supported by engram-focused approaches to examine engram cells that are reactivated in states with poor and good object recognition recall.
Weaknesses:<br /> • For the environmental enrichment, authors seem to suggest objects in the homecage are similar to (or reminiscent of) the familiar object. Thus, the effect of improved memory may not be related to enrichment per se as much as it may be related to the preservation of an object's memory through multiple retrievals, not the enriching experiences of the environment itself. This would be consistent with the brief retrieval figure. Authors should include a more thorough discussion of this.
• Authors should justify the marginally increased number of engram cells in the non-enrichment group that did not show object discrimination at test, especially relative to other figures. More specific cell counting criteria may be helpful for this. For example, was the DG region counted for engram and cfos cells or only a subsection?
• It is unclear why the authors chose a reactivation time point of 1hr prior to testing. While this may be outside of the effective time window for pharmacological interference with reconsolidation for most compounds, it is not necessarily outside of the structural and functional neuronal changes accompanied by reconsolidation-related manipulations.
• Figure 5: Levels of exploration at test are inconsistent between manipulations. This is problematic, as context-only reexposures seem to increase exploration for objects overall in a manner that I'm unsure resembles 'forgetting'. Instead, cross-group comparisons would likely reveal increased exploration time for familiar and novel objects. While I understand 'forgetting' may be accompanied by greater exploration towards objects, this is inconsistent across and within the same figure. Further, this effect is within the period of time that rodents should show intact recognition. Instead, context-only exposures may form a competing (empty context) memory for the familiar object in that particular context.
• I am concerned at the interpretation that a memory is 'forgotten' across figures, especially considering the brief reminder experiments. Typically, if a reminder session can trigger the original memory or there is rapid reacquisition, then this implies there is some savings for the original content of the memory. For instance, multiple context retrievals in the absence of an object reminder may be more consistent with procedures that create a distinct memory and subsequently recruit a distinct engram.
• Authors state that spine density decreases over time. While that may be generally true, there is no evidence that mature mushroom spines are altered or that this is consistent across figures. Additionally, it's unclear if spine volume is consistently reduced in reactivated and non-reactivated engram cells across groups. This would provide evidence that there is a functionally distinct aspect of engram cells that is altered consistently in procedures resulting in poor recognition memory (e.g. increased spine density relative to spine density of non-reactivated engram cells and non-engram cells)
• Authors should discuss how the enrichment-neurogenesis results here are compatible with other neurogenesis work that supports forgetting.
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www.researchsquare.com www.researchsquare.com
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Reviewer #1 (Public Review):
This manuscript provides an important case study for in-depth research on the adaptability of vertebrates in deep-sea environments. Through analysis of the genomic data of the hadal snailfish, the authors found that this species may have entered and fully adapted to extreme environments only in the last few million years. Additionally, the study revealed the adaptive features of hadal snailfish in terms of perceptions, circadian rhythms and metabolisms, and the role of ferritin in high-hydrostatic pressure adaptation. Besides, the reads mapping method used to identify events such as gene loss and duplication avoids false positives caused by genome assembly and annotation. This ensures the reliability of the results presented in this manuscript. Overall, these findings provide important clues for a better understanding of deep-sea ecosystems and vertebrate evolution.
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Reviewer #1 (Public Review):
Summary:<br /> This valuable study analyzes the contribution of fungal and bacterial microbiota species to the growth and development of Drosophila. The authors use bacterial and fungal species associated with Drosophila in the wild to analyze their respective contributions in promoting larval growth in a decaying banana, mimicking the natural niche of fruit fly. They found that some fungal species and some fungus/bacteria combinations effectively promote growth by supplementing key branched amino acids in the food substratum. Production of these amino acids by Drosophila itself is not sufficient, and only fungal species that secrete these amino acids into the medium can sustain Drosophila growth. Thus, the authors clarify how facultative symbionts contribute to host growth in a natural setting by changing the food substratum in a dynamic manner.
Strengths:<br /> The natural setting developed by the authors to analyze the impact of the microbiota is clearly valuable, as is the focus on the role of fungal microbiota species. This complements studies of Drosophila microbiota that have previously focused on bacterial species and used a lab setting.
While there has been an extensive focus on obligate endosymbionts or gut symbionts, this study analyzes how facultative symbionts shape the food substratum and influence host growth.<br /> A last strength of this study is that it analyzes the contribution of Drosophila microbiota over a dynamic timeframe, analyzing how microbial species change in decaying fruit over time.
Weaknesses:<br /> 1) The author should better review what we know of fungal Drosophila microbiota species as well as the ecology of rotting fruit. Are the microbiota species described in this article specific to their location/setting? It would have been interesting to know if similar species can be retrieved in other locations using other decaying fruits. The term 'core' in the title suggests that these species are generally found associated with Drosophila but this is not demonstrated. The paper is written in a way that implies the microbiota members they have found are universal. What is the evidence for this? Have the fungal species described in this paper been found in other studies? Even if this is not the case, the paper is interesting, but there should be a discussion of how generalizable the findings are.
2) Can the author clearly demonstrate that the microbiota species that develop in the banana trap are derived from flies? Are these species found in flies in the wild? Did the authors check that the flies belong to the D. melanogaster species and not to the sister group D. simulans?
3) Did the microarrays highlight a change in immune genes (ex. antibacterial peptide genes)? Whatever the answer, this would be worth mentioning. The authors described their microarray data in terms of fed/starved in relation to the Finke article. This is fine they should clarify if they observed significant differences between species (differences between species within bacteria or fungi, and more generally differences between bacteria versus fungi).
4) The whole paper - and this is one of its merits - points to a role of the Drosophila larval microbiota in processing the fly food. Are these bacterial and fungal species found in the gut of larvae/adults? Are these species capable of establishing a niche in the cardia of adults as shown recently in by the Ludington lab (Dodge et al.,)? Previous studies have suggested that microbiota members stimulate the Imd pathway leading to an increase in digestive proteases (Erkosar/Leulier). Are the microbiota species studied here affecting gut signaling pathways beyond providing branched amino acids?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study explores the relationship between guanine-quadruplex (G4) structures and pathogenicity islands (PAIs) in 89 pathogenic strains. G4 structures were found to be non-randomly distributed within PAIs and conserved within the same strains. Positive correlations were observed between G4s and GC content across various genomic features, suggesting a link between G4 structures and GC-rich regions. Differences in GC content between PAIs and the core genome underscored the unique nature of PAIs. High-confidence G4 structures in Escherichia coli's regulatory regions were identified, influencing DNA integration within PAIs. These findings shed light on the molecular mechanisms of G4-PAI interactions, enhancing our understanding of bacterial pathogenicity and G4 structures in infectious diseases.
Strengths:
The findings of this study hold significant implications for our understanding of bacterial pathogenicity and the role of guanine-quadruplex (G4) structures.
Molecular Mechanisms of Pathogenicity: The study highlights that G4 structures are not randomly distributed within pathogenicity islands (PAIs), suggesting a potential role in regulating pathogenicity. This insight into the uneven distribution of G4s within PAIs provides a basis for further research into the molecular mechanisms underlying bacterial pathogenicity.
Conservation of G4 Structures: The consistent conservation of G4 structures within the same pathogenic strains suggests that these structures might play a vital and possibly conserved role in the pathogenicity of these bacteria. This finding opens doors for exploring how G4s influence virulence across different pathogens.
Unique Nature of PAIs: The differences in GC content between PAIs and the core genome underscore the unique nature of PAIs. This distinction suggests that factors such as DNA topology and G4 structures might contribute to the specialized functions and characteristics of PAIs, which are often associated with virulence genes.
Regulatory Role of G4s: The identification of high-confidence G4 structures within regulatory regions of Escherichia coli implies that these structures could influence the efficiency or specificity of DNA integration events within PAIs. This finding provides a potential mechanism by which G4s can impact the pathogenicity of bacteria.
Weaknesses:
No weaknesses were identified by this reviewer.
Overall, the study provides fundamental insights into the pathogenicity island and conservation of G4 motifs.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this study, the authors examined the role of IBTK, a substrate-binding adaptor of the CRL3 ubiquitin ligase complex, in modulating the activity of the eiF4F translation initiation complex. They find that IBTK mediates the non-degradative ubiquitination of eiF4A1, promotes cap-dependent translational initiation, nascent protein synthesis, oncogene expression, and tumor cell growth. Correspondingly, phosphorylation of IBTK by mTORC1/ S6K1 increases eIF4A1 ubiquitination and sustains oncogenic translation.
Strengths:
This study utilizes multiple biochemical, proteomic, functional, and cell biology assays to substantiate their results. Importantly, the work nominates IBTK as a unique substrate of mTORC1, and further validates eiF4A1 ( a crucial subunit of the ei44F complex) as a promising therapeutic target in cancer. Since IBTK interacts broadly with multiple members of the translational initial complex - it will be interesting to examine its role in eiF2alpha-mediated ER stress as well as eiF3-mediated translation. Additionally, since IBTK exerts pro-survival effects in multiple cell types, it will be of relevance to characterize the role of IBTK in mediating increased mTORC1 mediated translation in other tumor types, thus potentially impacting their treatment with eiF4F inhibitors.
Limitations/Weaknesses:
The findings are mostly well supported by data, but some areas need clarification and could potentially be enhanced with further experiments:
1) Since eiF4A1 appears to function downstream of IBTK1, can the effects of IBTK1 KO/KD in reducing puromycin incorporation (in Fig 3A), cap-dependent luciferase reporter activity (Fig 3G), reduced oncogene expression ( Fig 4A) or 2D growth/ invasion assays (Fig 4) be overcome or bypassed by overexpressing eiF4A1? These could potentially be tested in future studies.
2) The decrease in nascent protein synthesis in puromycin incorporation assays in Figure 3A suggest that the effects of IBTK KO are comparable to and additive with silvesterol. It would be of interest to examine whether silvesterol decreases nascent protein synthesis or increases stress granules in the IBTK KO cells stably expressing IBTK as well.
3) The data presented in Figure 5 regarding the role of mTORC1 in IBTK-mediated eiF4A1 ubiquitination needs further clarification on several points:
- It is not clear if the experiments in Figure 5F with Phos-tag gels are using the FLAG-IBTK deletion mutant or the peptide containing the mTOR sites as it is mentioned on line 517, page 19 "To do so, we generated an IBTK deletion mutant (900-1150 aa) spanning the potential mTORC1-regulated phosphorylation sites" This needs further clarification.
-It may be of benefit to repeat the Phos tag experiments with full-length FLAG-IBTK and/or endogenous IBTK with molecular weight markers indicating the size of migrated bands.
-Additionally, torin or Lambda phosphatase treatment may be used to confirm the specificity of the band in separate experiments.
-Phos-tag gels with the IBTK CRISPR KO line would also help confirm that the non-phosphorylated band is indeed IBTK.
-It is unclear why the lower, phosphorylated bands seem to be increasing (rather than decreasing) with AA starvation/ Rapa in Fig 5H.
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Reviewer #1 (Public Review):
The manuscript investigates the binding of PHD-BD, a tandem of reader domains in the C-terminus of BPTF, to modified histone tail peptides and nucleosomes. It focuses on the differences in binding affinity between peptide and nucleosome substrates for BPTF PHD-BD. Using the dCypher approach, they find that multi-modified peptide substrates (both acetylation and methylation) do not increase PHD-BD binding affinity. They argue that histone peptide substrates do not support the histone code model, which champions that multivalent engagement by PHD-BD with a multi-modified substrate would lead to stronger binding when compared to the engagement of each domain alone. In contrast, when using nucleosome substrates, even though the overall affinity is reduced, the affinity for H3K4me3triac (double modification) is tighter than either modification on its own. This is consistent with the histone code model.
A strength of the manuscript is that it further delineates the contribution of each domain by again using dCypher to compare peptide and nucleosome binding of the PHD and BD domains alone, as well as tandem domain constructs where each domain has been inactivated by a point mutation (W2891A for the PHD and N3007A for the BD). PHD alone had a lower affinity for nucleosomes than peptides overall. With peptide substrates, PHD had the highest affinity for H3K4me3 and reduced affinity for H3K4me3triac; while with nucleosomes this trend was reversed. BD alone showed an affinity for acetylated H3 and H4 peptides but surprisingly was unable to bind nucleosomes. PHD requires the combination of H3K4 methylation and H3 tail acetylation for binding, and when partnered with BD, which is not able to bind nucleosomes alone, interestingly confers specificity for K14ac and K18ac. The in vivo relevance is argued using CUT&RUN analysis.
NMR spectroscopy is further used to show that PHD-BD binds acetylated H3 in a multivalent manner while forming a unique complex with H3K4me3triac. Deleting the N-terminal A1 region of H3 abolishes the binding of PHD-BD, implying its importance for recognition. The authors also discuss a "fuzzy complex" that forms between H3 and DNA, as well as H4 and DNA, which explains the occlusion of histone tail accessibility in the nucleosome. By changing the sidechain charge, such as with PTMs, this interaction can be weakened and allow PHD in this case to bind to the modified H3 tail. Comparisons between spectra of the H4 tail, H4 tail with DNA, and the H4 tail in the nucleosome are made and used to argue for H4-DNA interactions in the nucleosome.
The conclusions of the manuscript are very well-supported by the data and reveal a lot of insight into how the two reader domains of BPTF interact with modified nucleosomes. In many places, however, the manuscript is written more generally as if the conclusions apply in all cases (e.g. the title, abstract, and introduction) and this remains to be determined. It is also overstated that there is a belief that peptides perfectly recapitulate nucleosomes. It should also be pointed out that the nucleosomes are multi-valent and the data cannot discriminate binding of a single PHD-BD to single or multiple tails, and that the work is limited as it is using a construct of BPTF and in fact, there is at least one other reader domain involved.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This paper combines an array of techniques to study the role of cholecystokinin (CCK) in motor learning. Motor learning in a pellet reaching task is shown to depend on CCK, as both global and locally targeted CCK manipulations eliminate learning. This learning deficit is linked to reduced plasticity in the motor cortex, evidenced by both slice recordings and two-photon calcium imaging. Furthermore, CCK receptor agonists are shown to rescue motor cortex plasticity and learning in knockout mice. While the behavioral results are clear, the specific effects on learning are not directly tested, nor is the specificity pathway between rhinal CCK neurons and the motor cortex. In general, the results present interesting clues about the role of CCK in motor learning, though the specificity of the claims is not fully supported.
Since all CCK manipulations were performed throughout learning, rather than after learning, it is not clear whether it is learning that is affected or if there is a more general motor deficit. Related to this point, Figure 1D appears to show a general reduction in reach distance in CCK-/- mice. A general motor deficit may be expected to produce decreased success on training day 1, which does not appear to be the case in Figure 1C and Figure 2B, but may be present to some degree in Figure 5B. Or, since the task is so difficult on day 1, a general motor deficit may not be observable. It is therefore inconclusive whether the behavioral effect is learning-specific.
The paper implicates motor cortex-projecting CCK neurons in the rhinal cortex as being a key component in motor learning. However, the relative importance of this pathway in motor learning is not pinned down. The necessity of CCK in the motor cortex is tested by injecting CCK receptor antagonists into the contralateral motor cortex (Figure 2), though a control brain region is not tested (e.g. the ipsilateral motor cortex), so the specificity of the motor cortex is not demonstrated. The learning-related source of CCK in the motor cortex is also unclear, since even though it is demonstrated that CCK neurons in the rhinal cortex project to the motor cortex in Figure 4D, Figure 4C shows that there is also a high concentration of CCK neurons locally within the motor cortex. Likewise, the importance of the projection from the rhinal cortex to the motor cortex is not specifically tested, as rhinal CCK neurons targeted for inactivation in Figure 5 include all CCK cells rather than motor cortex-projecting cells specifically.
CCK is suggested to play a role in producing reliable activity in the motor cortex through learning through two-photon imaging experiments. This is useful in demonstrating what looks like normal motor cortex activity in the presence of CCK receptor antagonist, indicating that the manipulations in Figure 2 are not merely shutting off the motor cortex. It is also notable that, as the paper points out, the activity appears less variable in the CCK manipulations (Figure 3G). However, this could be due to CCK manipulation mice having less-variable movements throughout training. The Hausdorff distance is used for quantification against this point in Figure 1E, though the use of the single largest distance between trajectories seems unlikely to give a robust measure of trajectory similarity, which is reinforced by the CCK-/- traces looking much less variable than WT traces in Figure 1D. The activity effects may therefore be expected from a general motor deficit if that deficit prevented the mice from normal exploratory movements and restricted the movement (and activity) to a consistently unsuccessful pattern.
Finally, slice experiments are used to demonstrate the lack of LTP in the motor cortex following CCK knockout, which is rescued by CCK receptor agonists. This is a nice experiment with a clear result, though it is unclear why there are such striking short-term depression effects from high-frequency stimulation observed in Figure 6A that are not observed in Figure 1H. Also, relating to the specificity of the proposed rhinal-motor pathway, these experiments do not demonstrate the source of CCK in the motor cortex, which may for example originate locally.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This is a review of the manuscript entitled "Pharmacologic hyperstabilisation of the HIV-1 capsid lattice induces capsid failure" by Faysal et al., in this manuscript the authors used an elegant single virion fluorescence assay based on TIRF to measure the stability of mature HIV cores. Virions were biotinylated and captured onto glass coverslips through specific Biotin-Avidin interactions. Immobilized virions were then introduced to the imaging buffer which contained the pore-forming protein DLY, and fluorescently labeled CypA. Mature virions were identified through the binding of CypA which had a red fluorescent tag allowing them to measure the dynamics of GFP trapped within the mature cores as well as the CypA bound outside the core. The authors show that the addition of LEN starting from about 50nM stabilized the mature cores even after cores have ruptured and released their internal GFP. Higher concentration of Len results in ultrastabilization of the cores and rapid rupture leading to the release of GFP at an earlier timepoint. A biochemical assembly assay was performed which showed uM quantities of Len synergized with IP6 to promote CA assembly. Purified mature virions were also treated with 700nM of Len and analyzed by CryoET, this analysis showed an increased representation of irregular cores within the Len-treated sample. Putting all of this together, the authors concluded that Len facilitates core rupture through hyperstabilization of HIV cores, as described in the title.
While I have found this work technically well performed and well explained, I do not believe that the presented data supports the conclusions reached by the authors.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Through a series of psychophysical experiments, Merkel et al examined the process of feature-based resource allocation during parallel feature value tracking, where subjects are asked to simultaneously track changing but spatially inseparable color streams. They find that tracking precision is highly imbalanced between streams, and the tracking precision changes over time by alternating between streams at a rate of ~1Hz.
Strengths:
The study addresses an intriguing research question that fills a gap in existing literature, and was carefully designed and well-executed, with a series of experiments and control experiments.
Weaknesses:
1. My main concern is the null effect of precision estimation pattern between cued and un-cued trials. It is well established that relative to the un-cued stimuli, the cued stimuli obtain more attentional resource and this study claimed serial attentional resource allocation during parallel feature value tracking. However, all Experiments 3a-c did not find any difference in precision estimates between these two types of trials.<br /> 2. Results of Exp.1 in the main text were different from those in Figure.<br /> 3. It would be helpful to add more details for the assignation of response 1 and response 2 to target 1 and target 2, respectively, in all experiments.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Mohibi et al. utilized genetic approaches to determine the role of FDX1 in the regulation of development, oncogenesis, and metabolism. The strengths of the current study are the utilization of both in vivo and in vitro methods coupled to classical biochemical/molecular biology tools and lipidomic screening. The data provided is convincing demonstrating genetic loss of even one allele of FDX1 promotes premature death, increased incidence of adenocarcinoma, and dysregulated lipid metabolism. The authors provide further mechanistic evidence showing enhanced SREBP2 activation, which could potentially be underlying the altered lipid metabolism observed in their model. These findings are likely to provide a novel target for the amelioration to lipid metabolic disorders as the authors show genetic overexpression of FDX1 can reduce intracellular lipid accumulation.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Weber et al. collect locus coeruleus (LC) tissue blocks from 5 neurotypical European men, dissect the dorsal pons around the LC, and prepare 2-3 tissue sections from each donor on a slide for 10X spatial transcriptomics. From three of these donors, they also prepared an additional section for 10x single nucleus sequencing. Overall, the results validate well-known marker genes for the LC (e.g. DBH, TH, SLC6A2), and generate a useful resource that lists genes that are enriched in LC neurons in humans, with either of these two techniques. A comparison with publicly available mouse and rat datasets identifies genes that show reliable LC enrichment across species. Their analyses also support recent rodent studies that have identified subgroups of interneurons in the region surrounding the LC, which show enrichment for different neuropeptides. In addition, the authors claim that some LC neurons co-express cholinergic markers and that a population of serotonin (5-HT) neurons is located within or near the LC. These last two claims must be taken with great caution, as several technological limitations restrict the interpretation of these results. Technical limitations currently limit the ability to integrate spatial and single-nucleus sequencing, yet the manuscript presents a valuable resource on the gene expression landscape of the human LC.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The work by Ohigashi and colleagues addresses the developmental and lineage relationship of a newly characterized thymus epithelial cell (TEC) progenitor subset. The authors take advantage of an elegant and powerful set of experimental approaches to demonstrate that CCL21-expressing TECs appear early in thymus organogenesis and that these cells, which are centrally located, go on to give rise to medullary (m)TECs. What makes the findings intriguing is that these CCL21-expressing mTECs are a distinct subset, which do not express RANK or AIRE, and transcriptomic and lineage tracing approaches point to these cells as potential mTEC progenitor-like cells. Of note, using in vitro and in vivo precursor-product cell transfer experiments, the authors show that this subset has a developmental potential to give rise to AIRE+ self-antigen-displaying mTECs, revealing that CCL21-expressing mTECs can give rise to distinct mTEC subsets. This functional duality provides an attractive rationale for the necessary function of mTECs, which is to attract CCR7+ thymocytes that have just undergone positive selection in the thymus cortex to enter the medulla to undergo tolerance-induction against self-antigen-displaying mTECs. Overall, the work is well supported and offers new insights into the diverse functions of the medullary compartment, and how two distinct subsets of mTECs can achieve it.
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
Summary: A description of a modern protocol for cervical screening that likely could be used in any country of the world, based on self-sampling, extended HPV genotypinng and AI-assisted visual inspection - which is probably the best available combination today.
Strengths: Modern, optimised protocol, designed for global use. Innovative.
Weaknesses: The protocol is not clear. I could not even find how many women were going to be enrolled, the timelines of the study, the statistical methods ("comparing" is not statistics) or the power calculations.<br /> Tables 2 and 3 are too schematic - surely the authors must have an approximate idea of what the actual numbers are behind the green, red and yellow colors.<br /> Figure 1 comparing screening and vaccination is somewhat misleading. They screen 20 birth cohorts but vaccinate only 5 birth cohorts. Furthermore, the theoretical gains of screening has not really been attained in any country in practise. Modelling can be a difficult task and the commentary does not provide any detail on how to evaluate what was done. It just seems unnecessary to attack vaccination as a motivation on why screening needs to be modernised.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The presented study focuses on the role of formin-like 2 (FMNL2) in oocyte meiosis. The authors assessed FMNL2 expression and localization in different meiotic stages and subsequently, by using siRNA, investigated the role of FMNL2 in spindle migration, polar body extrusion, and distribution of mitochondria and endoplasmic reticulum (ER) in mouse oocytes.
Strengths:<br /> Novelty in assessing the role of formin-like 2 in oocyte meiosis.
Weaknesses:<br /> Methods are not properly described.<br /> Overstating presented data.<br /> It is not clear what statistical tests were used.
My main concern is that there are missing important details of how particular experiments and analyses were done. The material and methods section is not written in the way that presented experiments could be repeated - it is missing basic information (e.g., used mouse strain, timepoints of oocytes harvest for particular experiments, used culture media, image acquisition parameters, etc.). Some of the presented data are overstated and incorrectly interpreted. It is not clear to me how the analysis of ER and mitochondria distribution was done, which is an important part of the presented data interpretation. I'm also missing important information about the timing of particular stages of assessed oocytes because the localization of both ER and mitochondria differs at different stages of oocyte meiosis. The data interpretation needs to be justified by proper analysis based on valid parameters, as there is considerable variability in the ER and mitochondria structure and localization across oocytes based on their overall quality and stage.
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www.researchsquare.com www.researchsquare.com
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Reviewer #1 (Public Review):
Summary:<br /> Fukuhara, Maenaka, and colleagues report a crystal structure of the canine distemper virus (CDV) attachment hemagglutinin protein globular domain. The structure shows a dimeric organization of the viral protein and describes the detailed amino-acid side chain interactions between the two protomers. The authors also use their best judgement to comment on predicted sites for the two cellular receptors - Nectin-4 and SLAM - and thus speculate on the CDV host tropism. A complementary AFM study suggests a breathing movement at the hemagglutinin dimer interface.
Strengths:<br /> The study of CDV and related Paramyxoviruses is significant for human/animal health and is very timely. The crystallographic data seem to be of good quality.
Weaknesses:<br /> While the recent CDV hemagglutinin cryo-EM structure is mentioned, it is not compared to the present crystal structure, and thus the context of the present study is poorly justified. Additionally, the results of the AFM experiment are not unexpected. Indeed, other paramyxoviral RBP/G proteins also show movement at the protomer interface.
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
In the manuscript, the authors tried to explore the molecular alterations of adipose tissue and skeletal muscle in PCOS by global proteomic and phosphorylation site analysis. In the study, the samples are valuable, while there are no repeats for MS and there are no functional studies for the indicted proteins, phosphorylation sites. The authors achieved their aims to some extent, but not enough.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this revised preprint the authors investigate whether a presumably allosteric P2RX7 activating compound that they previously discovered reduces fibrosis in a bleomycin mouse model. They chose this particular model as publicly available mRNA data indicate that the P2RX7 pathway is downregulated in idiopathic pulmonary fibrosis patients compared to control individuals. In their revised manuscript, the authors use three proxies of lung damage, Ashcroft score, collagen fibers, and CD140a+ cells, to assess lung damage following the administration of bleomycin. These metrics are significantly reduced on HEI3090 treatment. Additional data implicate specific immune cell infiltrates and cytokines, namely inflammatory macrophages and damped release of IL-17A, as potential mechanistic links between their compound and reduced fibrosis. Finally, the researchers transplant splenocytes from WT, NLRP3-KO, and IL-18-KO mice into animals lacking the P2RX7 receptor to specifically ascertain how the transplanted splenocytes, which are WT for P2RX7 receptor, respond to HEI3090 (a P2RX7 agonist). Based on these results, the authors conclude that HEI3090 enhanced IL-18 production through the P2RX7-NLRP3 inflammasome axis to dampen fibrosis.
These findings could be interesting to the field, as there are conflicting results as to whether NLRP3 activation contributes to fibrosis and if so, at what stage(s) (e.g., acute damage phase versus progression). The revised manuscript is more convincing in that three orthogonal metrics for lung damage were quantified. However, major weaknesses of the study still include inconsistent and small effect sizes of HEI3090 treatment versus either batch effects from transplanted splenocytes or the effects of different genetic backgrounds. Moreover, the fundamental assumption that HEI3090 acts specifically and functionally through the P2RX7 pathway in this model cannot be directly tested, as the authors now provide results indicating that P2RX7 knockout mice do not establish lung fibrosis on bleomycin treatment.
In order to provide clear evidence that HEI3090 functions through P2RX7, a different lung fibrosis model that does not require P2RX7 would be necessary. For example, in such a system the authors could demonstrate a lack of HEI3090-mediated therapeutic effect on P2RX7 knockout. Molecularly, additional evidence on specificity, such as thermal proteome profiling and direct biophysical binding experiments, would also enhance the authors' argument that the compound indeed binds P2RX7 directly and specifically. Since all small molecules have some degree of promiscuity, the absence of an additional P2RX7 modulator, or direct recombinant IL-18 administration (as suggested by another reviewer), is needed to orthogonally validate the functional importance of this pathway. Another way the authors could probe pathway specificity would involve co-administering α-IL-18 with HEI3090 in several key experiments (similar to Figure 4L).
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors observed that miR-199b-5p is elevated in osteoarthritis (OA) patients. They also found that overexpression of miR-199b-5p induced OA-like pathological changes in normal mice and inhibiting miR-199b-5p alleviated symptoms in knee OA mice. They concluded that miR-199b-5p is not only a potential micro-target for knee OA but also provides a potential strategy for the future identification of new molecular drugs.
Strengths:
The data are generated from both human patients and animal models.
Weaknesses:
The data presented in this manuscript is not solid enough to support their conclusions. There are several questions that need to be addressed to improve the quality of this study.
The following questions that need to be addressed to improve the quality of the study.
1. Exosomes were characterized by electron microscopy and western blot analysis (for CD9, 264 CD63, and CD81). However, figure S1 only showed two sample WB results and there is no positive and negative control as well as the confused not clear WB figure.
2. The sequencing of miRNAs in serum exosomes showed that 88 miRNAs were upregulated and 89 miRNAs were downregulated in KOA patients compared with the control group based on fold change > 1.5 and p < 0.05. Figure 2 legend did not clearly elucidate what those represent and why the authors chose those five miRNAs to further validate although they did mention it with several words in line 108 'based on the p-value and exosomal'.
3. In Figure 3 legend and methods, the authors did not mention how they performed the cell viability assay. What cell had been used? How long were they treated and all the details? Other figure legends have the same problem without detailed information.
4. The authors claimed that Gcnt2 and Fzd6 are two target genes of miR-199b-5p. However, there is no convincing evidence such as western blot to support their bioinformatics prediction.
5. To verify the binding site on 3'UTR of two potential targets, the authors designed a mouse sequence for luciferase assay, but not sure if it is the same when using a human sequence.
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- Nov 2023
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The Eph receptor tyrosine kinase family plays a critical function in multiple physiological and pathophysiological processes. Hence, understating the regulation of these receptors is a highly important question. Through extensive experiments in cell lines and cultured neurons Chang et.al show that the signaling hub protein, MYCBP2 positively regulates the overall stability of a specific member of the family, EPHB2, and by that the cellular response to ephrinBs.<br /> Overall, this work sheds light on the divergent in the regulatory mechanisms of the Eph receptors family. Although the physiological importance of this new regularly mechanism in mammals awaits to be discovered, the authors provide genetic evidence using C.elegans that it is evolutionarily conserved.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study demonstrated a novel exciting link between the conserved miRNA-target axis of miR-182-Lrp6 in liver metabolism which causatively contributes to type 2 diabetes and NAFLD in mice and, potentially, humans.
Strengths:
The direct interaction and inhibition of Lrp6 by miR-182 are convincingly shown. The effects of miR-182-5p on insulin sensitivity are also credible for the in vivo and in vitro gain-of-function experiments.
Weaknesses:
However, the DIO cohorts lack key assays for insulin sensitivity such as ITT or insulin-stimulated pAKT, as well as histological evidence to support their claims and strengthen the link between miR-182-5p and T2D or NAFLD. Besides, the lack of loss-of-function experiments limits its aptitude as a potential therapeutic target.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The study presented in this manuscript presents very convincing evidence that purifying selection is the main force shaping the landscape of TE polymorphisms in B. distachyon, with only a few putatively adaptive variants detected, even though most conclusions are based on the 10% of polymorphisms contributed by retrotransposons. That first conclusion is not novel, however, as it had already been clearly established in natural A. thaliana strains (Baduel et al. Genome Biol 2021) and in experimental D. simulans lines (Langmüller et al. NAR 2023), two studies that the authors do not mention, or improperly mention. In contrast to the conclusions reached in A. thaliana, however, Horvath et al. report here a seemingly deleterious effect of TE insertions even very far away from genes (>5kb), a striking observation for a genome of relatively similar size. If confirmed, as a caveat of this study is the lack of benchmarking of the TE polymorphisms calls by a pipeline known for a high rate of false positives (see detailed Private Recommendations #1), this set of observations would make an important addition to the knowledge of TE dynamics in the wild and questioning our understanding of the main molecular mechanisms through which TEs can impact fitness.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The paper carries out an impressive and exhaustive non-sense mutagenesis using deep mutational scanning (DMS) of the gonadotropin-releasing hormone receptor for the WT protein and two single point mutations that I) influence TM insertion (V267T) and ii) influence protein stability (W107A), and then measures the effect of these mutants on correct plasma membrane expression (PME).
Overall, most mutations decreased mGnRHR PME levels in all three backgrounds, indicating poor mutational tolerance under these conditions. The W107A variant wasn't really recoverable with low levels of plasma membrane localisation. For the V267T variant, most additional mutations were more deleterious than WT based on correct trafficking, indicating a synergistic effect. As one might expect, there was a higher degree of positive correlation between V267T/W107A mutants and other mutants located in TM regions, confirming that improper trafficking was a likely consequence of membrane protein co-translational folding. Nevertheless, context is important, as positive synergistic mutants in the V27T could be negative in the W107A background and vice versa. Taken together, this important study highlights the complexity of membrane protein folding in dissecting the mechanism-dependent impact of disease-causing mutations related to improper trafficking.
Strengths:<br /> This is a novel and exhaustive approach to dissecting how receptor mutations under different mutational backgrounds related to co-translational folding, could influence membrane protein trafficking.
Weaknesses:<br /> The premise for the study requires an in-depth understanding of how the single-point mutations analysed affect membrane protein folding, but the single-point mutants used seem to lack proper validation. Furthermore, plasma membrane expression has been used as a proxy for incorrect membrane protein folding, but this not necessarily be the case, as even correctly folded membrane proteins may not be trafficked correctly, at least, under heterologous expression conditions. In addition, mutations can affect trafficking and potential post-translational modifications, like glycosylation.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The regulation of motor autoinhibition and activation is essential for efficient intracellular transport. This manuscript used biochemical approaches to explore two members in the kinesin-3 family. They found that releasing UNC-104 autoinhibition triggered its dimerization whereas unlocking KLP-6 autoinhibition is insufficient to activate its processive movement, which suggests that KLP-6 requires additional factors for activation, highlighting the common and diverse mechanisms underlying motor activation. They also identified a coiled-coil domain crucial for the dimerization and processive movement of UNC-104. Overall, these biochemical and single-molecule assays were well performed, and their data support their statements. The manuscript is also clearly written, and these results will be valuable to the field.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The study examines how hemocytes control whole-body responses to oxidative stress. Using single cell sequencing they identify several transcriptionally distinct populations of hemocytes, including one subset that show altered immune and stress gene expression. They also find that knockdown of DNA Damage Response (DDR) genes in hemocytes increases expression of the immune cytokine, upd3, and that both upd3 overexpression in hemocytes and hemocyte knockdown of DDR genes leads to increased lethality upon oxidative stress. And they find that the PQ-induced lethality seen when the DDR is disrupted can be rescued in upd3 null background, suggesting links between proper regulation of DDR in hemocytes, modulation of systemic upd3 signaling, and the control of oxidative stress survival.
The paper has two key strengths:
1, The single cell analyses provide a clear description of how oxidative stress can cause distinct transcriptional changes in different populations of hemocytes. These results add to the emerging them in the field that there functionally different subpopulations of hemocytes that can control organismal responses to stress.<br /> 2, The discovery that DDR genes are required upon oxidative stress to modulate upd3 cytokine production and lethality provides interesting new insight into the DDR may play non-canonical roles in controlling organismal responses to stress.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study is one of several around the world to investigate how urban wildlife responded to changes in human activity during the lockdowns associated with the COVID-19 pandemic. Unlike several other studies on the topic that used observational data from citizen science programs, this project relied on passive acoustic monitoring to record bird vocalizations during and after stringent lockdown periods in an urban environment. The authors focused on three species that differ in their level of adaptation to human presence, providing an ecologically relevant comparison that highlights the importance of micro-habitats for species living in close proximity to humans.
Strengths:
The element that sets this study apart from most others examining avian responses to COVID-19 lockdowns is the use of passive acoustic monitoring. As the authors describe, this method offers several advantages over other methods (though, it does come with some limitations on what questions can be addressed). Perhaps the most relevant advantage is that it offers the ability to concurrently measure anthropogenic noise in the environment, which is one of the most likely mechanisms for effects on wildlife from changes to human activity. These authors were, therefore, able to show local-scale differences in bird responses to human activity measured at the same scale. To my knowledge, only one other study (Derryberry et al. Science. 2020) has used recordings of vocalizations to examine the influence of COVID-19 lockdowns on a bird species.
It was encouraging to see a study that focused on the local-scale impacts of lockdowns, with methods that could investigate effects within micro-habitats. Logistics prevented many other projects from operating at such fine scales, making the results from this study particularly useful for the examination of rapid changes in bird behavior. This does mean that comparisons between this study and others examining the effects of COVID-19 lockdowns on birds should be done with care, as the effects described here may have been the result of different processes, operating at different spatial and temporal scales. However, that also means this study fills an important gap in our knowledge of how wildlife reacts to human activity in urban spaces.
Weaknesses:
One drawback of the approach is that the acoustic sampling only occurred during the pandemic: samples were taken during several lockdown periods in the early spring (March through early May) of 2020 and then for a period of 10 days after the end of the final lockdown period in late May of 2020. Unfortunately, this means that the interpretation of the effect of lockdowns could have been affected by any shifts in the birds' vocal behavior that resulted from unmodeled environmental factors or normal seasonal phenology during that three-month period. However, the authors chose focal species that would be less prone to seasonal changes in vocal behavior and their approach did account for several factors to minimize any such effects.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> This important study nicely integrates a breadth of experimental and computational data to address fundamental aspects of RNA methylation by an important for biology and health RNA methyltransferases (MTases).
Strengths:<br /> The authors offer compelling and strong evidence, based on carefully performed work with appropriate and well-established techniques to shed light on aspects of the methyl transfer mechanism of the methyltransferase-like protein 3 (METTL3), which is part of the methyltransferase-like proteins 3 & 14 (METTL3-14) complex.
Weaknesses:<br /> The significance of this foundational work is somewhat diminished mostly due to mostly efficient communication of certain aspects of this work. Parts of the manuscript are somewhat uneven and don't quite mesh well with one another. The manuscript could be enhanced by careful revision and significant textual and figure edits. Examples of recommended edits that would improve clarity and allow accessibility to a broader audience are highlighted in some detail below.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The manuscript by Dr. Shinkai and colleagues is about the posttranslational modification of a highly important protein, MT3, also known as the growth inhibitory factor. Authors postulate that MT3, or generally all MT isoforms, are sulfane sulfur binding proteins. The presence of sulfane sulfur at each Cys residue has, according to the authors, a critical impact on redox protein properties and almost does not affect zinc binding. They show a model in which 20 Cys residues with sulfane sulfur atoms can still bind seven zinc ions in the same clusters as unmodified protein. They also show that recombinant MT3 (but also MT1 and MT2) protein can react with HPE-IAM, an efficient trapping reagent of persulfides/polysulfides. This reaction performed in a new approach (high temperature and high reagent concentration) resulted in the formation of bis-S-HPE-AM product, which was quantitatively analyzed using LC-MS/MS. This analysis indicated that all Cys residues of MT proteins are modified by sulfane sulfur atoms. The authors performed a series of experiments showing that such protein can bind zinc, which dissociates in the reaction with hydrogen peroxide or SNAP. They also show that oxidized MT3 is reduced by thioredoxin. It gives a story about a new redox-dependent switching mechanism of zinc/persulfide cluster involving the formation of cystine tetrasulfide bridge.
The whole story is hard to follow due to the lack of many essential explanations or full discussion. What needs to be clarified is the conclusion (or its lack) about MT3 modification proven by mass spectrometry. Figure 1B shows the FT-ICR-MALDI-TOF/MS spectrum of recombinant MT3. It clearly shows the presence of unmodified MT3 protein without zinc ions. Ions dissociate in acidic conditions used for MALDI sample preparation. If the protein contained all Cys residues modified, its molecular weight would be significantly higher. Then, they show the MS spectrum (low quality) of oxidized protein (Fig. 1C), in which new signals (besides reduced apo-MT3) are observed. They conclude that new signals come from protein oxidation and modification with one or two sulfur atoms. If the conclusion on Cys residue oxidation is reasonable, how this protein contains sulfur is unclear. What is the origin of the sulfur if apo-MT does not contain it? Oxidized protein was obtained by acidification of the protein, leading to zinc dissociation and subsequent neutralization and air oxidation. Authors should perform a detailed isotope analysis of the isotopic envelope to prove that sulfur is bound to the protein. They say that the +32 mass increase is not due to the appearance of two oxygen donors. They do not provide evidence. This protein is not a sulfane sulfur binding protein, or its minority is modified. Moreover, it is unacceptable to write that during MT3 oxidation are "released nine molecules of H2". How is hydrogen molecule produced? Moreover, zinc is not "released", it dissociates from protein in a chemical process.
Another important point is a new approach to the HPE-IAM application. Zinc-binding MT3 was incubated with 5 mM reagent at 60oC for 36 h. Authors claim that high concentration was required because apoMT3 has stable conformation. Figure 2B shows that product concentration increases with higher temperature, but it is unclear why such a high temperature was used. Figure 1D shows that at 37oC, there is almost no reaction at 5 mM reagent. Changing parameters sounds reasonable only when the reaction is monitored by mass spectrometry. In conclusion, about 20 sulfane sulfur atoms present in MT3 would be clearly visible. Such evidence was not provided. Increased temperature and reagent concentration could cause modification of cysteinyl thiol/thiolates as well, not only persulfides/polysulfides. Therefore, it is highly possible that non-modified MT3 protein could react with HPE-IAM, giving false results. Besides mass spectrometry, which would clearly prove modifications of 20 Cys, authors should use very important control, which could be chemically synthesized beta- or alfa-domain of MT3 reconstituted with zinc (many protocols are present in the literature). Such models are commonly used to test any kind of chemistry of MTs. If a non-modified chemically obtained domain would undergo a reaction with HPE-IAM under such rigorous conditions, then my expectation would be right.
- The remaining experiments provided in the manuscript can also be applied for non-modified protein (without sulfane sulfur modification) and do not provide worthwhile evidence. For instance, hydrogen peroxide or SNAP may interact with non-modified MTs. Zinc ions dissociate due to cysteine residue modification, and TCEP may reduce oxidized residue to rescue zinc binding. Again, mass spectrometry would provide nice evidence.
- The same is thioredoxin (Fig. 7) and its reaction with oxidized MT3. Nonmodified and oxidized MT3 would react as well.
- If HPE-IAM reacts with Cys residues with unmodified MT3, which is more likely the case under used conditions, the protein product of such reaction will not bind zinc. It could be an explanation of the cyanolysis experiment (Fig. 6).
- Figure 4 shows the reactivity of (pol)sulfides with TCEP and HPE-IAM. What are redox potentials? Do they correlate with the obtained results?
- Raman spectroscopy experiments would illustrate the presence of sulfane sulfur in MT3 only if all Cys were modified.
- The modeling presented in this study is very interesting and confirms the flexibility of metallothioneins. MT domains are known to bind various metal ions of different diameters. They adopt in this way to larger size the ions. The same mechanism could be present from the protein site. The presence of 9 or 11 sulfur atoms in the beta or alfa domain would increase the size of the domains without changing the cluster structure.
- Comment to authors. Apo-MT is not present in the cell. It exists as a partially metallated species. The term "apo-MT" was introduced to explain that MTs are not fully saturated by metals and function as a metal buffer system. Apo-MT comes from old ages when MT was considered to be present only in two forms: apo-form and fully saturated forms.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The authors sought to establish a biochemical strategy to study ESAT-6 and CFP-10 biochemistry. They established recombinant reagents to study these protein associations in vitro revealing an unexpected relationship at low pH. They next develop much-needed reagents to study these proteins in an infection context and reveal that treatment with an ESAT-6 nanobody enhances Mtb control.
Strengths:<br /> The biochemical conclusions are supported by multiple configurations of the experiments. They combine multiple approaches to study a complex problem.
Weaknesses:<br /> It would be valuable to understand if the nanobody is disrupting the formation of the ESAT6-CFP10 complex. It is unclear how the nanobody is functioning to enhance control in the infection context. More detail or speculation in the discussion would have been valuable. Where is the nanobody in the cell during infection?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Exposure to cranial irradiation (IR) leads to cognitive deficits in the survivors of brain cancer. IR upregulates miR-206-3p, which in turn reduces the PAK3-LIMK1 axis leading to the loss of F and G-actin ratio and, thereby, mature dendritic spine loss. Silencing miR-206-3p reverses these degenerative consequences.
Strengths:<br /> The authors show compelling data indicating a clear correlation between PAK3 knockdown and the loss of mature dendritic spine density. In contrast, overexpression of PAK3 in the irradiated neurons restored mature spine types and recovered the F/G ratio. These in vitro results support the authors' hypotheses that PAK3 and LIMK1-mediated downstream signaling impact neuronal structure and reorganization in vitro. These data were supported by similar experiments using differentiated human neurons. Importantly, silencing miR-206-30 using antagonist miR also reverses IR-induced downregulation of the PAK3-LIMK1 axis, preventing spine loss and cognitive deficits.
Weaknesses:
All the miR-206-3p data are presented from in vitro cortical neurons or human stem cell-derived neuron cultures. This data (IR-induced elevation of miR-206-3p) should also be confirmed in vivo using an irradiated mouse brain to correlate the cognitive dysfunction timepoint.
Antago-miR-206-3p reversed Ir-induced upregulation of miR-206 (in vitro), and prevent reductions in PAK3 and downstream markers. Importantly, it reversed cognitive deficits induced by IR. This data should be supported by in vivo staining for important dendritic markers, including cofillin, p-cofilin, PSD-95, F- and G-actin within the hippocampal and PFC regions.
Other neuronal and non-neuronal targets of miR-206-3p should be discussed and looked into as a downstream impact of IR-induced functional and physiological impairments in the brain.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this study, authors performed multiple sets of mesoscale chromatin simulations at nucleosome resolution to study the effects of TF binding on chromatin structures. Through simulations at various conditions, authors performed systemically analysis to investigate how linker histone, tail acetylation, and linker DNA length can operate together with TFs to regulate chromatin architecture. Using gene Eed as one example, authors found that binding of Myc:Max could repress the gene expression by increasing fiber folding and compaction and this repression can be reversed by the linker histone. Understanding how transcription factors bind to regulatory DNA elements and modulate chromatin structure and accessibility is an essential question in epigenetics. Through modelling of TF binding to chromatin structures at nucleosome levels, authors demonstrated that TF binding could create microdomains that are visible in the ensemble-based contact maps and short DNA linkers prevent the formation microdomains. It has also been shown that tail acetylation and TF binding have opposite effects on chromatin compaction and linker histone can compete for the linker DNA with TF binding to impair the effect of TF binding. This study improves our knowledge on how TFs collaborate with different epigenetic marks and chromatin features to regulate chromatin structure and accessibility, which will be of broad interest to the community.
For this reviewer, there were a few notable limitations. One was the implicit model of TF binding, which is modelled by adding harmonic restraints at two DNA beads. The model is very simple and it lacks kind of validation of how the results can be extended to many other TFs. In addition, the results of TF binding creating microdomains are very interesting but it requires further quantitative analysis of how microdomains was affected under different conditions. Also, some definitions and protocols demand further elucidation.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This revised study follows up on previous work showing a female-specific enhancer region of PAX1 is associated with adolescent idiopathic scoliosis (AIS). This new analysis combines human GWAS analysis from multiple countries to identify a new AIS-associated coding variant in the COL11A1 gene (COL11A1P1335L). Using a Pax1 knockout mouse they go on to find that PAX1 and Collagen XI protein are expressed in the intervertebral discs (IVDs) and robustly in the growth plate, showing that COL11A1 expression is reduced in Pax1 mutant growth plate. Moreover, other AIS-associated genes, Gpr126 and Sox6, were also reduced in Pax1 mutant mice, suggesting a common pathway is involved in AIS.
Using SV40 immortalized costal cartilage cells, derived from floxed Col11a1 mice primary rib cage cartilage, they go to show that removal of Col11a1 leads to reduction of Mmp3 expression. In this context, the expression of wild-type Col11a1 restored regular levels of Mmp3 expression, while expression of the AIS-associated Col11a1P1335L allele failed to restore normal Mmp3 expression. This supports a model that the AIS-associated Col11a1P1335L allele leads to the dysregulation of ECM in vivo.
Using this culture system, they go on to test the role of the estrogen receptor ESR2, showing that loss of this receptor leads to reduced Mmp3 and Pax1 expression, and increased Col11a1 expression. They support this by showing similar gene expression changes and estrogen receptor function in Rat cartilage endplate cell culture.
Altogether, this study nicely brings together an impressive number of human genetic data from multi-ethnic AIS cohorts and controls from across the globe and functionally tests these findings in cell culture and animal models. This study wonderfully integrates other findings from other human and mouse work in AIS and supports a new molecular mechanism by which estrogen can interact and synergize with COL11A1/PAX1/MMP3 signaling to change ECM development and dynamics, thus providing a tangible model for mutations and dysregulation of this pathway can increase the susceptibility of scoliosis.
Strengths:
This work integrates a large cohort of human genetic data from AIS patient and control from diverse ethnic backgrounds, across the globe. This work attempts to functionally test their findings in vivio and by use of cell culture.
Weaknesses:
Many of the main functional work was done in cell culture and not in vivo.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> The study's abstract, introduction, and conclusions are not supported by the methods and results conducted. In fact, the results presented suggest that Arabidopsis could easily adapt to an extremely high CO2 environment.
This study offers good evidence pointing to a genetic basis for Arabidopsis thaliana's response to elevated CO2 (eCO2) levels and its subsequent impact on the leaf ionome. The natural variation analyses in the study support the hypothesis that genetic factors, rather than local adaptation, guide the influence of eCO2 on the ionome of rosette leaves in Arabidopsis. However, the manuscript's claim regarding its role in "the development of biofortified crops adapted to a high-CO2 world" (line 23) is overstated, especially given the absence of any analysis on the influence of eCO2 on the seed ionome and Arabidopsis is a poor model for harvest index for any crop. The manuscript, in its current form, necessitates massive revisions, particularly in clarifying its broader implications and in providing more substantial evidence for some of its assertions.
Major Drawbacks and Questions:
1. Evidence for the Central Premise:<br /> The foundational premise of the study is the assertion that rising atmospheric CO2 levels result in a decline in plant mineral content. This phenomenon is primarily observed in C3 plants, with C4 plants seemingly less affected. The evidence provided on this topic is scant and, in some instances, contradicts the authors' own references. The potential reduction of certain minerals, especially in grains, can be debated. For instance, reduced nitrogen (N) and phosphorus (P) content in grains might not necessarily be detrimental for human and animal consumption. In fact, it could potentially mitigate issues like nitrogen emissions and phosphorus leaching. Labeling this as a "major threat to food security" (line 30) is exaggerated. While the case for microelements might be more compelling, the introduction fails to articulate this adequately. Furthermore, the introduction lacks any discussion on how eCO2 might influence nutrient allocation to grains, which would be crucial in substantiating the claim that eCO2 poses a threat to food security. A more comprehensive introduction that clearly delineates the adverse effects of eCO2 and its implications for food security would greatly enhance the manuscript.
2. Exaggerated Concerns:<br /> The paper begins with the concern that carbon fertilization will lead to carbon dilution in our foods. While we indeed face numerous genuine threats in the coming decades, this particular issue is manageable. The increase in CO2 alone offers many opportunities for boosting yield. However, the heightened heat and increased evapotranspiration will pose massive challenges in many environments.
Figure 4 in fact suggests that 43% of the REGMAP panel (cluster 3) is already pre-adapted to very high CO2 levels. This suggests annual species could adapt very rapidly.
3. Assumptions on CO2 Levels:<br /> The assumption of 900ppm seems to be based on a very extreme climate change scenario. Most people believe we will overshoot the 1.5{degree sign}C scenario, however, it seems plausible that 2.5 to 3{degree sign}C scenarios are more likely. This would correspond to around 500ppm of CO2. https://www.nature.com/articles/s41597-022-01196-7/tables/4
4. Focus on Real Challenges:<br /> We have numerous real challenges, such as extreme heat and inconsistent rainfall, to address in the context of climate change. However, testing under extreme CO2 conditions and then asserting that carbon dilution will negatively impact nutrition is exaggerated.
In contrast, the FACE experiments are fundamental and are conducted at more realistic eCO2 levels. Understanding the interaction between a 20% increase in CO2 and new precipitation patterns is key for global carbon flux prediction.
As I look at the literature on commercial greenhouse tomato production, 1000ppm of eCO2 is common, but it also looks like the breeders and growers have already solved for flavor and nutrition under these conditions.
Conclusion:<br /> While the study provides valuable insights into the genetic underpinnings of Arabidopsis thaliana's response to elevated CO2 levels, it requires an entirely revised writeup, especially in its abstract, broader claims and implications. The manuscript would benefit from a more thorough introduction, a clearer definition of its scope, and a clear focus on the limits of this study.
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Reviewer #1 (Public Review):
Peng et al develop a computational method to predict/rank transcription factors (TFs) according to their likelihood of being pioneer transcription factors--factors that are capable of binding nucleosomes--using ChIP-seq for 225 human transcription factors, MNase-seq and DNase-seq data from five cell lines. The authors developed relatively straightforward, easy to interpret computational methods that leverage the potential for MNase-seq to enable relatively precise identification of the nucleosome dyad. Using an established smoothing approach and local peak identification methods to estimate positions together with identification of ChIP-seq peaks and motifs within those peaks which they referred to as "ChIP-seq motifs", they were able to quantify "motif profiles" and their density in nucleosome regions (NRs) and nucleosome depleted regions (NDRs) relative to their estimated nucleosome dyad positions. Using these profiles, they arrived at an odd-ratio based motif enrichment score along with a Fisher's exact test to assess the odds and significance that a given transcription factor's ChIP-seq motifs are enriched in NRs compared to NDRs, hence, its potential to be a pioneer transcription factor. They showed that known pioneer transcription factors had among the highest enrichment scores, and they could identify a number of relatively novel pioneer TFs with high enrichment scores and relatively high expression in their corresponding cell line. They used multiple validation approaches including (1) calculating the ROC-AUC and Matthews correlation coefficient (MCC) and generating ROC and precision-recall curves associated with their enrichment score based on 32 known pioneer TFs among their 225 TFs which they used as positives and the remaining TFs (among the 225) as negatives; (2) use of the literature to note that known pioneer TFs that acted as key regulators of embryonic stem cell differentiation had a highest enrichment scores; (3) comparison of their enrichment scores to three classes of TFs defined by protein microarray and electromobility shift assays (1. strong binder to free and nucleosomal DNA, 2. weak binder to free and nucleosomal DNA, 3. strong binding to free but not nucleosomal DNA); and (4) correlation between their calculated TF motif nucleosome end/dyad binding ratio and relevant data from an NCAP-SELEX experiment. They also characterize the spatial distribution of TF motif binding relative to the dyad by (1) correlating TF motif density and nucleosome occupancy and (2) clustering TF motif binding profiles relative to their distance from the dyad and identifying 6 clusters.
The strengths of this paper are the use of MNase-seq data to define relatively precise dyad positions and ChIP-seq data together with motif analysis to arrive at relatively accurate TF binding profiles relative to dyad positions in NRs as well as in NDRs. This allowed them to use a relatively simple odds ratio based enrichment score which performs well in identifying known pioneer TFs. Moreover, their validation approaches either produced highly significant or reasonable, trending results.
The weaknesses of the paper are relatively minor, and the authors do a good job describing the limitations of the data and approach.
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Reviewer #1 (Public Review):
Summary:
Mainali and colleagues provide evidence for Itaconate stabilising Cpt1a via a decrease in ubiquitination. This in turn likely regulates fatty acid oxidation which in turn would appear to be involved in thermoregulation in the context of sepsis.
Strengths:
These findings add to our knowledge of the role of Itaconate in sepsis and its rather complex effects on metabolism, specifically lipid metabolism.
Weaknesses:
1. This is a complex paper and would benefit from a schematic depicting the key findings.
2. The paper would benefit from additional supporting evidence. Would it be possible to measure fatty acid oxidation by metabolic tracing here, in IRG-deficient cells or in response to 4-OI? Although changes in protein level for Cpt1A are seen, this is correlated with fatty acid oxidation rather than direct demonstration. This may be challenging but would strengthen the manuscript.
3. The aspect concerning body temperature regulation is confusing. Would Itaconate not promote fatty acid oxidation to increase or maintain body temperature? Itaconate must therefore not be involved in the hypothermic response? Bringing UCP1 into the finding is confusing and needs to be better explained. Again a diagram would help, but enhanced BAT fatty acid oxidation and UCP1 expression appear linked here, with both being affected by Itaconate. This needs clarifying.
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Reviewer #1 (Public Review):
The association of vitamin D supplementation in reducing Asthma risk is well studied, although the mechanistic basis for this remains unanswered. In the presented study, Kilic and co-authors aim to dissect the pathway of Vitamin D-mediated amelioration of allergic airway inflammation. They use initial leads from bioinformatic approaches, which they then associate with results from a clinical trial (VDAART) and then validate them using experimental approaches in murine models. The authors identify a role of VDR in inducing the expression of the key regulator Ikzf3, which possibly suppresses the IL-2/STAT5 axis, consequently blunting the Th2 response and mitigating allergic airway inflammation.
The major strength of the paper lies in its interdisciplinary approach, right from hypothesis generation, and linkage with clinical data, as well as in the use of extensive ex vivo experiments and in vivo approaches using knock-out mice. The study presents some interesting findings including an inducible baseline absence/minimal expression of VDR in lymphocytes, which could have physiological implications and needs to be explored in future studies.<br /> However, the study presents a potential for further dissection of relevant pathophysiological parameters using additional techniques, to explain certain seemingly associative results, and allow for a more effective translation.
Several results in the study suggest multiple factors and pathways influencing the phenotype seen, which remain unexplored. The inferences of this study also need to be read in the context of the different sub-phenotypes and endotypes of Asthma, where the Th2 response may not be predominant. While this does not undermine the importance of this elegant study, it is essential to emphasise a holistic picture while interpreting the results.
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Reviewer #1 (Public Review):
The manuscript by Geurrero and colleagues introduces two new metrics that extend the concept of "druggability"- loosely speaking, the potential suitability of a particular drug, target, or drug-target interaction for pharmacological intervention-to collections of drugs and genetic variants. The study draws on previously measured growth rates across a combinatoriality complete mutational landscape involving 4 variants of the TEM-50 (beta lactamase) enzyme, which confers resistance to commonly used beta-lactam antibiotics. To quantify how growth rate - in this case, a proxy for evolutionary fitness - is distributed across allelic variants and drugs, they introduce two concepts: "variant vulnerability" and "drug applicability".
Variant vulnerability is the mean vulnerability (1-normalized growth rate) of a particular variant to a library of drugs, while drug applicability measures the mean across the collection of genetic variants for a given drug. The authors rank the drugs and variants according to these metrics. They show that the variant vulnerability of a particular mutant is uncorrelated with the vulnerability of its one-step neighbors, and analyze how higher-order combinations of single variants (SNPs) contribute to changes in growth rate in different drug environments.
The work addresses an interesting topic and underscores the need for evolution-based metrics to identify candidate pharmacological interventions for treating infections. The authors are clear about the limitations of their approach - they are not looking for immediate clinical applicability - and provide simple new measures of druggability that incorporate an evolutionary perspective, an important complement to the orthodoxy of aggressive, kill-now design principles.
As I said in my initial review, I think the work could be improved with additional analysis that tie the new metrics to evolutionary outcomes. Without this evidence, or some other type of empirical or theoretical support for the utility of these metrics, I am not fully convinced that these concepts have substantial impact. The new metrics could indeed be useful--and they have intuitive appeal--but the current revisions stop short of demonstrating that these intuitive notions hold up under "realistic" conditions (whether in simulation, theory, or experiment).
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Reviewer #1 (Public Review):
Summary:<br /> The study follows the role of yeast eIF2A protein as a potential translation initiation factor engaged in the non-canonical translation initiation under stress conditions and as a substitute for eIF2. Using ribosome profiling, RNA-Seq and reporter-based assays authors evaluated the role of eIF2A protein under regular or stress conditions (cells starved for branched amino acids). The authors found that yeast cells depleted of eIF2A protein do not change significantly their translation initiation, or translation in general. In contrast to previously reported data for human homolog, yeast eIF2A does not significantly contribute to the regulation of the uORFs, regardless of whether they start with canonical AUG or near cognate start codons. eIF2A is not involved in the repression of IRES element in the URE2 gene or has a role in purine biosynthesis. It appears that in yeast eIF2A contributes to the regulation of a very limited number of mRNAs (32 with significant changes in translation efficiency), where only 17 of such messages indeed are consistent with eIF2A deletion, and single mRNA (HKR1) could be validated in reporter assay.
Strengths:<br /> The main strength of the manuscript is a complete analysis and unbiased approach using genomic analysis methods (ribosome profiling and RNA-seq) as well as reporter validation studies. Additional strengths of the manuscript are scientific rigor and statistics associated with data analyses, clear data presentation, and discussion of the results in the context of the previous studies and results.
Weaknesses:<br /> No weaknesses were noted by this reviewer.
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Reviewer #1 (Public Review):
Summary and Strengths:
Zhang et al. conducted a study in which they isolated and characterized a Marburg virus (MARV) glycoprotein-specific antibody, AF-03. The antibody was obtained from a phage-display library. The study shows that AF-03 competes with the previously characterized MARV-neutralizing antibody MR78, which binds to the virus's receptor binding site. The authors also performed GP mutagenesis experiments to confirm that AF-03 binds near the receptor binding site. In addition, the study confirmed that AF-03, like MR78, can neutralize Ebola viruses with cleaved glycoproteins. Finally, the authors demonstrated that NPC2-fused AF-03 was effective in neutralizing several filovirus species.
Weaknesses:
1. The main premise of this study is unclear. Flyak et al. in 2015 described the isolation and characterization of a large panel of neutralizing antibodies from a Marburg survivor (Flyak et al., Cell, 2015). Based on biochemical and structural characterization, Flyak proposed that the Marburg neutralizing antibodies bind to the NPC1 receptor binding side. In the same study, it has been shown that several MARV-neutralizing antibodies can bind to cleaved Ebola glycoproteins that were enzymatically treated to remove the mucin-like domain and glycan cap. In the following study, it has been shown that the bispecific-antibody strategy can be used to deliver Marburg-specific antibodies into the endosome, where they can neutralize Ebola viruses (Wec et al., Science 2016). Finally, the use of lysosome-resident protein NPC2 to deliver antibody cargos to late endosomes has been previously described (Wirchnianski et al., Front. Immunol, 2021)
The above-mentioned studies are not referenced in the introduction. The authors state that "there is no licensed treatment or vaccine for Marburg [virus] infection." While this is true, there are human antibodies that recognize neutralizing epitopes - that information can't be excluded while providing the rationale for the study. Furthermore, the authors use the word "novel" to describe the AF-03 antibody. How novel is AF-03 if multiple Marburg-neutralizing antibodies were previously characterized in multiple studies? Since AF-03 competes with previously characterized MR78, it binds to the same antigenic region as MR78. AF-03 also has comparable neutralization potency as MR78.
2. Without the AF-03-MARV GP crystal structure, it's unclear how van der Waals interactions, H-bonds, and polar and electrostatic interactions can be evaluated. While authors use computer-guided homology modeling, this technique can't be used to determine critical interactions. Furthermore, Flyak et al. reported that binding to the NPC1 receptor binding site is the main mechanism of Marburg virus neutralization by human monoclonal antibodies. Since both AF-03 (this study) and MR78 (Flyak study) competed with each other, that information alone was sufficient for GP mutagenesis experiments that identified the NPC1 receptor binding site as the main region for mutagenesis.
3. The AF-03-GP affinity measurements were performed using bivalent IgG molecules and trimeric GP molecules. This format does not allow accurate measurements of affinity due to the avidity effect. The reported KD value is abnormally low due to avidity effects. The authors need to repeat the affinity experiments by immobilizing trimeric GPs and then adding monovalent AF-03 Fab.
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Reviewer #1 (Public Review):
Summary:
Mandal et al build upon their earlier work in CD 4 T cells to address the role of WASP in cytotoxic T cell mechanosensing. As shown previously by this group and others, the authors present evidence that tumour cell lysis is stiffness dependent and requires CTL WASP expression. They proceed to show that CTLs engaging targets form actin-rich foci, that the formation of these structures is dependent upon tumour cell stiffness and WASP dependent actin nucleation. Traction force measurements show that WASP is involved in force generation, and evidence that WASP plays a role in mechanosensing comes from studies showing that stiffness dependent phosphorylation of early TCR signalling intermediates (but not the later stages of T cell activation) is WASP dependent, as is phosphorylation of the tension sensor CasL. Finally, the authors provide in vivo data that WASP-deficient T cells kill tumours inefficiently.
Strengths:
The paper is well-written and brings together a range of well-established techniques for measuring T cell stiffness responses, force production, signalling, and effector function. Although some of the findings are necessarily correlative, the authors have largely achieved their aims. One particularly interesting observation is that stiffness dependent phosphorylation of ZAP70 requires WASP expression. Evidence that ZAP70 phosphorylation is WASP dependent is important, as it suggests that forces exerted by WASP are needed for some of the earliest stages of TCR signalling, perhaps TCR deformation itself. This observation, made in CD8 T cells, is particularly interesting given that previous work from this group [Kumari et al eLife 2015] showed that ZAP70 phosphorylation was intact in WASP-/- CD4 T cell blasts. In that study, the first clear differences in TCR signaling were seen at the level of PLCγ phosphorylation. This could represent an interesting difference between CD4 and CD8 T cells, but supplemental data from Figure S2 also show WASP dependence for CD3ζ and ZAP70 phosphorylation in naïve CD4 T cells. Unfortunately, this interesting issue was not discussed or pursued experimentally.
Weaknesses:
While the study is well executed, it is rather limited in scope, and many of the observations have been reported previously in other systems. These weaknesses limit the impact of the study. In particular, the authors have previously shown in CD4 T cells that the nucleation promoting activity of WASP is responsible for the formation of actin foci, for early TCR signalling events associated with T cell activation, for traction force generation and for CasL phosphorylation [Kumari et al eLife 2015, Kumari et al EMBO J 2020]. It could be argued that this paper extends findings made originally in CD4 cells to include CD8 T cells. But the authors did not make this clear, and the advance is rather incremental. Moreover, similar studies have been done in CD8 T cells by other labs. Most notably, the Huse group has conducted highly relevant work investigating the mechanobiology of CTL function in vitro and in vivo [Basu et al Cell 2016, Wang et al Nat Comms 2022, Tamzalit et al Sci Immunol 2019, Tello-Lafoz et al Immunity 2021, de Jesus et al bioRxiv Preprint 2023]. Indeed, one study showed that WASP depletion impairs the formation of protrusions that deform the target cell surface and promote target lysis [Tamzalit et al Sci Immunol 2019]. Mandal et al cite this work and argue that what they show differs from the mechanopotentiation shown in Tamzalit et al, but they don't explore the issue further. They also fail to cite work from Tello-Lafoz et al showing that regulated changes in target cell stiffness contribute to CTL vulnerability. Finally, Mandal et al. fail to deal with evidence that WASP participates in many phases of the CTL response, including adhesion, migration, granule release, and serial killing. All of these are likely contributors to the in vivo phenotypes shown in Figure 4.
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