- May 2024
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
I have read the re-submission of the manuscript "The optimal clutch size revisited: separating individual quality from the parental survival costs of reproduction" by LA Winder and colleagues.
I have to say that I am quite disappointed not to see any formalisation of the mechanism that the authors have in mind to explain the results they have and to draw general conclusions from it. In my original review, I strongly recommended "improving the theoretical component of the analysis by providing a solid theoretical framework before, from it, drawing conclusions. This, at a minimum, requires [...] most importantly a mechanistic model describing the assumed relationships."
Without it, it is impossible to follow, agree or disagree with the authors and learn something from the meta-analysis other than: the clutch size-annual survival relationship has opposite slopes for manipulated and natural populations. Such a set of equations (would replace pages of verbose and) is not only necessary for the readers to be able to understand the authors' points and to clearly understand the simplifying assumptions, but also for the authors to ensure they conclusions are sound. For these reasons this is a central part of such studies, see, e.g. (Walker et al., 2008). This is supposedly replaced here by a figure (figure 5), which top-left part reads: "Parental survival costs of reproduction constrain intra-specific reproduction" - "no the effect size on fig 4 is too small". Figure 4 is the output of simulations where the authors have incorporated the mean effect on survival rate per egg from the manipulated populations into a model where they compute R0 for various increases in the annual fertility rate, and related decreases in annual survival rates, showing that along the slow-fast gradient, for balanced survival-reproduction (certainly not far from R0=1), R0 is not affected (or very little) by change in fertility-survival along the trade-off. Nowhere on this figure, do we have any information inferring that survival costs of reproduction do not constrain intra-specific reproduction. It is actually possible to build a simple mechanistic model with a trade-off mechanism that strongly affects the LRS and its variance between individuals and to would produce the exact same figure.
This is compounded in this manuscript by the constant verbose, imprecisions, outright mistakes, with a general confusion between magnitudes and variation of magnitudes, which makes it very hard to read. Let us just look at two examples illustrating my points. In the abstract, I read: " ... revealed that reproduction presented negligible costs, except when reproductive effort was forced beyond the level observed within species, to that seen between species" means nothing: what is the level of reproductive effort seen between species? I suppose the authors mean "forced beyond the maximum level observed within species, to that seen between species" or something like that. Caption figure 4:" Selection differentials (i.e., the difference in lifetime reproductive output between hypothetical control and brood-manipulated populations)" It cannot be how this was calculated however: the difference between equal things is 0, not 1. These errors and all the other imprecisions, lengthy definitions that are for some almost impossible to fathom are the direct result of trying at all costs not to use a single equation, the most important tool in the study of ecology and trade-offs in particular, in a paper on costs of reproduction.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The manuscript of Davidsen and Sullivan describes an improved tRNA-seq protocol to determine aminoacyl-tRNA levels. The improvements include: (i) optimizing the Whitfeld or oxidation reaction to select aminoacyl-tRNAs from oxidation-sensitive non-acylated tRNAs; (ii) using a splint-assisted ligation to modify the tRNAs' ends for the following RT-PCR reaction; (iii) using an error-tolerating mapping algorithm to map the tRNA sequencing reads that contain mismatches at modified nucleotides.
The revised manuscript of Davidsen and Sullivan has addressed my concerns in the previous review. The authors performed a end-to-end comparison, which I requested - Fig. 2 and Fig S2. This is exactly what I meant, albeit the differences in each method to perform the comparison of the detectability. The manuscript is a strong methodological improvement of the tRNA quantification protocols!
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
In this study, Kashio et al examined the role of TRPV4 in regulating perspiration in mice. They find coexpression of TRPV4 with the chloride channel ANO1 and aquaporin 5, which implies possible coupling of heat sensing through TRPV4 to ion and water excretion through the latter channels. Calcium imaging of eccrine gland cells revealed that the TRPV4 agonist GSK101 activates these cells in WT mice, but not in TRPV4 KO. This effect is reduced with cold-stimulating menthol treatment. Temperature-dependent perspiration in mouse skin, either with passive heating or with ACh stimulation, was reduced in TRPV4 KO mice. Functional studies in mice - correlating the ability to climb a slippery slope to properly regulate skin moisture levels - reveal potential dysregulation of foot pad perspiration in TRPV4 KO mice, which had fewer successful climbing attempts. Lastly, a correlation of TRPV4 to hypohydrosis in humans was shown, as anhidrotic skin showed reduced levels of TRPV4 expression compared to normohidrotic or control skin.
Overall this is an interesting study on how TRPV4 regulates perspiration.
(1) The functional relationship between TRPV3 and ANO1 remains correlative.
(2) Littermate controls were not used, but TRPV4ko were backcrossed onto the WT strain.
(3) In general, the results support the authors' claims that TRPV4 activity is a necessary component of sweat gland secretion, which may have important implications for controlling perspiration; secretion from other glands where TRPV4 may be expressed remains a possibility given the lack of us of exocrine-specific knockouts.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this study, Gu at al., investigated the role of the central noradrenaline system from LC to VLPO in the recovery of consciousness induced by midazolam. Combining pharmacology, optogenetics/chemogenetics, they found that the LC to VLPO NE circuits are essential for consciousness rebooting after midazolam, activation of this circuit strongly speeded up the recovery process, dependent on alpha1 adrenergic receptors in the VLPO neurons. The topic is important and their findings are of some interest.<br /> However, substantial improvements are needed in the language, for grammar, clarity, and layout. There are significant experimental errors (see below 1-2). Further experiments are required to support their main conclusions.
(1) One major issue arises in Figure 4, the recording of VLPO Ca2+ activity. In Lines 211-215, they stated that they injected AAV2/9-DBH-GCaMP6m into the VLPO, while activating LC NE neurons. As they claimed in line 157, DBH is a specific promoter for NE neurons. This implies an attempt to label NE neurons in the VLPO, which is problematic because NE neurons are not present in the VLPO. This raises concerns about their viral infection strategy since Ca activity was observed in their photometry recording. This means that DBH promoter could randomly label some non-NE neurons. Is DBH promoter widely used? The authors should list references. Additionally, they should quantify the labeling efficiency of both DBH and TH-cre throughout the paper.<br /> (2) A similar issue arises with chemogenetic activation in Fig. 5 L-R, the authors used TH-cre and DIO-Gq virus to label VLPO neurons. Were they labelling VLPO NE or DA neurons for recording? The authors have to clarify this.<br /> (3) Another related question pertains to the specificity of LC NE downstream neurons in the VLPO. For example, do they preferentially modulate GABAergic or glutamatergic neurons?<br /> (4) In Figure 1A-D, in the measurement of the dosage-dependent effect of Mida in LORR, were they only performed one batch of testing? If more than one batch of mice were used, error bar should be presented in 1B. Also, the rationale of testing TH expression levels after Mid is not clear. Is TH expression level change related to NE activation specifically? If so, they should cite references.<br /> (5) Regarding the photometry recording of LC NE neurons during the entire process of midazolam injection in Fig. 2 and Fig. 4, it is unclear what time=0 stands for. If I understand correctly, the authors were comparing spontaneous activity during the four phases. Additionally, they only show traces lasting for 20s in Fig. 2F and Fig. 4L. How did the authors select data for analysis, and what criteria were used? The authors should also quantify the average Ca2+ activity and Ca2+ transient frequency during each stage instead of only quantifying Ca2+ peaks. In line 919, the legend for Figure 2D, they stated that it is the signal at the BLA; were they also recorded from the BLA?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this MS, Muenker and colleagues, explore the intracellular mechanics of a range of animal adherent cells. The study is based on the use of an optical tweezer set up, which allows to apply oscillatory forces on endocytosed/phagocytosed glass beads with a large frequency range (from ~1 to 1000 Hz) , allowing to probe cytoplasm material properties at multiple time scales. By switching off the laser trap, the authors also record the positional fluctuations of beads, to extract passive rheological signatures. The combination of both methods allow to fit 6 parameters (from power law fits) that allow to characterize the viscous and elastic nature of the cytoplasm material as well as an effective active energy driven by cellular metabolism. Using these methodologies, the authors first establish/confirm, using HeLa cells, that the cytoplasm is more solid like at short frequencies, and more fluid like at higher frequencies, and that these material states depend on both microtubules and actin cytoskeleton. The manuscript then go on to explore how these parameters evolve in other 6 cell types including muscles, highly migratory and epithelial cells. These results show for instance that muscle cells are much stiffer, while migratory cells are more fluid like with an increased active energy. Finally using statistical methods and principal component analysis, the authors establish some mechanical fingerprints (activity, fluidity and resistance) that allow to distinguish cell's mechanical state and relate it to their particular functions.
Strengths:
Overall this is a very well-executed work, which provides a large body of rigorous numbers and data to understand the regulation of cytoplasm mechanics and its relation to cell state/function.
Weaknesses:
A limit of the paper is that the biological mechanisms by which intracellular mechanics is modulated (e.g. among cell types) remains unexplored and only briefly discussed. Yet this limit is greatly offset by the rigor of the approach.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this study, Kroll et al. conduct an in-depth behavioral analysis of F0 knockouts of 4 genes associated with late-onset Alzheimer's Disease (AD), together with 3 genes associated with early-onset AD. Kroll and colleagues developed a web application (ZOLTAR) to compare sleep-associated traits between genetic mutants with those obtained from a panel of small molecules to promote the identification of affected pathways and potential therapeutic interventions. The authors make a set of potentially important findings vis-à-vis the relationship between AD-associated genes and sleep. First, they find that loss-of-function in late-onset AD genes universally results in nighttime sleep loss, consistent with the well-supported hypothesis that sleep disruption contributes to Alzheimer's-related pathologies. psen-1, an early-onset associated AD gene, which the authors find is principally responsible for the generation of AB40 and AB42 in zebrafish, also shows a slight increase in activity at night and slight decreases in nighttime sleep. Conversely, psen-2 mutations increase daytime sleep, while appa/appb mutations have no impact on sleep. Finally, using ZOLTAR, the authors identify serotonin receptor activity as potentially disrupted in sorl1 mutants, while betamethasone is identified as a potential therapeutic to promote reversal of psen2 knockout-associated phenotypes.
This is a highly innovative and thorough study, yet a handful of key questions remain. First, are nighttime sleep loss phenotypes observed in all knockouts for late-onset AD genes in the larval zebrafish a valid proxy for AD risk? For those mutants that cause nighttime sleep disturbances, do these phenotypes share a common underlying pathway? e.g. Do 5-HT reuptake inhibitors promote sleep across all 4 late-onset genes in addition to psen1? Can 5-HT reuptake inhibitors reverse other AD-related pathologies in zebrafish? Can compounds be identified that have a common behavioral fingerprint across all or multiple AD risk genes? Do these modify sleep phenotypes? Finally, the web-based platform presented could be expanded to facilitate comparison of other behavioral phenotypes, including stimulus-evoked behaviors. Finally, the authors propose but do not test the hypothesis that sorl1 might regulate localization/surface expression of 5-HT2 receptors. This could provide exciting / more convincing mechanistic support for the assertion that serotonin signaling is disrupted upon loss of AD-associated genes. Despite these important considerations, this study provides a valuable platform for high-throughput analysis of sleep phenotypes and correlation with small-molecule-induced sleep phenotypes.
Strengths:
- Provides a useful platform for comparison of sleep phenotypes across genotypes/drug manipulations.
- Presents convincing evidence that nighttime sleep is disrupted in mutants for multiple late-onset AD-related genes.
- Provides potential mechanistic insights for how AD-related genes might impact sleep and identifies a few drugs that modify their identified phenotypes
Weaknesses:
- Exploration of potential mechanisms for serotonin disruption in sorl1 mutants is limited.
- The pipeline developed can only be used to examine sleep-related / spontaneous movement phenotypes and stimulus-evoked behaviors are not examined.
- Comparisons between mutants/exploration of commonly affected pathways are limited.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this study, Hoops et al. showed that Netrin-1 and UNC5c can guide dopaminergic innervation from nucleus accumbens to cortex during adolescence in rodent models. They found that these dopamine axons project to the prefrontal cortex in a Netrin-1 dependent manner and knocking down Netrin-1 disrupted motor and learning behaviors in mice. Furthermore, the authors used hamsters, a seasonal model that is affected by the length of daylight, to demonstrate that the guidance of dopamine axons is mediated by the environmental factor such as daytime length and in sex dependent manner.
Regarding the cell type specificity of Netrin-1 expression, the authors began by stating "this question is not the focus of the study and we consider it irrelevant to the main issue we are addressing, which is where in the forebrain regions we examined Netrin-1+ cells are present." This statement contradicts the exact issue regarding the specificity issue I raised. They then went on to show the RNAscope data for Netriin-1 in Figure 2, which showed Netrin-1 mRNA was actually expressed quite ubiquitously in anterior cingulate cortex, dorsopeduncular cortex, infralimbic cortex, prelimbic cortex, etc. In addition, contrary to the authors' statement that Netrin-1 is a "secreted protein", the confocal images in Figure 1 in the rebuttal letter actually show Netrin-1 present in "granule-like" organelles inside the cytoplasm of neurons. Finally, the authors presented Figure 7 to indicate the location where virus expressing Netrin-1 shRNA might be located. Again, the brain region targeted was quite focal and most likely did not cover all the Netrin-1+ brain regions in Figure 2. Collectively, these results raised more questions regarding the specificity of Netrin-1 expression in brain regions that are behaviorally relevant to this study.
With respect to the effectiveness of Netrin-1 knockdown in the animals in this study, the authors cited data in HEK293 cells (Figure 5), which did not include any statistics, and previously published in vivo data in a separate, independent study (Figure 6). They do not provide any data regarding the effectiveness of Netrin-1 knockdown in THIS study.
Similar concerns regarding UNC5C knockdown (points #6, #7, and #8) were not adequately addressed.
In brief, while this study provides a potential role of Netrin-1-UNC5C in target innervation of dopaminergic neurons and its behavioral output in risk-taking, the data lack sufficient evidence to firmly establish the cause-effect relationship.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study trained a CNN for visual word classification and supported a model that can explain key functional effects of the evoked MEG response during visual word recognition, providing an explicit computational account from detection and segmentation of letter shapes to final word-form identification.
Strengths:
This paper not only bridges an important gap in modeling visual word recognition, by establishing a direct link between computational processes and key findings in experimental neuroimaging studies, but also provides some conditions to enhance biological realism.
Weaknesses:
The interpretation of CNN results, especially the number of layers in the final model and its relationship with the processing of visual words in the human brain, needs to be further strengthened.
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors are looking to assess fragmentomics effects using the Delfi method in exonic regions (Exome sequencing). They argue that this is to make the test more cost effective by extracting this information from exome sequencing.
Strengths:
Well written and explained. Different ML approaches tried.
Weaknesses:
To assess fragmentomics in WES, it doesn't seem valid to downsample WGS. WES is generated by a different library preparations so to answer this question, it would be necessary to try this in WES samples. The coverage of WES is generally done much higher because this is necessary to assess mutation calls therefore the approach of combining seems flawed because these were not generated by the same experiment.
The authors do not really show why they included longer fragment sizes in their model that had previously been excluded from the original Delfi publication
As a proof of concept this is a good idea but really needs a bit of a rethink on the utility and impact.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The study used root tips from semi-hydroponic tea seedlings. The strategy followed sequential steps to draw partial conclusions.
Initially, protoplasts obtained from root tips were processed for scRNA-seq using the 10x Genomics platform. The sequencing data underwent pre-filtering at both the cell and gene levels, leading to 10,435 cells. These cells were then classified into eight clusters using t-SNE algorithms. The present study scrutinised cell typification through protein sequence similarity analysis of homologs of cell type marker genes. The analysis was conducted to ensure accuracy using validated genes from previous scRNA-seq studies and the model plant Arabidopsis thaliana. The cluster cell annotation was confirmed using in situ RT-PCR analyses. This methodology provided a comprehensive insight into the cellular differentiation of the sample under study. The identified clusters, spanning 1 to 8, have been accurately classified as xylem, epidermal, stem cell niche, cortex/endodermal, root cap, cambium, phloem, and pericycle cells.
Then, the authors performed a pseudo-time analysis to validate the cell cluster annotation by examining the differentiation pathways of the root cells. Lastly, they created a differentiation heatmap from the xylem and epidermal cells and identified the biological functions associated with the highly expressed genes.
Upon thoroughly analysing the scRNA-seq data, the researchers delved into the cell heterogeneity of nitrate and ammonium uptake, transport, and nitrogen assimilation into amino acids. The scRNA-seq data was validated by in situ RT-PCR. It allows the localisation of glutamine and alanine biosynthetic enzymes along the cell clusters and confirms that both constituent the primary amino acid metabolism in the root. Such investigation was deemed necessary due to the paramount importance of these processes in theanine biosynthesis since this molecule is synthesised from glutamine and alanine-derived ethylamine.
Afterwards, the authors analysed the cell-specific expression patterns of the theanine biosynthesis genes, combining the same molecular tools. They concluded that theanine biosynthesis is more enriched in cluster 8 "pericycle cells" than glutamine biosynthesis (Lines 271-272). However, the statement made in Line 250 states that the highest expression levels of genes responsible for glutamine biosynthesis were observed in Clusters 1, 3, 4, 6, and 8, leading to an unclear conclusion.
The regulation of theanine biosynthesis by the MYB transcription factor family is well-established. In particular, CsMYB6, a transcription factor expressed specifically in roots, has been found to promote theanine biosynthesis by binding to the promoter of the TSI gene responsible for theanine synthesis. However, their findings indicate that CsMYB6 expression is present in Cluster 3 (SCN), Cluster 6 (cambium cells), and Cluster 1 (xylem cells) but not in Cluster 8 (pericycle cells), which is known for its high expression of CsTSI. Similarly, their scRNA-seq data indicated that CsMYB40 and CsHHO3, which activate and repress CsAlaDC expression, respectively, did not show high expression in Cluster 1 (the cell cluster with high CsAlaDC expression). Based on these findings, the authors hypothesised that transcription factors and target genes are not necessarily always highly expressed in the same cells. Nonetheless, additional evidence is essential to substantiate this presumption.
Lastly, the authors have discovered a novel transcription factor belonging to the Lateral Organ Boundaries Domain (LBD) family known as CsLBD37 that can co-regulate the synthesis of theanine and the development of lateral roots. The authors observed that CsLBD37 is located within the nucleus and can repress the CsAlaDC promoter's activity. To investigate this mechanism further, the authors conducted experiments to determine whether CsLBD37 can inhibit CsAlaDC expression in vivo. They achieved this by creating transiently CsLBD37-silenced or over-expression tea seedlings through antisense oligonucleotide interference and generation of transgenic hairy roots. Based on their findings, the authors hypothesise that CsLBD37 regulates CsAlaDC expression to modulate the synthesis of ethylamine and theanine.
Additionally, the available literature suggests that the transcription factors belonging to the Lateral Organ Boundaries Domain (LBD) family play a crucial role in regulating the development of lateral roots and secondary root growth. Considering this, they confirmed that pericycle cells exhibit a higher expression of CsLBD37. A recent experiment revealed that overexpression of CsLBD37 in transgenic Arabidopsis thaliana plants led to fewer lateral roots than the wild type. From this observation, the researchers concluded that CsLBD37 regulates lateral root development in tea plants. I respectfully submit that the current conclusion may require additional research before it can be considered definitive.
Further efforts should be made to investigate the signalling mechanisms that govern CsLBD37 expression to arrive at a more comprehensive understanding of this process. In the context of Arabidopsis lateral root founder cells, the establishment of asymmetry is regulated by LBD16/ASL18 and other related LBD/ASL proteins, as well as the AUXIN RESPONSE FACTORs (ARF7 and ARF19). This is achieved by activating plant-specific transcriptional regulators such as LBD16/ASL18 (Go et al., 2012, https://doi.org/10.1242/dev.071928). On the other hand, other downstream homologues of LBD genes regulated by cytokinin signalling play a role in secondary root growth (Ye et al., 2021, https://doi.org/10.1016/j.cub.2021.05.036). It is imperative to shed light on the hormonal regulation of CsLBD37 expression in order to gain a comprehensive understanding of its involvement in the morphogenic process.
Strength:
The manuscript showcases significant dedication and hard work, resulting in valuable insights that serve as a fundamental basis for generating knowledge. The authors skillfully integrated various tools available for this type of study and meticulously presented and illustrated every step involved in the survey. The overall quality of the work is exceptional, and it would be a valuable addition to any academic or professional setting.
Weaknesses:
In its current form, the article presents certain weaknesses that need to be addressed to improve its overall quality. Specifically, the authors' conclusions appear to have been drawn in haste without sufficient experimental data and a comprehensive discussion of the entire plant. It is strongly advised that the authors devote additional effort to resolving the abovementioned issues to bolster the article's credibility and dependability. This will ensure that the article is of the highest quality, providing readers with reliable and trustworthy information.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this study, Sarver and colleagues carried out an exhaustive analysis of the functioning of various components (Complex I/II/IV) of the mitochondrial electron transport chain (ETC) using a real-time cell metabolic analysis technique (commonly referred as Seahorse oxygen consumption rate (OCR) assay). The authors aimed to generate an atlas of ETC function in about 3 dozen tissue types isolated from all major mammalian organ systems. They used a recently published improvised method by which ETC function can be quantified in freshly frozen tissues. This method enabled them to collect data from almost all organ systems from the same mouse and use many biological replicates (10 mice/experiment) required for an unbiased and statistically robust analysis. Moreover, they studied the influence of sex (male and female) and aging (young adult and old age) on ETC function in these organ systems. The main findings of this study are (1) cells in the heart and kidneys have very active ETC complexes compared to other organ systems, (2) the sex of the mice has little influence on the ETC function, and (3) aging undermined the mitochondrial function in most tissue, but surprisingly in some tissue aging promoted the activity of ETC complexes (e.g., Quadriceps, plantaris muscle, and Diaphragm). Although this study provides a comprehensive outlook on the ETC function in various tissues, the main caveat is that it's too technical and descriptive. The authors didn't invest much effort in putting their findings in the context of the biological function of the tissue analyzed, i.e., some tissues might be more glycolytic than others and have low ETC activity. Also, it is unclear what slight changes in the activity of one or the other ETC complex mean in terms of mitochondrial ATP production. Likely, these small changes reported do not affect the mitochondrial respiration. With such a detailed dataset, the study falls short of deriving more functionally relevant conclusions about the heterogeneity of mitochondrial function in various tissues. In the current format, the readers get lost in the large amount of data presented in a technical manner. Also, it is highly recommended that all the raw data and the values be made available as an Excel sheet (or other user-friendly formats) as a resource to the community.
Major concerns
(1) In this study, the authors used the method developed by Acin-Perez and colleagues (EMBO J, 2020) to analyze ETC complex activities in mitochondria derived from the snap-frozen tissue samples. However, the preservation of cellular/mitochondrial integrity in different types of tissues after being snap-frozen was not validated. Additionally, the conservation of mitochondrial respiration in snap-frozen tissues might differ, especially in those derived from old mice. For example, quadriceps (young male/female), plantaris (young male/female), intestinal segments (duodenum), and pancreas preparations show almost no activity (nearly flat OCR in Seahorse assays). For such a comprehensive study, the author must at least validate those tissues where the OCR plots looked suboptimal with the mitochondrial preparations derived from the fresh tissue. Since aging has been identified as the most important effector in this study, it is essential to validate how aging affects respiration in various fresh frozen tissues. Such analysis will ensure that the results presented are not due to the differential preservation of the mitochondrial respiration in the frozen tissue. In addition, such validations will further strengthen the conclusions and promote the broad usability of this "new" method.
(2) In this study, the authors sampled the maximal activity of ETC complex I, II, and IV, but throughout the manuscript, they discussed the data in the context of mitochondrial function. However, it is unclear how the changes in CI, CII, and CIV activity affect overall mitochondrial function (if at all) and how small changes seen in the maximal activity of one or more complexes affect the efficiency and efficacy of ATP production (OxPhos). The authors report huge variability between the activity of different complexes - in some tissues all three complexes (CI, CII, and CIV) and often in others, just one complex was affected. For example, as presented in Figure 4, there is no difference in CI activity in the hippocampus and cerebellum, but there is a slight change in CII and CIV activity. In contrast, in heart atria, there is a change in the activity of CI but not in CII and CIV. However, the authors still suggest that there is a significant difference in mitochondrial activity (e.g., "Old males showed a striking increase in mitochondrial activity via CI in the heart atria....reduced mitochondrial respiration in the brain cortex..." - Lines 5-7, Page 9). Until and unless a clear justification is provided, the authors should not make these broad claims on mitochondrial respiration based on small changes in the activity of one or more complexes (CI/CII/CIV). With such a data-heavy and descriptive study, it is confusing to track what is relevant and what is not for the functioning of mitochondria.
(3) What do differences in the ETC complex CI, CII, and CIV activity in the same tissue mean? What role does the differential activity of these complexes (CI, CII, and CIV) play in mitochondrial function? What do changes in Oxphos mean for different tissues? Does that mean the tissue (cells involved) shift more towards glycolysis to derive their energy? In the best world, a few experiments related to the glycolytic state of the cells would have been ideal to solidify their finding further. The authors could have easily used ECAR measurements for some tissues to support their key conclusions.
(4) The authors further analyzed parameters that significantly changed across their study (Figure 7, 98 data points analyzed). The main caveat of such analysis is that some tissue types would be represented three or even more times (due to changes in the activity of all three complexes - CI, CII, and CIV, and across different ages and sexes), and some just once. Such a method of analysis will skew the interpretation towards a few over-represented organ/tissue systems. Perhaps the authors should separately analyze tissue where all three complexes are affected from those with just one affected complex.
(5) The current protocol does not provide cell-type-specific resolution and will be unable to identify the cellular source of mitochondrial respiration. This becomes important, especially for those organ systems with tremendous cellular heterogeneity, such as the brain. The authors should discuss whether the observed changes result from an altered mitochondria respiratory capacity or if changes in proportions of cell types in the different conditions studied (young vs. aged) might also contribute to differential mitochondrial respiration.
(6) Another critical concern of this study is that the same datasets were repeatedly analyzed and reanalyzed throughout the study with almost the same conclusion - namely, aging affects mitochondrial function, and sex-specific differences are limited to very few organs. Although this study has considerable potential, the authors missed the chance to add new insights into the distinct characteristics of mitochondrial activity in various tissue and organ systems. The author should invest significant efforts in putting their data in the context of mitochondrial function.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary
Type 1 diabetes mellitus (T1DM) progression is accelerated by oxidative stress and apoptosis. Eugenol (EUG) is a natural compound previously documented as anti-inflammatory, anti-oxidative, and anti-apoptotic. In this manuscript by Jiang et al., the authors study the effects of EUG on T1DM in MIN6 insulinoma cells and a mouse model of chemically induced T1DM. The authors show that EUG increases nuclear factor E2-related factor 2 (Nrf2) levels. This results in a reduction of pancreatic beta-cell damage, apoptosis, oxidative stress markers, and a recovery of insulin secretion. The authors highlight these effects as indicative of the therapeutic potential of EUG in managing T1DM.
Strengths
Relevant, timely, and addresses an interesting question in the field. The authors consistently observe enhanced beta cell functionality following EUG treatment, which makes the compound a promising candidate for T1DM therapy.
Weaknesses
The in vivo experiments have too few biological replicates. With an n=3 (as all figure legends indicate) in complex mouse studies such as these, drawing robust conclusions becomes challenging. It is important to reproduce these results in a larger cohort, to validate the conclusions of the authors. Another big concern is the lack of quantifications and statistical analysis throughout the manuscript. Although the authors claim statistical significance in various experiments, the limited information provided makes it difficult to verify. The authors use vague and minimal descriptions of their experiments, which further reduces the reader's comprehension and the reproducibility of the experiments. Finally, the use of Min6 cells as a model for pancreatic beta cells is a strong limitation of this study. Future studies should seek to reproduce these findings in a more translational model and use more relevant in vitro cell systems (eg. Islets).
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors used a coarse-grained DNA model (cgNA+) to explore how DNA sequences and CpG methylation/hydroxymethylation influence nucleosome wrapping energy and the probability density of optimal nucleosomal configuration. Their findings indicate that both methylated and hydroxymethylated cytosines lead to increased nucleosome wrapping energy. Additionally, the study demonstrates that methylation of CpG islands increases the probability of nucleosome formation.
Strengths:
The major strength of this method is that the model explicitly includes elastic constraints on the positions of phosphate groups facing a histone octamer, as DNA-histone binding site constraints. The authors claim that their model enhances the accuracy and computational efficiency and allows comprehensive calculations of DNA mechanical properties and deformation energies.
Weaknesses:
A significant limitation of this study is that the parameter sets for the methylated and hydroxymethylated CpG steps in the cgNA+ model are derived from all-atom molecular dynamics (MD) simulations that suggest that both methylated and hydroxymethylated cytosines increase DNA stiffness and nucleosome wrapping energy (Pérez A, et al. Biophys J. 2012; Battistini, et al. PLOS Comput Biol. 2021). It could predispose the coarse-grained model to replicate these findings. Notably, conflicting results from other all-atom MD simulations, such as those by Ngo T in Nat. Commun. 2016, shows that hydroxymethylated cytosines increase DNA flexibility, contrary to methylated cytosines. If the cgNA+ model was trained on these later parameters or other all-atom force fields, different conclusions might be obtained regarding the effects of methylated and hydroxymethylation on nucleosome formation.
Despite the training parameters of the cgNA+ model, the results presented in the manuscript indicate that methylated cytosines increase both DNA stiffness and nucleosome wrapping energy. However, when comparing nucleosome occupancy scores with predicted nucleosome wrapping energies and optimal configurations, the authors find that methylated CGIs exhibit higher nucleosome occupancies than unmethylated ones, which seems to contradict their findings from the same paper which showed that increased stiffness should reduce nucleosome formation affinity. In the manuscript, the authors also admit that these conclusions "apparently runs counter to the (perhaps naive) intuition that high nucleosome forming affinity should arise for fragments with low wrapping energy". Previous all-atom MD simulations (Pérez A, et al. Biophys J. 2012; Battistini, et al. PLOS Comput Biol. 202; Ngo T, et al. Nat. Commun. 20161) show that the stiffer DNA upon CpG methylation reduces the affinity of DNA to assemble into nucleosomes or destabilizes nucleosomes. Given these findings, the authors need to address and reconcile these seemingly contradictory results, as the influence of epigenetic modifications on DNA mechanical properties and nucleosome formation are critical aspects of their study.<br /> Understanding the influence of sequence-dependent and epigenetic modifications of DNA on mechanical properties and nucleosome formation is crucial for comprehending various cellular processes. The authors' study, focusing on these aspects, will definitely garner interest from the DNA methylation research community.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
HMGCS1, 3-hydroxy-3-methylglutaryl-CoA synthase1 is predicted to be involved in Acetyl-CoA metabolic process and mevalonate-cholesterol pathway. To induce diet-induced diabetes, they fed wild-type littermates either a standard chow (Control) or a high fat-high sucrose (HFHG) diet, where the diet composition consisted of 60% fat, 20% protein, and 20% carbohydrate (H10060, Hfkbio, China). The dietary regimen was maintained for 14 weeks. Throughout this period, body weight and fasting blood glucose (FBG) levels were measured on a weekly basis. Although the authors induced diabetes with a diet also rich in fat, the cholesterol concentration or metabolism was not investigated. After the treatment, were the animals with endothelial dysfunction? How was the blood pressure of the animals?
Strengths:
To explore the potential role of circHMGCS1 in regulating endothelial cell function, the authors cloned exons 2-7 of HMGCS1 into lentiviral vectors for ectopic overexpression of circHMGCS1 (Figure S2). The authors could use this experiment as a concept proof and investigate the glucose concentration in the cell culture medium. Is the pLV-circ HMGCS1 transduction in HUVEC increasing the glucose release? (Line 163)
Weaknesses:
(1) Pg 20. The cells were transfected with miR-4521 mimics, miR-inhibitor, or miR-NC and incubated for 24 hours. Subsequently, the cells were treated with PAHG for another 24 hours.
Were the cells transfected with lipofectanine? The protocol or the lipofectamine kit used should be described. The lipofectamine protocol suggests using an incubation time of 72 hours. Why did the authors incubate for only 24 hours?
If the authors did the mimic and inhibitor curves, these should be added to the supplementary figures. Please, describe the miRNA mimic and antagomir concentration used in cell culture.
(2) Pg 20, line 507. What was the miR-4521 agomiR used to treatment of the animals?
(3) Figure 1B. The results are showing the RT-qPCR for only 5 circRNA, however, the results show 48 circRNAs were upregulated, and 18 were downregulated (Figure S1D). Why were the other cicRNAs not confirmed? The circRNAs upregulated with high expression are not necessarily with the best differential expression comparing control vs. PAHG groups. Furthermore, Figure 1A and S1D show circRNAs downregulated also with high expression. Why were these circRNAs not confirmed?
(4) Figure 1B shows the relative circRNAs expression. Were host genes expressed in the same direction?
(5) Line 128. The circRNA RT-qPCR methodology was not described. The methodology should be described in detail in the Methods Session.
(6) Line 699. The relative gene expression was calculated using the 2-ΔΔCt method. This is not correct, the expression for miRNA and gene expression are represented in percentage of control.
(7) Line 630. Detection of ROS for tissue and cells. The methodology for tissue was described, but not for cells.
(8) Line 796. RNA Fluorescent In Situ Hybridization (RNA-FISH). Figure 1F shows that the RNA-Fluorescence in situ hybridization (RNA-FISH) confirmed the robust expression of cytoplasmic circHMGCS1 in HUVECs (Figure 1F). However, in the methods, lines 804 and 805 described the probes targeting circMAP3K5 and miR-4521 were applied to the sections. Hybridization was performed in a humid chamber at 37{degree sign}C overnight. Is it correct?
(9) Line 14. Fig 1-H. The authors discuss qRT-PCR demonstrated that circHMGCS1 displayed a stable half-life exceeding 24 h, whereas the linear transcript HMGCS1 mRNA had a half-life less than 8 h (Figure 1H).<br /> Several of the antibodies may contain trace amounts of RNases that could degrade target RNA and could result in loss of RNA hybridization signal or gene expression. Thus, all of the solutions should contain RNase inhibitors. The HMGCS1 mRNA expression could be degraded over the incubation time (0-24hs) leading to incorrect results. Moreover, in the methods is not mentioned if the RNAse inhibitor was used. Please, could the authors discuss and provide information?
(10) Further experiments demonstrated that the overexpression of circHMGCS1 stimulated the expression of adhesion molecules (VCAM1, ICAM1, and ET-1) (Figures 2B and 2C), suggesting that circHMGCS1 is involved in VED. How were these genes expressed in the RNA-seq?
(11) Line 256. By contrast, the combined treatment of circHMGCS1 and miR-4521 agomir did not significantly affect the body weight and blood glucose levels. OGTT and ITT experiments demonstrated that miR-4521 agomir considerably enhanced glucose tolerance and insulin resistance in diabetic mice (Figures 5C, 5D, and Figures S5B and S5C). Why didi the miR-4521 agomir treatment considerably enhance glucose tolerance and insulin resistance in diabetic mice, but not the blood glucose levels?
(12) In the experiments related to pull-down, the authors performed Biotin-coupled miR-4521 or its mutant probe, which was employed for circHMGCS1 pull-down. This result only confirms the Luciferase experiments shown in Figure 4A. The experiment that the authors need to perform is pull-down using a biotin-labeled antisense oligo (ASO) targeting the circHMGCS1 backsplice junction sequence followed by pulldown with streptavidin-conjugated magnetic beads to capture the associated miRNAs and RNA binding proteins (RBPs). Also, the ASO pulldown assay can be coupled to miRNA RT-qPCR and western blotting analysis to confirm the association of miRNAs and RBPs predicted to interact with the target circRNA.
(13) In Figure 5, the authors showed that the results suggest that miR-4521 can inhibit the occurrence of diabetes, whereas circHMGCS1 specifically dampens the function of miR-4521, weakening its protective effect against diabetes. In this context, what are the endogenous target genes for the miR-4521 that could be regulating diabetes?
(14) In the western blot of Figure 5, the β-actin band appears to be different from the genes analyzed. Was the same membrane used for the four proteins? The Ponceau S membrane should be provided.
(15) Why did the authors use AAV9, since the AAV9 has a tropism for the liver, heart, skeletal muscle, and not to endothelial vessels?
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
Summary:
This is a well-written and detailed manuscript showing important results on the molecular profile of 4 different cohorts of female patients with lung cancer.
The authors conducted comprehensive multi-omic profiling of air-pollution-associated LUAD to study the roles of the air pollutant BaP. Utilizing multi-omic clustering and mutation-informed interface analysis, potential novel therapeutic strategies were identified.
Strengths:
The authors used several different methods to identify potential novel targets for therapeutic interventions.
Weaknesses:
Statistical test results need to be provided in comparisons between cohorts.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This manuscript reports the substrate-bound structure of SiaQM from F. nucleatum, which is the membrane component of a Neu5Ac-specific Tripartite ATP-dependent Periplasmic (TRAP) transporter. Until recently, there was no experimentally derived structural information regarding the membrane components of the TRAP transporter, limiting our understanding of the transport mechanism. Since 2022, there have been 3 different studies reporting the structures of the membrane components of Neu5Ac-specific TRAP transporters. While it was possible to narrow down the binding site location by comparing the structures to proteins of the same fold, a structure with substrate bound has been missing. In this work, the authors report the Na+-bound state and the Na+ plus Neu5Ac state of FnSiaQM, revealing information regarding substrate coordination. In previous studies, 2 Na+ ion sites were identified. Here, the authors also tentatively assign a 3rd Na+ site. The authors reconstitute the transporter to assess the effects of mutating the binding site residues they identified in their structures. Of the 2 positions tested, only one of them appears to be critical to substrate binding.
Strengths:
The main strength of this work is the capture of the substrate-bound state of SiaQM, which provides insight into an important part of the transport cycle.
Weaknesses:
The main weakness is the lack of experimental validation of the structural findings. The authors identified the Neu5Ac binding site, but only tested 2 residues for their involvement in substrate interactions, which was very limited. The authors tentatively identified a 3rd Na+ binding site, which if true would be an impactful finding, but this site was not tested for its contribution to Na+ dependent transport, and the authors themselves report that the structural evidence is not wholly convincing. This lack of experimental validation undermines the confidence of the findings. However, the reporting of these new data is important as it will facilitate follow-up studies by the authors or other researchers.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this manuscript by Wu et al., the authors present the high resolution cryoEM structures of the WT Kv1.2 voltage-gated potassium channel. Along with this structure the authors have solved several structures of mutants or experimental conditions relevant to the slow inactivation process that these channels undergo and which is not yet completely understood.
One of the main findings is the determination of the structure of a mutant (W366F) that is thought to correspond to the slow inactivated state. These experiments confirm results in similar mutants in different channels from Kv1.2 that indicate that inactivation is associated with an enlarged selectivity filter.
Another interesting structure is the complex of Kv1.2 with the pore blocking toxin Dendrotoxin 1. The results shown in the revised version indicate that the mechanism of block is similar to that of related blocking-toxins, in which a lysine residue penetrates in the pore. Surprisingly, in these new structures, the bound toxin results in a pore with empty external potassium binding sites.
The quality of the structural data presented in this revised manuscript is very high and allows for unambiguous assignment of side chains. The conclusions are supported by the data. This is an important contribution that should further our understanding of voltage-dependent potassium channel gating. In the revised version, the authors have addressed my previous specific comments, which are appended below.
(1) In the main text's reference to Figure 2d residues W18' and S22' are mentioned but are not labeled in the insets.
(2) On page 8 there is a discussion of how the two remaining K+ ions in binding sites S3 and S4 prevent permeation K+ in molecular dynamics. However, in Shaker, inactivated W434F channels can sporadically allow K+ permeation with normal single-channel conductance but very reduced open times and open probability at not very high voltages.
(3) The structures of WT in the absence of K+ shows a narrower selectivity filter, however Figure 4 does not convey this finding. In fact, the structure in Figure 4B is constructed in such an angle that it looks as if the carbonyl distances are increased, perhaps this should be fixed. Also, it is not clear how the distances between carbonyls given in the text on page 12 are measured. Is it between adjacent or kitty-corner subunits?
(4) It would be really interesting to know the authors opinion on the driving forces behind slow inactivation. For example, potassium flux seems to be necessary for channels to inactivate, which might indicate a local conformational change is the trigger for the main twisting events proposed here.
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munk.org munk.org
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Taming The Tinny Typing Sound of a Brother JP-1 Typewriter (Part 3 – the Finale?) by [[Theodore Munk]]
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munk.org munk.org
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Adjusting Ring & Cylinder on a Brother JP-1 by [[Theodore Munk]]
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
General comments:
This paper investigates the pH-specific enzymatic activity of mouse acidic mammalian chitinase (AMCase) and aims to elucidate its function's underlying mechanisms. The authors employ a comprehensive approach, including hydrolysis assays, X-ray crystallography, theoretical calculations of pKa values, and molecular dynamics simulations to observe the behavior of mouse AMCase and explore the structural features influencing its pH-dependent activity.
The study's key findings include determining kinetic parameters (Kcat and Km) under a broad range of pH conditions, spanning from strong acid to neutral. The results reveal pH-dependent changes in enzymatic activity, suggesting that mouse AMCase employs different mechanisms for protonation of the catalytic glutamic acid residue and the neighboring two aspartic acids at the catalytic motif under distinct pH conditions.<br /> The novelty of this research lies in the observation of structural rearrangements and the identification of pH-dependent mechanisms in mouse AMCase, offering a unique perspective on its enzymatic activity compared to other enzymes. By investigating the distinct protonation mechanisms and their relationship to pH, the authors reveal the adaptive nature of mouse AMCase, highlighting its ability to adjust its catalytic behavior in response to varying pH conditions. These insights contribute to our understanding of the pH-specific enzymatic activity of mouse AMCase and provide valuable information about its adaptation to different physiological conditions.<br /> Overall, the study enhances our understanding of the pH-dependent activity and catalytic properties of mouse AMCase and sheds light on its adaptation to different physiological pH environments.
Comments on revised version:
In their revised manuscript, the authors have made significant efforts to address the reviewers' comments.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.
However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts. Additionally, it is noted that the main claims put forth in the manuscript are only partially supported by the data presented.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This manuscript presents a pipeline incorporating a deep generative model and peptide property predictors for the de novo design of peptide sequences with dual antimicrobial/antiviral functions. The authors synthesized and experimentally validated three peptides designed by the pipeline, demonstrating antimicrobial and antiviral activities, with one leading peptide exhibiting antimicrobial efficacy in animal models. However, the manuscript as it stands, has several major limitations on the computational side.
Major issues:
(1) The choice of GAN as the generative model. There are multiple deep generative frameworks (e.g., language models, VAEs, and diffusion models), and GANs are known for their training difficulty and mode collapse. Could the authors elaborate on the specific rationale behind choosing GANs for this task?
(2) The pipeline is supposed to generate peptides showing dual properties. Why were antiviral peptides not used to train the GAN? Would adding antiviral peptides into the training lead to a higher chance of getting antiviral generations?
(3) For the antimicrobial peptide predictor, where were the contact maps of peptides sourced from?
(4) Morgan fingerprint can be used to generate amino acid features. Would it be better to concatenate ESM features with amino acid-level fingerprints and use them as node features of GNN?
(5) Although the number of labeled antiviral peptides may be limited, the input features (ESM embeddings) should be predictive enough when coupled with shallow neural networks. Have the authors tried simple GNNs on antiviral prediction and compared the prediction performance to those of existing tools?
(6) Instead of using global alignment to get match scores, the authors should use local alignment.
(7) How novel are the validated peptides? The authors should run a sequence alignment to get the most similar known AMP for each validated peptide, and analyze whether they are similar.
(8) Only three peptides were synthesized and experimentally validated. This is too few and unacceptable in this field currently. The standard is to synthesize and characterize several dozens of peptides at the very least to have a robust study.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This work shows, based on basic laboratory investigations of in vitro grown bacteria as well as human bone samples, that conventional bacterial culture can substantially underrepresent the quantity of bacteria in infected tissues. This has often been mentioned in the literature, however, relatively limited data has been provided to date. This manuscript compares culture to a digital droplet PCR approach, which consistently showed greater levels of bacteria across the experiments (and for two different strains).
Strengths:
Consistency of findings across in vitro experiments and clinical biopsies. There are real-world clinical implications for the findings of this study.
Weaknesses:<br /> No major weaknesses. Only 3 human samples were analyzed, although the results are compelling.
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
Summary:
This manuscript by Vuong and colleagues reports a study that pooled data from 3 separate longitudinal study that collectively spanned an observation period of over 15 years. The authors examined for correlation between viraemia measured at various days from illness onset with thrombocytopaenia and severe dengue, according to the WHO 2009 classification scheme. The motivation for this study is both to support the use of viraemia measurement as a prognostic indicator of dengue and also to, when an antiviral drug becomes licensed for use, guide the selection of patients for antiviral therapy. They found that the four DENVs show differences in peak and duration of viraemia and that viraemia levels before day 5 but not those after from illness onset correlated with platelet count and plasma leakage at day 7 onwards. They concluded that the viraemia kinetics call for early measurement of viraemia levels in the early febrile phase of illness.
Strengths:
This is a unique study due to the large sample size and longitudinal viraemia measurements in the study subjects. The data addresses a gap in information in the literature, where although it has been widely indicated that viraemia levels are useful when collected early in the course of illness, this is the first time anyone has systematically examined this notion. The inclusion of correlation between rate of viraemia decline and risk of severe dengue/plasma leakage further strengthens the relevance of this paper to those interested in anti-dengue therapeutic research and development.
Weaknesses:
The study only analysed data from dengue patients in Vietnam. Moreover, the majority of these patients had DENV-1 infection; few had DENV-4 infection. The data could thus be skewed by the imbalance in the prevalence of the different types of DENV during the period of observation. The use of patient-reported time of symptom onset as a reference point for viraemia measurement is pragmatic although there is subjectivity and thus noise in the data.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Authors previously demonstrated that species-specific variation in primate CD4 impacts its ability to serve as a functional receptor for diverse SIVs. Here, Warren and Barbachano-Guerrero et al. perform population genetics analyses and functional characterization of great ape CD4 with a particular focus on gorillas, which are natural hosts of SIVgor. They first used ancestral reconstruction to derive the ancestral hominin and hominid CD4. Using pseudotyped viruses representing a panel of envelopes from SIVcpz and HIV strains, they find that these ancestral reconstructions of CD4 are more similar to human CD4 in terms of being a broadly susceptible entry receptor (in the context of mediating entry into Cf2Th cells stably expressing human CCR5). In contrast, extant gorilla and chimpanzee CD4 are functional entry receptors for a narrower range of HIV and SIVcpz isolates. Based on these differences, authors next surveyed gorilla sequences and identified several CD4 haplotypes, specifically in the region encoding the CD4 D1 domain, which directly contacts the viral glycoprotein and thus may impact the interaction. Consistent with this possibility, authors demonstrated that gorilla CD4 haplotypes are, on average, less capable of supporting entry than human CD4, and that some are largely unable to function as SIV entry receptors. Interestingly, individual residues found at key positions in the gorilla CD4 D1 when tested in the context of human CD4 reduce entry of some virions pseudotyped with diverse SIVcpz envelopes, suggesting that individual amino acids can in part explain the observed differences across gorilla CD4 haplotypes. Finally, the authors perform statistical tests to infer that CD4 from great apes with endemic SIV (i.e., chimpanzees and gorillas) but not non-reservoirs (i.e., orangutans, bonobos) or recent spillover hosts (i.e., humans), have been subject to selection as a result of pressure from endemic SIV.
The conclusions of this paper are mostly well supported by data.
Strengths:
(1) The functional assays are appropriate to test the stated hypothesis, and the authors use a broad diversity of envelopes from HIV and SIVcpz strains. Authors also partially characterize one potential mechanism of gorilla CD4 resistance - receptor glycosylation at the derived N15 found in 5/6 gorilla haplotypes.
(2) Ancestral reconstruction provides a particularly interesting aspect of the study, allowing authors to infer the ancestral state of hominid CD4 relative to modern CD4 from gorillas and chimpanzees. This, coupled with evidence supporting SIV-driven selection of gorilla CD4 diversity and the characterization of functional diversity of extant haplotypes provides several interesting findings.
Weaknesses:
(3). The major inference of the work is that SIV infection of gorillas drove the observed diversity in gorilla CD4. This is supported by the majority of SNPs being localized to the CD4 D1, which directly interacts with envelope, and the demonstrated functional consequences of that diversity for viral entry. However, SIVgor (to the best of my knowledge) only infects Western lowland gorillas (Gorilla gorilla gorilla), and one Gorilla gorilla diehli and three Gorilla beringei graueri individuals were included in the haplotype and allele frequency analyses. The presence of these haplotypes or the presence of similar allele frequencies in Eastern lowland and mountain gorillas would impact this conclusion. It would be helpful for the authors to clarify this point.
(4) The authors appear to use a somewhat atypical approach to assess intra-population selection to compensate for relatively small numbers of NHP sequences (Fig. 6). However, they do not cite precedence for the robustness of the approach or the practice of grouping sequences from multiple species for the endemic vs other comparison. They also state in the methods that some genes encoded in the locus were removed from the analysis "because they have previously been shown to directly interact with a viral protein." This seems to undercut the analysis, and prevents alternative explanations for the observed diversity in CD4 (e.g., passenger mutations from selection at a neighboring locus).
(5) Data in Figure 5 is graphed as % infected cells instead of virus titer (TDU/mL). It's unclear why this is the case, and prevents a comparison to data in Figure 2 and Figure 4.
(6) The lack of pseudotyping with SIVgor envelope is a surprising omission from this study, that would help to contextualize the findings. Similarly, building gorilla CD4 haplotype SNPs onto the hominin ancestor (as opposed to extant human CD4) may provide additional insights that are meaningful towards understanding the evolutionary trajectory of gorilla CD4.
Comments on revised version:
In the revised manuscript, the authors more appropriately contextualize conclusions that can be made based on their data versus inferences, which are now much more clearly described in the discussion. The authors also included more references to substantiate claims, additional description of methodology, and provided well-reasoned responses to the weaknesses described in my primary review.
Re: #3. As the authors point out, we do not know if eastern gorillas were at one time exposed to SIV. The authors use a variety of phylogenetic and functional approaches to infer that SIVcpz is the selective pressure-shaping gorilla CD4. While I agree this is a highly likely scenario, the allelic diversity of CD4 across gorilla subpopulations raises multiple evolutionary scenarios consistent with the data.
Re: #4. The explanation provided by the authors is reasonable. However, a demonstration that this approach is robust to potential factors that might skew the data (e.g., recombination) is argued but not tested. Part of the concern here is that the study is limited by very small sample sizes, and to the best of my knowledge, grouping sequences from multiple species to make claims about selection is not an established practice. The authors note in their response that they confirmed the existence of CD4 alleles in this study with those identified in 100 gorilla individuals from Russell et al. 2021 (unavailable to the authors at the time of submission) - a re-analysis that includes that data from Russell et al. 2021 would have strengthened the analyses.
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Joint Public Review:
The premise of this work carries great potential. Namely, developing a humanized mouse system in which features of adaptive immunity that contribute to inflammatory demyelination can be interrogated will allow for traction into therapeutics currently unavailable to the field. Immediate questions stemming from the current study include the potential effect of ex vivo activation of PBMCs (or individual T and B cells) in vitro prior to transfer as well as the TCR and BCR repertoire of CNS vs peripheral lymphocytes before and after immunization. This group has been thoughtful and clever about their approach (e.g. use of subjects treated with natalizumab), which gives hope that fundamental aspects of pathogenesis will be uncovered by this form of modeling MS disease.
Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system where immune cells play an important role in disease pathobiology. Increased incidence of disease in individuals carrying certain HLA class-II genes plus studies in animal models suggests that HLA-DRB1*15 restricted CD4 T cells might be responsible for disease initiation, and other immune cells such as B cells, CD8 T cells, monocytes/macrophages, and dendritic cells (DC) also contribute to disease pathology. However, a direct role of human immune cells in disease is lacking to a lag between immune activation and the first sign of clinical disease. Therefore, there is an emphasis on understanding whether immune cells from HLA-DR15+ MS patients differ from HLA-DR15+ healthy controls in their phenotype and pro-inflammatory capacity. To overcome this, authors have used severely immunodeficient B2m-NOG mice that lack B, T cells and NK cells and have defective innate immune responses and engrafted PBMCs from 3 human donors (HLA-DR15+ MS and HI donors, HLA-DR13+ MS donor) in these B2m-NOG mice to determine whether they can induce CNS inflammation and demyelination like MS.
The study's strength is the use of PBMCs from HLADRB1-typed MS subjects and healthy control, the use of NOG mice, the characterization of immune subsets (revealing some interesting observations), CNS pathology etc. Weaknesses are lack of phenotype in mice and no disease phenotype even in humanized mice immunized for disease using standard disease induction protocol employed in an animal model of MS, and lack of mechanistic data on why CD8 T cells are more enriched than CD4+ T cells. The last point is important as postmortem human MS patients' brain tissue had been shown to have more CD8+ T cells than CD4+ T cells.
Thus, this work is an important step in the right direction as previous humanized studies have not used HLA-DRB1 typed PBMCs however the weaknesses as highlighted above are limitations in the model.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
I'll begin by summarizing what I understand from the results presented, and where relevant how my understanding seems to differ from the authors' claims. I'll then make specific comments with respect to points raised in my previous review (below), using the same numbering. Because this is a revision I'll try to restrict comments here to the changes made, which provide some clarification, but leave many issues incompletely addressed.
As I understand it the main new result here is that certain recurrent network architectures promote emergence of coordinated grid firing patterns in a model previously introduced by Kropff and Treves (Hippocampus, 2008). The previous work very nicely showed that single neurons that receive stable spatial input could 'learn' to generate grid representations by combining a plasticity rule with firing rate adaptation. The previous study also showed that when multiple neurons were synaptically connected their grid representations could develop a shared orientation, although with the recurrent connectivity previously used this substantially reduced the grid scores of many of the neurons. The advance here is to show that if the initial recurrent connectivity is consistent with that of a line attractor then the network does a much better job of establishing grid firing patterns with shared orientation.
Beyond this point, things become potentially confusing. As I understand it now, the important influence of the recurrent dynamics is in establishing the shared orientation and not in its online generation. This is clear from Figure S3, but not from an initial read of the abstract or main text. This result is consistent with Kropff and Treves' initial suggestion that 'a strong collateral connection... from neuron A to neuron B... favors the two neurons to have close-by fields... Summing all possible contributions would result in a field for neuron B that is a ring around the field of neuron A.' This should be the case for the recurrent connections now considered, but the evidence provided doesn't convincingly show that attractor dynamics of the circuit are a necessary condition for this to arise. My general suggestion for the authors is to remove these kind of claims and to keep their interpretations more closely aligned with what the results show.
Major (numbered according to previous review)
(1) Does the network maintain attractor dynamics after training? Results now show that 'in a trained network without feedforward Hebbian learning the removal of recurrent collaterals results in a slight increase in gridness and spacing'. This clearly implies that the recurrent collaterals are not required for online generation of the grid patterns. This point needs to be abundantly clear in the abstract and main text so the reader can appreciate that the recurrent dynamics are important specifically during learning.<br /> (2) Additional controls for Figure 2 to test that it is connectivity rather than attractor dynamics (e.g. drawing weights from Gaussian or exponential distributions). The authors provide one additional control based on shuffling weights. However, this is far from exhaustive and it seems difficult on this basis to conclude that it is specifically the attractor dynamics that drive the emergence of coordinated grid firing.<br /> (3) What happens if recurrent connections are turned off? The new data clearly show that the recurrent connections are not required for online grid firing, but this is not clear from the abstract and is hard to appreciate from the main text.<br /> (4) This is addressed, although the legend to Fig. S2D could provide an explanation / definition for the y-axis values.<br /> (5) Given the 2D structure of the network input it perhaps isn't surprising that the network generates 2D representations and this may have little to do with its 1D connectivity. The finding that the networks maintain coordinated grids when recurrent connections are switched off supports my initial concern and the authors explanation, to me at least, remain confusing. I think it would be helpful to consider that the connectivity is specifically important for establishing the coordinated grid firing, but that the online network does not require attractor dynamics to generate coordinated grid firing.<br /> (6) Clarity of the introduction. This is somewhat clearer, but I wonder if it would be hard for someone not familiar with the literature to accurately appreciate the key points.<br /> (7) Remapping. I'm not sure why this is ill posed. It seems the proposed model can not account for remapping results (e.g. Fyhn et al. 2007). Perhaps the authors could just clearly state this as a limitation of the model (or show that it can do this).
Previous review:
This study investigates the impact of recurrent connections on grid fields generated in networks trained by adjusting the strength of feedforward spatial inputs. The main result is that if the recurrent connections in the network are given a 1D continuous attractor architecture, then aligned grid firing patterns emerge in the network following training. Detailed analyses of the low dimensional dynamics of the resulting networks are then presented. The simulations and analyses appear carefully carried out.
The feedforward model investigated by the authors (previously introduced by Kropff & Treves, 2008) is an interesting and important alternative to models that generate grid firing patterns through 2-dimensional continuous attractor network (CAN) dynamics. However, while both classes of model generate grid fields, in making comparisons the manuscript is insufficiently clear about their differences. In particular, in the CAN models grid firing is a direct result of their 2-D architecture, either a torus structure with a single activity bump (e.g. Guanella et al. 2007, Pastoll et al. 2013), or sheet with multiple local activity bumps (Fuhs & Touretzky, Burak & Fiete, 2009). In these models, spatial input can anchor the grid representations but is not necessary for grid firing. By contrast, in the feedforward models neurons transform existing spatial inputs into a grid representation. Thus, the two classes of model implement different computations; CANs path integrate, while the feedforward models transform spatial representations. A demonstration that a 1D CAN generates coordinated 2D grid fields would be surprising and important, but its less clear why coordination between grids generated by the feedforward mechanism would be surprising. As written, it's unclear which of these claims the study is trying to make. If the former, then the conclusion doesn't appear well supported by the data as presented, if the latter then the results are perhaps not so unexpected, and the imposed attractor dynamics may still not be relevant.
Whichever claim is being made, it could be helpful to more carefully evaluate the model dynamics given predictions expected for the different classes of model. Key questions that are not answered by the manuscript include:
- At what point is the 1D attractor architecture playing a role in the models presented here? Is it important specifically for training or is it also contributing to computation in the fully trained network?
- Is an attractor architecture required at all for emergence of population alignment and gridness? Key controls missing from Figure 2 include training on networks with other architectures. For example, one might consider various architectures with randomly structured connectivity (e.g. drawing weights from exponential or Gaussian distributions).
- In the trained models do the recurrent connections substantially influence activity in the test conditions? Or after training are the 1D dynamics drowned out by feedforward inputs?
- What is the low dimensional structure of the input to the network? Can the apparent discrepancy between dimensionality of architecture and representation be resolved by considering structure of the inputs, e.g. if the input is a 2 dimensional representation of location then is it surprising that the output is too?
- What happens to representations in the trained networks presented when place cells remap? Is the 1D manifold maintained as expected for CAN models, or does it reorganise?
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
Summary:
This study examined the role of statistical learning in pain perception, suggesting that individuals' expectations about a sequence of events influence their perception of pain intensity. They incorporated the components of volatility and stochasticity into their experimental design and asked participants (n = 27) to rate the pain intensity, their prediction, and their confidence level. They compared two different inference strategies: Bayesian inference vs. heuristic-employing Kalman filters and model-free reinforcement learning. They showed that the expectation-weighted Kalman filter best explained the temporal pattern of participants' ratings. These results provide evidence for a Bayesian inference perspective on pain, supported by a computational model that elucidates the underlying process.
Strengths:
- Their experimental design included a wide range of input intensities and the levels of volatility and stochasticity. With elaborated computational models, they provide solid evidence that statistical learning shapes pain.
Weaknesses:
- Relevance to clinical pain: While the authors underscore the relevance of their findings to chronic pain, they did not include data pertaining to clinical pain.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This paper investigates the neural population activity patterns of the medial frontal cortex in rats performing a nose poking timing task using in vivo calcium imaging. The results showed neurons that were active at the beginning and end of the nose poking and neurons that formed sequential patterns of activation that covaried with the timed interval during nose poking on a trial-by-trial basis. The former were not stable across sessions, while the latter tended to remain stable over weeks. The analysis on incorrect trials suggests the shorter non-rewarded intervals were due to errors in the scaling of the sequential pattern of activity.
Strengths:
This study measured stable signals using in vivo calcium imaging during experimental sessions that were separated by many days in animals performing a nose poking timing task. The correlation analysis on the activation profile to separate the cells in the three groups was effective and the functional dissociation between beginning and end, and duration cells was revealing. The analysis on the stability of decoding of both the nose poking state and poking time was very informative. Hence, this study dissected a neural population that formed sequential patterns of activation that encoded timed intervals.
Weaknesses:
It is not clear whether animals had enough simultaneously recorded cells to perform the analyzes of Figures 2-4. In fact, rat 3 had 18 responsive neurons which probably is not enough to get robust neural sequences for the trial-by-trial analysis and the correct and incorrect trial analysis. In addition, the analysis of behavioral errors could be improved. The analysis in Figure 4A could be replaced by a detailed analysis on the speed, and the geometry of neural population trajectories for correct and incorrect trials. In the case of Figure 4G is not clear why the density of errors formed two clusters instead of having a linear relation with the produce duration. I would be recommendable to compute the scaling factor on neuronal population trajectories and single cell activity or the computation of the center of mass to test the type III errors.
Due to the slow time resolution of calcium imaging, it is difficult to perform robust analysis on ramping activity. Therefore, I recommend downplaying the conclusion that: "Together, our data suggest that sequential activity might be a more relevant coding regime than the ramping activity in representing time under physiological conditions."
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This work studies spatio-temporal patterns of structure-function coupling in developing brains, using a large set of imaging data acquired from children aged 5-22. Magnetic resonance imaging data of brain structure and function were obtained from a publicly available database, from which structural and functional features and measures were derived. The authors examined the spatial patterns of structure-function coupling and how they evolve with brain development. This work further sought correlations of brain structure-function coupling with behavior and explored evolutionary, microarchitectural and genetic bases that could potentially account for the observed patterns.
Strength:
The strength of this work is the use of currently available state-of-the-art analysis methods, along with a large set of high-quality imaging data, and comprehensive examinations of structure-function coupling in developing brains. The results are comprehensive and illuminating.
Weakness:
As with most other studies, transcriptomic and cellular architectures of structure-function coupling were characterized only on the basis of a common atlas in this work.
The authors have achieved their aims in this study, and the findings provide mechanistic insights into brain development, which will inspire further basic and clinical studies along this line.
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Reviewer #1 (Public Review):
Summary:
In this study, the authors provide a new computational platform called Vermouth to automate topology generation, a crucial step that any biomolecular simulation starts with. Given a wide arrange of chemical structures that need to be simulated, varying qualities of structural models as inputs obtained from various sources, and diverse force fields and molecular dynamics engines employed for simulations, automation of this fundamental step is challenging, especially for complex systems and in case that there is a need to conduct high-throughput simulations in the application of computer-aided drug design (CADD). To overcome this challenge, the authors develop a programing library composed of components that carry out various types of fundamental functionalities that are commonly encountered in topological generation. These components are intended to be general for any type of molecules and not to depend on any specific force field and MD engines. To demonstrate the applicability of this library, the authors employ those components to re-assemble a pipeline called Martinize2 used in topology generation for simulations with a widely used coarse-grained model (CG) MARTINI. This pipeline can fully recapitulate the functionality of its original version Martinize but exhibit greatly enhanced generality, as confirmed by the ability of the pipeline to faithfully generate topologies for two high-complexity benchmarking sets of proteins.
Strengths:
The main strength of this work is the use of concepts and algorithms associated with induced subgraph in graph theory to automate several key but non-trivial steps of topology generation such as the identification of monomer residue units (MRU), the repair of input structures with missing atoms, the mapping of topologies between different resolutions, and the generation of parameters needed for describing interactions between MRUs. In addition, the documentation website provided by the authors is very informative, allowing users to get quickly started with Vermouth.
Weaknesses:
Although the Vermouth library is designed as a general tool for topology generation for molecular simulations, only its applications with MARTINI have been demonstrated in the current study. Thus, the claimed generality of Vermouth remains to be exmained. The authors may consider to point out this in their manuscript.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors demonstrate with a simple stochastic model that the initial composition of the community is important in achieving a target frequency during the artificial selection of a community.
Strengths:
To my knowledge, the intra-collective selection during artificial selection has not been seriously theoretically considered. However, in many cases, the species dynamics during the incubation of each selection cycle are important and relevant to the outcome of the artificial selection experiment. Stochasticity from birth and death (demographic stochasticity) plays a big role in these species' abundance dynamics. This work uses a simple framework to tackle this idea meticulously.
This work may or may not be related to hysteresis (path dependency). If this is true, maybe it would be nice to have a discussion paragraph talking about how this may be the case. Then, this work would even attract the interest of people studying dynamic systems.
Weaknesses:
(1) Connecting structure and function
In typical artificial selection literature, most of them select the community based on collective function. Here in this paper, the authors are selecting a target composition. Although there is a schematic cartoon illustrating the relationship between collective function (y-axis) and the community composition in the main Figure 1, there is no explicit explanation or justification of what may be the origin of this relationship. I think giving the readers a naïve idea about how this structure-function relationship arises in the introduction section would help. This is because the conclusion of this paper is that the intra-collective selection makes it hard to artificially select a community that has an intermediate frequency of f (or s). If there is really evidence or theoretical derivation from this framework that indeed the highest function comes from the intermediate frequency of f, then the impact of this paper would increase because the conclusions of this stochastic model could allude to the reasons for the prevalent failures of artificial selection in literature.
(2) Explain intra-collective and inter-collective selection better for readers.
The abstract, the introduction, and the result section use these terms or intra-collective and inter-collective selection without much explanation. A clear definition in the beginning would help the audience grasp the importance of this paper, because these concepts are at the core of this work.
(3) Achievable target frequency strongly depending on the degree of demographic stochasticity.
I would expect that the experimentalists would find these results interesting and would want to consider these results during their artificial selection experiments. The main Figure 4 indicates that the Newborn size N0 is a very important factor to consider during the artificial selection experiment. This would be equivalent to how much bottleneck is imposed on the artificial selection process in every iteration step (i.e., the ratio of serial dilution experiment). However, with a low population size, all target frequencies can be achieved, and therefore in these regimes, the initial frequency now does not matter much. It would be great for the authors to provide what the N0 parameter actually means during the artificial selection experiments. Maybe relative to some other parameter in the model. I know this could be very hard. But without this, the main result of this paper (initial frequency matters) cannot be taken advantage of by the experimentalists.
(4) Consideration of environmental stochasticity.
The success (gold area of Figure 2d) in this framework mainly depends on the size of the demographic stochasticity (birth-only model) during the intra-collective selection. However, during experiments, a lot of environmental stochasticity appears to be occurring during artificial selection. This may be out of the scope of this study. But it would definitely be exciting to see how much environmental stochasticity relative to the demographic stochasticity (variation in the Gaussian distribution of F and S) matters in succeeding in achieving the target composition from artificial selection.
(5) Assumption about mutation rates
If setting the mutation rates to zero does not change the result of the simulations and the conclusion, what is the purpose of having the mutation rates \mu? Also, is the unidirectional (S -> F -> FF) mutation realistic? I didn't quite understand how the mutations could fit into the story of this paper.
(6) Minor points
In Figure 3b, it is not clear to me how the frequency difference for the Intra-collective and the Inter-collective selection is computed.
In Figure 5b, the gold region (success) near the FF is not visible. Maybe increase the size of the figure or have an inset for zoom-in. Why is the region not as big as the bottom gold region?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this study, Kume et al examined the role of the protein Semaphorin 4a in steady-state skin homeostasis and how this relates to skin changes seen in human psoriasis and imiquimod-induced psoriasis-like disease in mice. The authors found that human psoriatic skin has reduced expression of Sema4a in the epidermis. While Sema4a has been shown to drive inflammatory activation in different immune populations, this finding suggested Sema4a might be important for negatively regulating Th17 inflammation in the skin. The authors go on to show that Sema4a knockout mice have skin changes in key keratinocyte genes, increased gdT cells, and increased IL-17 similar to differences seen in non-lesional psoriatic skin, and that bone marrow chimera mice with WT immune cells and Sema4a KO stromal cells develop worse IMQ-induced psoriasis-like disease, further linking expression of Sema4a in the skin to maintaining skin homeostasis. The authors next studied downstream pathways that might mediate the homeostatic effects of Sema4a, focusing on mTOR given its known role in keratinocyte function. As with the immune phenotypes, Sema4a KO mice had increased mTOR activation in the epidermis in a similar pattern to mTOR activation noted in non-lesional psoriatic skin. The authors next targeted the mTOR pathway and showed rapamycin could reverse some of the psoriasis-like skin changes in Sema4a KO mice, confirming the role of increased mTOR in contributing to the observed skin phenotype.
Strengths:
The most interesting finding is the tissue-specific role for Sema4a, where it has previously been considered to play a mostly pro-inflammatory role in immune cells, this study shows that when expressed by keratinocytes, Sema4a plays a homeostatic role that when missing leads to the development of psoriasis-like skin changes. This has important implications in terms of targeting Sema4a pharmacologically. It also may yield a novel mouse model to study mechanisms of psoriasis development in mice separate from the commonly used IMQ model. The included experiments are well-controlled and executed rigorously.
Weaknesses:
A weakness of the study is the lack of tissue-specific Sema4a knockout mice (e.g. in keratinocytes only). The authors did use bone marrow chimeras, but only in one experiment. This work implies that psoriasis may represent a Sema4a-deficient state in the epidermal cells, while the same might not be true for immune cells. Indeed, in their analysis of non-lesional psoriasis skin, Sema4a was not significantly decreased compared to control skin, possibly due to compensatory increased Sema4a from other cell types. Unbiased RNA-seq of Sema4a KO mouse skin for comparison to non-lesional skin might identify other similarities besides mTOR signaling. Indeed, targeting mTOR with rapamycin reveres some of the skin changes in Sema4a KO mice, but not skin thickness, so other pathways impacted by Sema4a may be better targets if they could be identified. Utilizing WTKO chimeras in addition to global KO mice in the experiments in Figures 6-8 would more strongly implicate the separate role of Sema4a in skin vs immune cell populations and might more closely mimic non-lesional psoriasis skin.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study investigated the role of CD47 and TSP1 in extramedullary erythropoiesis by utilization of both global CD47-/- mice and TSP1-/- mice.
Strengths:
Flow cytometry combined with spleen bulk and single cell transcriptomics were employed. The authors found that stress-induced erythropoiesis markers were increased in CD47-/- spleen cells, particularly genes that are required for terminal erythroid differentiation. Moreover, CD47 dependent erythroid precursors population was identified by spleen scRNA sequencing. In contrast, the same cells were not detected in TSP1-/- spleen. These findings provide strong evidence to support the conclusion that differential role of CD47 and TSP1 in extramedullary erythropoiesis in mouse spleen. Furthermore, the relevance of the current finding to the prevalent side effect (anemia) of anti-CD47 mediated cancer therapy has been discussed in the Discussion section.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study by Wang et al. identifies a new type of deacetylase, CobQ, in Aeromonas hydrophila. Notably, the identification of this deacetylase reveals a lack of homology with eukaryotic counterparts, thus underscoring its unique evolutionary trajectory within the bacterial domain.
Strengths:
The manuscript convincingly illustrates CobQ's deacetylase activity through robust in vitro experiments, establishing its distinctiveness from known prokaryotic deacetylases. Additionally, the authors elucidate CobQ's potential cooperation with other deacetylases in vivo to regulate bacterial cellular processes. Furthermore, the study highlights CobQ's significance in the regulation of acetylation within prokaryotic cells.
Weaknesses:
While the manuscript is generally well-structured, some clarification and some minor corrections are needed.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Plasmodium vivax can persist in the liver of infected individuals in the form of dormant hypnozoites, which cause malaria relapses and are resistant to most current antimalarial drugs. This highlights the need to develop new drugs active against hypnozoites that could be used for radical cure. Here, the authors capitalize on an in vitro culture system based on primary human hepatocytes infected with P. vivax sporozoites to screen libraries of repurposed molecules and compounds acting on epigenetic pathways. They identified a number of hits, including hydrazinophthalazine analogs. They propose that some of these compounds may act on epigenetic pathways potentially involved in parasite quiescence. To provide some support to this hypothesis, they document DNA methylation of parasite DNA based on 5-methylcytosine immunostaining, mass spectrometry, and bisulfite sequencing.
Strengths:<br /> -The drug screen itself represents a huge amount of work and, given the complexity of the experimental model, is a tour de force.<br /> -The screening was performed in two different laboratories, with a third laboratory being involved in the confirmation of some of the hits, providing strong support that the results were reproducible.<br /> -The screening of repurposing libraries is highly relevant to accelerate the development of new radical cure strategies.
Weaknesses:
-The manuscript is composed of two main parts, the drug screening itself and the description of DNA methylation in Plasmodium pre-erythrocytic stages. Unfortunately, these two parts are loosely connected. First, there is no evidence that the identified hits kill hypnozoites via epigenetic mechanisms. The hit compounds almost all act on schizonts in addition to hypnozoites, therefore it is unlikely that they target quiescence-specific pathways. At least one compound, colforsin, seems to selectively act on hypnozoites, but this observation still requires confirmation. Second, while the description of DNA methylation is per se interesting, its role in quiescence is not directly addressed here. Again, this is clearly not a specific feature of hypnozoites as it is also observed in P. vivax and P. cynomolgi hepatic schizonts and in P. falciparum blood stages. Therefore, the link between DNA methylation and hypnozoite formation is unclear. In addition, DNA methylation in sporozoites may not reflect epigenetic regulation occurring in the subsequent liver stages.
-The mode of action of the hit compounds remains unknown. In particular, it is not clear whether the drugs act on the parasite or on the host cell. Merely counting host cell nuclei to evaluate the toxicity of the compounds is probably acceptable for the screen but may not be sufficient to rule out an effect on the host cell. A more thorough characterization of the toxicity of the selected hit compounds is required.
-There is no convincing explanation for the differences observed between P. vivax and P. cynomolgi. The authors question the relevance of the simian model but the discrepancy could also be due to the P. vivax in vitro platform they used.
-Many experiments were performed only once, not only during the screen (where most compounds were apparently tested in a single well) but also in other experiments. The quality of the data would be increased with more replication.
-While the extended assay (12 days versus 8 days) represents an improvement of the screen, the relevance of adding inhibitors of core cytochrome activity is less clear, as under these conditions the culture system deviates from physiological conditions.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors established an in vitro triple co-culture BBB model and demonstrated its advantages compared with the mono or double co-culture BBB model. Further, the authors used their established in vitro BBB model and combined it with other methodologies to investigate the specific mechanism that co-culture with astrocytes but also neurons enhanced the integrity of endothelial cells.
Strengths:
The results persuasively showed the established triple co-culture BBB model well mimicked several important characteristics of BBB compared with the mono-culture BBB model, including better barrier function and in vivo/in vitro correlation. The human-derived immortalized cells used made the model construction process faster and more efficient, and have a better in vivo correlation without species differences. This model is expected to be a useful high-throughput evaluation tool in the development of CNS drugs.
Based on the previous experimental results, detailed studies investigated how co-culture with neurons and astrocytes promoted claudin-5 and VE-cadherin in endothelial cells, and the specific signaling mechanisms were also studied. Interestingly, the authors found that neurons also released GDNF to promote barrier properties of brain endothelial cells, as most current research has focused on the promoting effect of astrocytes-derived GDNF on BBB. Meanwhile, the author also validated the functions of GDNF for BBB integrity in vivo by silencing GDNF in mouse brains. Overall, the experiments and data presented support their claim that, in addition to astrocytes, neurons also have a promoting effect on the barrier function of endothelial cells through GDNF secretion.
Weaknesses:
Although the authors demonstrated a highly usable for predicting the BBB permeability, recorded TEER measurements are still far from the human BBB in vivo reported measurements of TEER, and expression of transporters was not promoted by co-culture, which may lead to the model being unsuitable for studying drug transport mediated by transporters on BBB.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The paper has shown the expression of RGS10 is related to the molecular subtype, distant metastasis, and survival status of breast cancer. The study utilizes bioinformatic analyses, human tissue samples, and in vitro and in vivo experiments which strengthen the data. RGS10 was validated to inhibit EMT through a novel mechanism dependent on LCN2 and miR-539-5p, thereby reducing cancer cell proliferation, colony formation, invasion, and migration. The study elaborated on the function of RGS10 in influencing the prognosis and biological behavior which could be considered as a potential drug target in breast cancer.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This paper is focused on the role of Cadherin Flamingo (Fmi) - also called Starry night (stan) - in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that express activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which makes continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind.
The authors show that clonal loss of Fmi by an allele or by RNAi in the RasV12, scrib-i tumors suppresses their growth in both the eye disc (continuous clones) and wing disc (discrete clones). The authors attributed this result to less killing of WT neighbors when Myc over-expressing clones lacking Fmi, but another interpretation (that Fmi regulates clonal growth) is equally as plausible with the current results. Next, the authors show that scrib-RNAi clones that are normally out-competed by WT cells prior to adult stages are present in higher numbers when WT cells are depleted for Fmi. They then examine death in RasV12, scrib-i ey-FLP clones, or in discrete hs-FLP UAS-Myc clones. They state that they see death in WT cells neighboring RasV12, scrib-i clones in the eye disc (Figures 4A-C). Next, they write that RasV12, scrib-I cells become losers (i.e., have apoptosis markers) when Fmi is removed. Neither of these results are quantified and thus are not compelling. They state that a similar result is observed for Myc over-expression clones that lack Fmi, but the image was not compelling, the results are not quantified and the controls are missing (Myc over-expressing clones alone and Fmi clones alone). They then want to test whether Myc over-expressing clones have more proliferation. They show an image of a wing disc that has many small Myc overexpressing clones with and without Fmi. The pHH3 results support their conclusion that Myc overexpressing clones have more pHH3, but I have reservations about the many clones in these panels (Figures 5L-N). They show that the cell competition roles of Fmi are not shared by another PCP component and are not due to the Cadherin domain of Fmi. The authors appear to interpret their results as Fmi is required for winner status. Overall, some of these results are potentially interesting and at least partially supported by the data, but others are not supported by the data.
Strengths:
Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.
Weaknesses:
(1) In the Myc over-expression experiments, the increased size of the Myc clones could be because they divide faster (but don't outcompete WT neighbors). If the authors want to conclude that the bigger size of the Myc clones is due to out-competition of WT neighbors, they should measure cell death across many discs of with these clones. They should also assess if reducing apoptosis (like using one copy of the H99 deficiency that removes hid, rpr, and grim) suppresses winner clone size. If cell death is not addressed experimentally and quantified rigorously, then their results could be explained by faster division of Myc over-expressing clones (and not death of neighbors). This could also apply to the RasV12, scrib-i results.
(2) This same comment about Fmi affecting clone growth should be considered in the scrib RNAi clones in Figure 3.
(3) I don't understand why the quantifications of clone areas in Figures 2D, 2H, 6D are log values. The simple ratio of GFP/RFP should be shown. Additionally, in some of the samples (e.g., fmiE59 >> Myc, only 5 discs and fmiE59 vs >Myc only 4 discs are quantified but other samples have more than 10 discs). I suggest that the authors increase the number of discs that they count in each genotype to at least 20 and then standardize this number.
(4) There is a typo when referring to Figures 3C-D. It should be Figure 2C-D.
(5) Figure 4 - shows examples of cell death. Cas3 is written on the figure but Dcp-1 is written in the results. Which antibody was used? The authors need to quantify these results. They also need to show that the death of cells is part of the phenotype, like an H99 deficiency, etc (see above).
(6) It is well established that clones overexpressing Myc have increased cell death. The authors should consider this when interpreting their results.
(7) A better characterization of discrete Fmi clones would also be helpful. I suggest inducing hs-flp clones in the eye or wing disc and then determining clone size vs twin spot size and also examining cell death etc. If such experiments have already been done and published, the authors should include a description of such work in the preprint.
(8) We need more information about the expression pattern of Fmi. Is it expressed in all cells in imaginal discs? Are there any patterns of expression during larval and pupal development?
(9) Overall, the paper is written for specialists who work in cell competition and is fairly difficult to follow, and I suggest re-writing the results to make it accessible to a broader audience.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study shows a new mechanism of GS regulation in the archaean Methanosarcina maze and clarifies the direct activation of GS activity by 2-oxoglutarate, thus featuring another way in which 2-oxoglutarate acts as a central status reporter of C/N sensing.
Mass photometry and single particle cryoEM structure analysis convincingly show the direct regulation of GS activity by 2-OG promoted formation of the dodecameric structure of GS. The previously recognized small proteins GlnK1 and Sp26 seem to play a subordinate role in GS regulation, which is in good agreement with previous data. Although these data are quite clear now, there remains one major open question: how does 2-OG further increase GS activity once the full dodecameric state is achieved (at 5 mM)? This point needs to be reconsidered.
Strengths:
Mass photometry reveals a dynamic mode of the effect of 2-OG on the oligomerization state of GS. Single particle Cryo-EM reveals the mechanism of 2-OG mediated dodecamer formation.
Weaknesses:
It is not entirely clear, how very high 2-OG concentrations activate GS beyond dodecamer formation.
The data presented in this work are in stark contrast to the previously reported structure of M. mazei GS by the Schumacher lab. This is very confusing for the scientific community and requires clarification. The discussion should consider possible reasons for the contradictory results.
Importantly, it is puzzling how Schumacher could achieve an apo-structire of dodecameeric GS? If 2-OG is necessary for dodecameric formation, this should be discussed. If GlnK1 doesn't form a complex with the dodecameric GS, how could such a complex be resolved there?
In addition, the text is in principle clear but could be improved by professional editing. Most obviously there is insufficient comma placement.
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Reviewer #1 (Public Review):
The paper meticulously explores various conformations and states of the ribosome-translocon complex. Employing advanced techniques such as cryoEM structural determination and AlphaFold modeling, the study delves into the dynamic nature of the ribosome-translocon complex. The findings from these analyses unveil crucial insights, significantly advancing our understanding of the co-translational translocation process in cellular mechanisms.
To begin with, the authors employed a construct comprising the first two transmembrane domains of rhodopsin as a model for studying protein translocation. They conducted in vitro translation, followed by the purification of the ribosome-translocon complex, and determined its cryoEM structures. An in-depth analysis of their ribosome-translocon complex structure revealed that the nascent chain can pass through the lateral gate of translocon Sec61, akin to the behavior of a Signaling Peptide. Additionally, Sec61 was found to interact with 28S rRNA helix 24 and the ribosomal protein uL24. In summary, their structural model aligns with the through-pore model of insertion, contradicting the sliding model.
Secondly, the authors successfully identified RAMP4 in their ribosome-translocon complex structure. Notably, the transmembrane domain of RAMP4 mimics the binding of a Signaling Peptide at the lateral gate of Sec61, albeit without unplugging. Intriguingly, RAMP4 is exclusively present in the non-multipass translocon ribosome-translocon complex, not in those containing multipass translocon. This observation suggests that co-translational translocation specifically occurs in the Sec61 channel that includes bound RAMP4. Additionally, the authors discovered an interaction between the C-tail of ribosomal proteins uL22 and the translocon Sec61, providing valuable insights into the nascent chain's behavior.
Moving on to the third point, the focused classification unveiled TRAP complex interactions with various components. The authors propose that the extra density observed in their novel ribosome-translocon complex can be attributed to calnexin, a major binder of TRAP according to previous studies. Furthermore, the new structure reveals a TRAP-OSTA interaction. This newly identified TRAP-OSTA interaction offers a potential explanation for why patients with TRAP delta defects exhibit congenital disorders of glycosylation.
In conclusion, this paper presents a robust contribution to the field with its thorough structural and modeling analyses. The significance of the findings is evident, providing valuable insights into the intricate mechanisms of protein co-translational translocation. The well-crafted writing, meticulous analyses, and clear figures collectively contribute to the overall strength of the paper.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors have previously described a way to boost WNT/CTNNB1 signaling in a tissue-specific manner, by directing an RSPO2 mutant protein (RSPO2RA) to a liver-specific receptor (ASGR1/2). This is done by fusing the RSPO2RA to an antibody that binds ASGR1/2.
Here the authors describe two new antibodies, 8M24 and 8G8, with similar effects. 8M24 shows specificity for ASGR1, while 8G8 has broader affinity for mouse/human ASGR1/2.<br /> The authors resolve and describe the crystal structure of the hASGR1CRD:8M24 complex and the hASGR2CRD:8G8 complex in great detail, which help explain the specificities of the 8M24 and 8G8 antibodies. Their epitopes are non-overlapping.<br /> Upon fusion of the antibodies to an RSPO2RA (an RSPO mutant), these antibodies are able to enhance WNT signaling by promoting the ASGR1-mediated clearance of ZNRF3/RNF43, thereby increasing cell surface expression of FZD. This has previously also been shown to be the case for RSPO2RA fused to an anti-ASGR1 antibody 4F3 - and the paper also tests how the antibodies compare to the 4F3 fusion.
Strengths:
(1) One challenge in treating diseases, is the fact that one would like therapeutics to be highly specific - not just in terms of their target (e.g. aimed at a specific protein of interest) but also in terms of tissue specificity (i.e. affecting only tissue X but leaving all others unaffected). This study broadens the collection of antibodies that can be used for this purpose and thus expands a potential future clinical toolbox.
(2) The authors have addressed questions raised after a first round of review, e.g. by showing that ASGR1 is itself indeed ubiquitinated.
Weaknesses:
(1) Some questions remain as to how 8M24 and 8G8 compare to 4F3.
(2) Some questions remain as to the specificity of the approach: the initial goal was not to also downregulate ASGR1 per se, so this targeting to a specific receptor/membrane protein is not trivial and/or neutral.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The manuscript presents novel results on the regulation of Drosophila wing growth by the protocadherins Ds and Fat. The manuscript performs a more careful analysis of disc volume, larval size, and the relationship between the two, in normal and mutant larvae, and after localized knockdown or overexpression of Fat and Ds. Not all of the results are equally surprising given the previous work on Fat, Ds, and their regulation of disc growth, pupariation, and the Hippo pathway, but the presentation and detail of the presented data is new. The most novel results concern the scaling of gradients of Fat and Ds protein during development, a largely unstudied gradient of Fat protein, and using overexpression of Ds to argue that changes in the Ds gradient do not underlie the slowing and halting of cell divisions during development.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Muscle models are important tools in the fields of biomechanics and physiology. Muscle models serve a wide variety of functions, including validating existing theories, testing new hypotheses, and predicting forces produced by humans and animals in health and disease. This paper attempts to provide an alternative to Hill-type muscle models that includes contributions of titin to force enhancement over multiple time scales. Due to the significant limitations of Hill-type models, alternative models are needed and therefore the work is important and timely.
The effort to include a role for titin in muscle models is a major strength of the methods and results. The results clearly demonstrate the weaknesses of Hill models and the advantages of incorporating titin into theoretical treatments of muscle mechanics. Another strength is to address muscle mechanics over a large range of time scales.
The authors succeed in demonstrating the need to incorporate titin in muscle models, and further show that the model accurately predicts in situ force of cat soleus (Kirsch et al. 1994; Herzog & Leonard, 2002) and rabbit posts myofibrils (Leonard et al. 2010). However, it remains unclear whether the model will be practical for use with data from different muscles or preparations. Several ad hoc modifications were described in the paper, and the degree to which the model requires parameter optimization for different muscles, preparations and experiment types remains unclear.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:<br /> In the paper by Choi et al., the authors aimed to develop base editing strategies to convert CAG repeats to CAA repeats in the huntingtin gene (HTT), which causes Huntington's disease (HD). They hypothesized that this conversion would delay disease onset by shortening the uninterrupted CAG repeat. Using HEK-293T cells as a model, the researchers employed cytosine base editors and guide RNAs (gRNAs) to efficiently convert CAG to CAA at various sites within the CAG repeat. No significant indels, off-target edits, transcriptome alterations, or changes in HTT protein levels were detected. Interestingly, somatic CAG repeat expansion was completely abolished in HD knock-in mice carrying CAA-interrupted repeats.
Strengths:<br /> This study represents the first proof-of-concept exploration of the cytosine base editing technique as a potential treatment for HD and other repeat expansion disorders with similar mechanisms.
Weaknesses:<br /> Given that HD is a neurodegenerative disorder, it is crucial to determine the efficiency of the base editing strategies tested in this manuscript and their feasibility in relevant cells affected by HD and the brain, which needed to be improved in this manuscript.
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Reviewer #1 (Public Review):
Summary:
In this study, the authors used a multi-alternative decision task and a multidimensional signal-detection model to gain further insight into the cause of perceptual impairments during the attentional blink. The model-based analyses of behavioural and EEG data show that such perceptual failures can be unpacked into distinct deficits in visual detection and discrimination, with visual detection being linked to the amplitude of late ERP components (N2P and P3) and discrimination being linked to the coherence of fronto-parietal brain activity.
Strengths:
The main strength of this paper lies in the fact that it presents a novel perspective on the cause of perceptual failures during the attentional blink. The multidimensional signal-detection modelling approach is explained clearly, and the results of the study show that this approach offers a powerful method to unpack behavioural and EEG data into distinct processes of detection and discrimination.
Weaknesses:
While the model-based analyses are compelling, the paper also features some analyses that seem misguided, or, at least, insufficiently motivated and explained. Specifically, in the introduction, the authors raise the suggestion that the attentional blink could be due to a reduction in sensitivity or a response bias. The suggestion that a response bias could play a role seems misguided, as any response bias would be expected to be constant across lags, while the attentional blink effect is only observed at short lags. Thus, it is difficult to understand why the authors would think that a response bias could explain the attentional blink.
A second point of concern regards the way in which the measures for detection and discrimination accuracy were computed. If I understand the paper correctly, a correct detection was defined as either correctly identifying T2 (i.e., reporting CW or CCW if T2 was CW or CCW, respectively, see Figure 2B), or correctly reporting T2's absence (a correct rejection). Here, it seems that one should also count a misidentification (i.e., incorrect choice of CW or CCW when T2 was present) as a correct detection, because participants apparently did detect T2, but failed to judge/remember its orientation properly in case of a misidentification. Conversely, the manner in which discrimination performance is computed also raises questions. Here, the authors appear to compute accuracy as the average proportion of T2-present trials on which participants selected the correct response option for T2, thus including trials in which participants missed T2 entirely. Thus, a failure to detect T2 is now counted as a failure to discriminate T2. Wouldn't a more proper measure of discrimination accuracy be to compute the proportion of correct discriminations for trials in which participants detected T2?
My last point of critique is that the paper offers little if any guidance on how the inferred distinction between detection and discrimination can be linked to existing theories of the attentional blink. The discussion mostly focuses on comparisons to previous EEG studies, but it would be interesting to know how the authors connect their findings to extant, mechanistic accounts of the attentional blink. A key question here is whether the finding of dissociable processes of detection and discrimination would also hold with more meaningful stimuli in an identification task (e.g., the canonical AB task of identifying two letters shown amongst digits). There is evidence to suggest that meaningful stimuli are categorized just as quickly as they are detected (Grill-Spector & Kanwisher, 2005; Grill-Spector K, Kanwisher N. Visual recognition: as soon as you know it is there, you know what it is. Psychol Sci. 2005 Feb;16(2):152-60. doi: 10.1111/j.0956-7976.2005.00796.x. PMID: 15686582.). Does that mean that the observed distinction between detection and discrimination would only apply to tasks in which the targets consist of otherwise meaningless visual elements, such as lines of different orientations?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This manuscript from So et al. describes what is suggested to be an improved protocol for single-nuclei RNA sequencing (snRNA-seq) of adipose tissue. The authors provide evidence that modifications to the existing protocols result in better RNA quality and nuclei integrity than previously observed, with ultimately greater coverage of the transcriptome upon sequencing. Using the modified protocol, the authors compare the cellular landscape of murine inguinal and perigonadal white adipose tissue (WAT) depots harvested from animals fed a standard chow diet (lean mice) or those fed a high-fat diet (mice with obesity).
Strengths:
Overall, the manuscript is well-written, and the data are clearly presented. The strengths of the manuscript rest in the description of an improved protocol for snRNA-seq analysis. This should be valuable for the growing number of investigators in the field of adipose tissue biology that are utilizing snRNA-seq technology, as well as those other fields attempting similar experiments with tissues possessing high levels of RNAse activity.
Moreover, the study makes some notable observations that provide the foundation for future investigation. One observation is the correlation between nuclei size and cell size, allowing for the transcriptomes of relatively hypertrophic adipocytes in perigonadal WAT to be examined. Another notable observation is the identification of an adipocyte subcluster (Ad6) that appears "stressed" or dysfunctional and likely localizes to crown-like inflammatory structures where pro-inflammatory immune cells reside.
Weaknesses:
Analogous studies have been reported in the literature, including a notable study from Savari et al. (Cell Metabolism). This somewhat diminishes the novelty of some of the biological findings presented here. Moreover, a direct comparison of the transcriptomic data derived from the new vs. existing protocols (i.e. fully executed side by side) was not presented. As such, the true benefit of the protocol modifications cannot be fully understood.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This paper by Beath et. al. identifies a potential regulatory role for proteins involved in cytoplasmic streaming and maintaining the grouping of paternal organelles: holding sperm contents in the fertilized embryos away from the oocyte meiotic spindle so that they don't get ejected into the polar body during meiotic chromosome segregation. The authors show that by time-lapse video, paternal mitochondria (used as a readout for sperm and its genome) is excluded from yolk granules and maternal mitochondria, even when moving long distances by cytoplasmic streaming. To understand how this exclusion is accomplished, they first show that it is independent of both internal packing and the engulfment of the paternal chromosomes by maternal endoplasmic reticulum creating an impermeable barrier. They then test whether the control of cytoplasmic steaming affects this exclusion by knocking down two microtubule motors, Katanin and kinesis I. They find that the ER ring, which is used as a proxy for paternal chromosomes, undergoes extensive displacement with these treatments during anaphase I and interacts with the meiotic spindle, supporting their hypothesis that the exclusion of paternal chromosomes is regulated by cytoplasmic streaming. Next, they test whether a regulator of maternal ER organization, ATX-2, disrupts sperm organization so that they can combine the double depletion of ATX-2 and KLP-7, presumably because klp-7 RNAi (unlike mei-1 RNAi) does not affect polar body extrusion and they can report on what happens to paternal chromosomes. They find that the knockdown of both ATX-2 and KLP-7 produces a higher incidence of what appears to be the capture of paternal chromosomes by the meiotic spindle (5/24 vs 1/25). However, this capture event appears to halt the cell cycle, preventing the authors from directly observing whether this would result in the paternal chromosomes being ejected into the polar body.
Strengths:
This is a useful, descriptive paper that highlights a potential challenge for embryos during fertilization: when fertilization results in the resumption of meiotic divisions, how are the paternal and maternal genomes kept apart so that the maternal genome can undergo chromosome segregation and polar body extrusion without endangering the paternal genome? In general, the experiments are well-executed and analyzed. In particular, the authors' use of multiple ways to knock down ATX-2 shows rigor.
Weaknesses:
The paper makes a case that this regulation may be important but the authors should do some additional work to make this case more convincing and accessible for those outside the field. In particular, some of the figures could include greater detail to support their conclusions, they could explain the rationale for some experiments better and they could perform some additional control experiments with their double depletion experiments to better support their interpretations. Also, the authors' inability to assess the functional biological consequences of the capture of the sperm genome by the oocyte spindle should be discussed, particularly in light of the cell cycle arrest that they observe.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Summary:
The study identified biallelic variants of DNAH3 in four unrelated Han Chinese infertile men through whole-exome sequencing, which contributes to abnormal sperm flagellar morphology and ultrastructure. To investigate the importance of DNAH3 in male infertility, the authors generated crispant DNAH3 knockout (KO) male mice. They observed that KO mice are also infertile, showing a severe reduction in sperm movement with abnormal IDA (inner dynein arms) and mitochondrion structure. Moreover, nonfunctional DNAH3 expression decreased the expression of IDA-associated proteins in the spermatozoa of patients and KO mice, which are involved in the disruption of sperm motility. Interestingly, the infertility of patients and KO mice was rescued by intracytoplasmic sperm injection (ICSI). Taken together, the authors propose that DNAH3 is a novel pathogenic gene for asthenoterozoospermia and male infertility.
Strengths:
This work investigates the role of DNAH3 in sperm mobility and male infertility and utilised gold-standard molecular biology techniques, showing strong evidence of its role in male infertility. All aspects of the study design and methods are well described and appropriate to address the main question of the manuscript. The conclusions drawn are consistent with the analyses conducted and supported by the data.
Weaknesses:
(1) The manuscript lacks a comparison with previous studies on DNAH3 in the Discussion section.
(2) The variants of DNAH3 in four infertile men were identified through whole-exome sequencing. Providing an overview of the WES data would be beneficial to offer additional insights into whether other variants may contribute the infertility. This could also help explain why ICSI only works for two out of four patients with DNAH3 variants.
(3) Quantification of images would help substantiate the conclusions, particularly in Figures 2, 3, 4, and 6. Improved images in Figures 3A, 4B, and 4C, would help increase confidence in the claims made.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The goal of Knudsen-Palmer et al. was to define a biological set of rules that dictate the differential RNAi-mediated silencing of distinct target genes, motivated by facilitating the long-term development of effective RNAi-based drugs/therapeutics. To achieve this, the authors use a combination of computational modeling and RNAi function assays to reveal several criteria for effective RNAi-mediated silencing. This work provides insights into how (1) cis-regulatory elements influence the RNAi-mediated regulation of genes; (2) it is determined that genes can "recover" from RNAi-silencing signals in an animal; and 3) pUGylation occurs exclusively downstream of the dsRNA trigger sequence, suggesting 3º siRNAs are not produced. In addition, the authors show that the speed at which RNAi-silencing is triggered does not correlate with the longevity of the silencing. These insights are significant because they suggest that if we understand the rules by which RNAi pathways effectively silence genes with different transcription/processing levels then we can design more effective synthetic RNAi-based therapeutics targeting endogenous genes. The conclusions of this study are mostly supported by the data, but there are some aspects that need to be clarified.
(1) The methods do not describe the "aged RNAi plates feeding assay" in Figure 2E. The figure legend states that "aged RNAi plates" were used to trigger weaker RNAi, but the detail explaining the experiment is insufficient. How aged is aged? If the goal was to effectively reduce the dsRNA load available to the animals, why not quantitatively titrate the dsRNA provided? Were worms previously fed on the plates, or was simply a lawn of bacteria grown until presumably the IPTG on the plate was exhausted?
(2) Is the data presented in Figure 2F completed using the "aged RNAi plates" to achieve the partial silencing of dpy-7 observed? Clarification of this point would be helpful.
(3) Throughout the manuscript the authors refer to "non-dividing cells" when discussing animals' ability to recover from RNA silencing. It is not clear what the authors specifically mean with the phrase "non-dividing cells", but as this is referred to in one of their major findings, it should be clarified. Do they mean the cells are somatic cells in aged animals, thus if they are "non-dividing" the siRNA pools within the cells cannot be diluted by cell division? Based on the methods, the animals of RNAi assays were L4/Young adults that were scored over 8 days after the initial pulse of dsRNA feeding. If this is the case, wouldn't these animals be growing into gravid adults after the feeding, and thus have dividing cells as they grew?
(4) What are the typical expression levels/turnover of unc-22 and bli-1? Based on the results from the altered cis-regulatory regions of bli-1 and unc-22 in Figure 5, it seems like the transcription/turnover rates of each of these genes could also be used as a proof of principle for testing the model proposed in Figure 4. The strength of the model would be further increased if the RNAi sensitivity of unc-22 reflects differences in its transcription/turnover rates compared to bli-1.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Clostridium thermocellum serves as a model for consolidated bioprocess (CBP) in lignocellulosic ethanol production, but yet faces limitations in solid contents and ethanol titers achieved by engineered strains thus far. The primary ethanol production pathway involves the enzyme aldehyde-alcohol dehydrogenase (AdhE), which forms long oligomeric structures known as spirosomes, previously characterized via the 3.5 Å resolution E. coli AdhE structure using single-particle cryo-EM. The present study describes the cryo-EM structure of the C. thermocellum ortholog, sharing 62% sequence identity with E. coli AdhE, resolved at 3.28 Å resolution. Detailed comparative structural analysis, including the Vibrio cholerae AdhE structure, was conducted. Integrating cryo-EM data with molecular dynamics simulations indicated that the aldehyde intermediate resides longer in the channel of the extended form, supporting the hypothesis that the extended spirosome represents the active form of AdhE.
Strengths:
The study conducts a comprehensive structural comparative analysis of oligomerization interfaces and the acetaldehyde channel across compact and extended conformations. Structural and computational results suggest the extended spirosome as the most likely active state of AdhE.
Weaknesses:
The overall resolution of the C. thermocellum structure is similar to the E. coli ortholog, which shares 62% sequence identity, and the oligomerization interfaces and the acetaldehyde channel were previously described.
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Reviewer #1 (Public Review):
In their paper, Kang et al. investigate rigidity sensing in amoeboid cells, showing that, despite their lack of proper focal adhesions, amoeboid migration of single cells is impacted by substrate rigidity. In fact, many different amoeboid cell types can durotax, meaning that they preferentially move towards the stiffer side of a rigidity gradient.
The authors observed that NMIIA is required for durotaxis and, building on this observation, they generated a model to explain how durotaxis could be achieved in the absence of strong adhesions. According to the model, substrate stiffness alters the diffusion rate of NMAII, with softer substrates allowing for faster diffusion. This allows for NMAII accumulation at the back, which, in turn, results in durotaxis.
The experiments support the main message of the paper regarding durotaxis by amoeboid cells. In my opinion, a few clarifications on the mechanism proposed to explain this phenomenon could strengthen this research:
(1) According to your model, the rear end of the cell, which is in contact with softer substrates, will have slower diffusion rates of MNIIA. Does this mean that bigger cells will durotax better than smaller cells because the stiffness difference between front and rear is higher? Is it conceivable to attenuate the slope of the durotactic gradient to a degree where smaller cells lose their ability to durotact, while longer cells retain their capacity for directional movement?
(2) Where did you place the threshold for soft, middle, and stiff regions (Figure 6)? Is it possible that you only have a linear rigidity gradient in the center of your gel and the more you approach the borders, the flatter the gradient gets? In this case, cells would migrate randomly on uniform substrates. Did you perform AFM over the whole length of the gel or just in the central part?
(3) In which region (soft, middle, stiff) did you perform all the cell tracking of the previous figures?
(4) What is the level of confinement experienced by the cells? Is it possible that cells on the soft side of the gels experience less confinement due to a "spring effect" whereby the coverslips descending onto the cells might exert diminished pressure because the soft hydrogels act as buffers, akin to springs? If this were the case, cells could migrate following a confinement gradient.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors address cellular mechanisms underlying the early stages of Sjogren's syndrome, using a mouse model in which 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA) is applied to stimulate the interferon gene (STING) pathway. They show that, in this model, salivary secretion in response to neural stimulation is greatly reduced, even though individual secretory cell calcium responses were enhanced. They attribute the secretion defect to reduced activation of Ca2+ -activated Cl- channels (TMEM16a), due to an increased distance between Ca2+ release channels (IP3 receptors) and TMEM16a which is expected to reduce the [Ca2+] sensed by TMEM16a. A variety of disruptions in mitochondria were also observed after DMXAA treatment, including reduced abundance, altered morphology, depolarization, and reduced oxygen consumption rate. The results of this study shed new light on some of the early events leading to the loss of secretory function in Sjogren's syndrome, at a time before inflammatory responses cause the death of secretory cells.
Strengths:
Two-photon microscopy enabled Ca2+ measurements in the salivary glands of intact animals in response to physiological stimuli (nerve stimulation). This approach has been shown previously by the authors as necessary to preserve the normal spatiotemporal organization of calcium signals that lead to secretion under physiological conditions.
Superresolution (STED) microscopy allowed precise measurements of the spacing of IP3R and TMEM16a and the cell membranes that would otherwise be prevented by the diffraction limit. The measured increase of distance (from 84 to 155 nm) would be expected to reduce [Ca2+] at the TMEM16a channel.
The authors effectively ruled out a variety of alternative explanations for reduced secretion, including changes in AQP5 expression, TMEM16a expression, localization, and Ca2+ sensitivity as indicated by Cl- current in response to defined levels of Ca2+.
Weaknesses:
While the Ca2+ distribution in the cells was less restricted to the apical region in DMXAA-treated cells, it is not clear that this is relevant to the reduced activation of TMEM16a. The way in which the change in Ca2+ distribution is quantified (apical/basal ratio) is not informative, as this is not what activates TMEM16a, but rather the local [Ca2+] at the channel.
Despite the decreased level of secretion, Ca2+ signal amplitudes were higher in the treated cells, raising the question of how much this might compensate for the increased distance between IP3R and TMEM16a. The authors assume that the increased separation of IP3R and TMEM16a (and the resulting decrease in local [Ca2+]) outweighed the effect of higher global [Ca2+], but this important point was not addressed.
The description of mitochondrial changes in abundance, morphology, membrane potential, and oxygen consumption rate were not well integrated into the rest of the paper. While they may be a facet of the multiple effects of STING activation and may occur during Sjogren's syndrome, their possible role in reducing secretion was not examined. As it stands, the mitochondrial results are largely descriptive and there is no evidence here that they contribute to the secretory phenotype.
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pressbooks.pub pressbooks.pub
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student-faculty contact in and out of classes is the most important factor in student motivation and involvement. Faculty concern helps students get through rough times and keep on working. Knowing a few faculty members well enhances students’ intellectual commitment and encourages them to think about their own values and future plans
Contact outside of the classroom is difficult for both commuters in on-ground courses and online environments. Also it's becoming an issue for traditional students who are working more and more hours to reduce overall costs of education.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The current manuscript provides an extensive in vivo analysis of two guidance pathways identifying multiple mechanisms that shape the bifurcation of DRG axons when forming the dorsal funiculus in the DREZ.
Strengths:
Multiple mouse mutant lines were used, together with complementary techniques; the results are very clear and compelling.<br /> The findings are very significant and clearly move forward our understanding of the regulation of axonal development at the DREZ.
Weaknesses:
No major weaknesses were found. As it is I have no recommendations that would increase the clarity or quality of the manuscript.
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
The authors sought to establish the role played by N343 glycosylation on the SARS-CoV-2 S receptor binding domain structure and binding affinity to the human host receptor ACE2 across several variants of concern. The work includes both computational analysis in the form of molecular dynamics simulations and experimental binding assays between the RBD and ganglioside receptors.
The work extensively samples the conformational space of the RBD beginning with atomic coordinates representing both the bound and unbound states and computes molecular dynamics trajectories until equilibrium is achieved with and without removing N343 glycosylation. Through comparison of these simulated structures, the authors are able to demonstrate that N343 glycosylation stabilizes the RBD. Prior work had demonstrated that glycosylation at this site plays an important role in shielding the RBD core and in this work the authors demonstrate that removal of this glycan can trigger a conformational change to reduce water access to the core without it. This response is variant dependent and variants containing interface substitutions which increase RBD stability, including Delta substitution L452R, do not experience the same conformational change when the glycan is removed. The authors also explore structures corresponding to Alpha and Beta in which no structure-reinforcing substitutions were identified and two Omicron variants in which other substitutions with an analogous effect to L452R are present.
The authors experimentally assessed these inferred structural changes by measuring the binding affinity of the RBD for the oligosaccharides of the monosialylated gangliosides GM1os and GM2os with and without the glycan at N343. While GM1os and GM2os binding is influenced by additional factors in the Beta and Omicron variants, the comparison between Delta and Wuhan-hu-1 is clear: removal of the glycan abrogated binding for Wuhan-hu-1 and minimally affected Delta as predicted by structural simulations.
In summary, these findings suggest, in the words of the authors, that SARS-CoV-2 has evolved to render the N-glycosylation site at N343 "structurally dispensable". This study emphasizes how glycosylation impacts both viral immune evasion and structural stability which may in turn impact receptor binding affinity and infectivity. Mutations which stabilize the antigen may relax the structural constraints on glycosylation opening up avenues for subsequent mutations which remove glycans and improve immune evasion. This interplay between immune evasion and receptor stability may support complex epistatic interactions which may in turn substantially expand the predicted mutational repertoire of the virus relative to expectations which do not take into account glycosylation.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The study by Seo et al highlights knowledge gaps regarding the role of cerebellar complex spike (CS) activity during different phases of learning related to optokinetic reflex (OKR) in mice. The novelty of the approach is twofold: first, specifically perturbing the activity of climbing fibers (CFs) in the flocculus (as opposed to disrupting communication between the inferior olive (IO) and its cerebellar targets globally); and second, examining whether disruption of the CS activity during the putative "consolidation phase" following training affects OKR performance.
The first part of the results provides adequate evidence supporting the notion that optogenetic disruption of normal CF-Purkinje neuron (PN) signaling results in the degradation of OKR performance. As no effects are seen in OKR performance in animals subjected to optogenetic irradiation during the memory consolidation or retrieval phases, the authors conclude that CF function is not essential beyond memory acquisition. However, the manuscript does not provide a sufficiently solid demonstration that their long-term activity manipulation of CF activity is effective, thus undermining the confidence of the conclusions.
Strengths:
The main strength of the work is the aim to examine the specific involvement of the CF activity in the flocculus during distinct phases of learning. This is a challenging goal, due to the technical challenges related to the anatomical location of the flocculus as well as the IO. These obstacles are counterbalanced by the use of a well-established and easy-to-analyse behavioral model (OKR), that can lead to fundamental insights regarding the long-term cerebellar learning process.
Weaknesses:
The impact of the work is diminshed by several methodological shortcomings.
Most importantly, the key finding that prolonged optogenetic inhibition of CFs (for 30 min to 6 hours after the training period) must be complemented by the demonstration that the manipulation maintains its efficacy. In its current form, the authors only show inhibition by short-term optogenetic irradiation in the context of electrical-stimulation-evoked CSs in an ex vivo preparation. As the inhibitory effect of even the eNpHR3.0 is greatly diminished during seconds-long stimulations (especially when using the yellow laser as is done in this work (see Zhang, Chuanqiang, et al. "Optimized photo-stimulation of halorhodopsin for long-term neuronal inhibition." BMC biology 17.1 (2019): 1-17. ), we remain skeptical of the extent of inhibition during the long manipulations. In short, without a demonstration of effective inhibition throughout the putative consolidation phase (for example by showing a significant decrease in CS frequency throughout the irradiation period), the main claim of the manuscript of phase-specific involvement of CF activity in OKR learning can not be considered to be based on evidence.
Second, the choice of viral targeting strategy leaves gaps in the argument for CF-specific mechanisms. CaMKII promoters are not selective for the IO neurons, and even the most precise viral injections always lead to the transfection of neurons in the surrounding brainstem, many of which project to the cerebellar cortex in the form of mossy fibers (MF). Figure 1Bii shows sparsely-labelled CFs in the flocculus, but possibly also MFs. While obtaining homogenous and strong labeling in all floccular CFs might be impossible, at the very least the authors should demonstrate that their optogenetic manipulation does not affect simple spiking in PNs.
Finally, while the paper explicitly focuses on the effects of CF-evoked complex spikes in the PNs and not, for example, on those mediated by molecular layer interneurons or via direct interaction of the CF with vestibular nuclear neurons, it would be best if these other dimensions of CF involvement in cerebellar learning were candidly discussed.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this paper, the authors used target agnostic MBC sorting and activation methods to identify B cells and antibodies against sexual stages of Plasmodium falciparum. While they isolated some Mabs against PFs48/45 and PFs230, two well-known candidates for "transmission blocking" vaccines, these antibodies' efficacies, as measured by TRA, did not perform as well as other known antibodies. They also isolated one cross-reactive mAb to proteins containing glutamic acid-rich repetitive elements, that express at different stages of the parasite life cycle. They then determined the structure of the Fab with the highest protein binder they could determine through protein microarray, RESA, and observed homotypic interactions.
Strengths:
- Target agnostic B cell isolation (although not a novel methodology).<br /> - New cross-reactive antibody and mechanism (homotypic interactions) as demonstrated by structural data and other biophysical data.
Weaknesses:
The paper lacks clarity at times and could benefit from more transparency (showing all the data) and explanations.<br /> In particular:<br /> -define SIFA<br /> -define TRAbs<br /> -it is not possible to read the Supplementary Figure 6B and C panels.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Throughout the paper, the authors do a fantastic job of highlighting caveats in their approach, from image acquisition to analysis. Despite this, some conclusions and viewpoints portrayed in this study do not appear well-supported by the provided data. Furthermore, there are a few technical points regarding the analysis that should be addressed.
(1) Analysis of signaling traces
- Relevance of "modeled signaling level": It is not clear whether this added complexity and potential for error (below) provides benefits over a more simple analysis such as taking the derivative (shown in Figure 3C). Could the authors provide evidence for the benefits? For example, does the "maximal response" given a simpler metric correlate less well with cell fate than that calculated from the fitted response?
- Assumptions for "modeled signaling level": According to equation (1) Kaede levels are monotonically increasing. This is assumed given the stability of the fluorescent protein. However, this only holds for the "totally produced Kaede/fluorescence". Other metrics such as mean fluorescence can very well decrease over time due to growth and division. Does "intensity" mean total fluorescence? Visual inspection of the traces shown in Figure 2 suggests that "fluorescence intensity" can decrease. What does this mean for the inferred traces?
- Estimation of Kaede reporter half-live: It is not clear how the mRNA stability of Kaede is estimated. It sounds like it was just assessed visually, which seems not entirely appropriate given the quantitative aspects of the rest of the study. Also, given that Shh signaling was inhibited on the level of Smoothened, it is not obvious how the dynamics of signaling shutdown affect the estimate. Most results in Figure 7 seem to be quite robust to the estimate of the half-live. That they are, might suggest that the whole analysis is unnecessary in the first place. However, not all are. Thus, it would be important to make this estimate more quantitative.
(2) Assignment of fates and correlations
- Error estimate for cell-type assignment: Trying to correlate signaling traces to cell fate decisions requires accurate cell fate assignment post-tracking. The provided protocol suggests a rather manual, expert-directed process of making those decisions. Can the authors provide any error-bound on those decisions, for example comparing the results obtained by two experts or something comparable? I am particularly concerned about the results regarding the higher degree of variability in the correlation between signaling dynamics and cell fate in the posterior neural tube. Here, the expression of Olig2 does not seem to segregate between different assigned fates, while it does so nicely in the anterior neural tube. This would suggest to me that cells in the posterior neural tube might not yet be fully committed to a fate or that there could be a relatively high error rate in assigning fates. Thus, the results could emerge from technical errors or differences in pure timing. Could the authors please comment on these possibilities?
- Clustering and fates: One approach the authors use to analyze the correlation between signaling and fate is clustering of cell traces and comparison of the fate distributions in those clusters. There is a large number of clusters with only single traces, suggesting that the data (number of traces) might not be sufficient for this analysis. Furthermore, I am skeptical about clustering cells of different anterior-posterior identities together, given potential differences in the timing of signal reception and signaling. I am not convinced that this analysis reveals enough about how signaling maps to fate given the heterogeneity in traces in large clusters and the prevalence of extremely small clusters.
- Signaling vector and hand-picked metrics: As an alternative approach, that might be better suited for their data, the authors then pick three metrics (based on their model-predicted signaling dynamics) and show that the maximal response is a very good predictor of fate for different anterior-posterior identities. Previous information-theoretic analysis of signaling dynamics has found that a whole time-vector of signaling can carry much more information than individual metrics (Selimkhanov et al, 2014, PMID: 25504722). Have the authors tried to use approaches that make use of the whole trace (such as simple classifiers (Granados et al, 2018, PMID: 29784812), or can comment on why this is not feasible for their data? The authors should at least make clear that their results present a lower bound to how accurately cells can make cell-fate decisions based on signaling dynamics.
(3) Consequences of signaling heterogeneity
The authors focus heavily on portraying that signaling dynamics are highly variable, which seems visually true at first glance. However, there is no metric used or a description given of what this actually means. Mainly, the variability seems to relate to the correlation between signaling and fate. However, given the data and analysis, I would argue that the decoding of signaling dynamics into fate is surprisingly accurate. So signaling dynamics that seem quite noisy and variable by visual inspection can actually be very well discriminated by cells, which to me appears very exciting.
Indeed, simple features of signaling traces can predict cell fate as well as position (for anterior progenitors). Given that signaling should be a function of position, it naively seems as if signaling read-out could be almost perfect. It might be interesting to plot dorsal-ventral position vs the signaling metrics, to also investigate how Shh concentration/position maps to signaling dynamics, this would give an even more comprehensive view of signal transmission.
There remains the discrepancy between signaling traces and fate in the posterior neural tube. The authors point towards differences in tissue architecture and difficulties in interpreting a "small" Shh gradient. However, the data seems consistent with differences in timing of cell-fate decisions between anterior and posterior cells. The authors show that fate does initially not correlate well with position in the posterior neural tube. So, signaling dynamics should likely also not, as they should rather be a function of position, given they are downstream of the Shh gradient. As mentioned above, not even Olig2 expression does segregate the assigned fates well. All this points towards a difference in the time of fate assignment between the anterior and posterior. Given likely delays in reporter protein production and maturation, it can thus not be expected that signaling dynamics correlate better with cell fate than the reporter "83%". Can the authors please discuss this possibility in the paper?
Thus, while this paper represents an example of what the community needs to do to gain a better understanding of robust patterning under variability, the provided data is not always sufficient to make clear conclusions regarding the functional consequences of signaling dynamics.
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Reviewer #1 (Public Review):
Summary:
There is a long-standing idea that choices influence evaluation: options we choose are re-evaluated to be better than they were before the choice. There has been some debate about this finding, and the authors developed several novel methods for detecting these re-evaluations in task designs where options are repeatedly presented against several alternatives. Using these novel methods the authors clearly demonstrate this re-evaluation phenomenon in several existing datasets.
Strengths:
The paper is well-written and the figures are clear. The authors provided evidence for the behaviour effect using several techniques and generated surrogate data (where the ground truth is known) to demonstrate the robustness of their methods.
Weaknesses:
The description of the results of the fMRI analysis in the text is not complete: weakening the claim that their re-evaluation algorithm better reveals neural valuation processes.
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Reviewer #1 (Public Review):
Summary:
Drosophila is one of the most studied model organisms to understand how neural circuits form and function to control intricate animal behaviors. The ventral nerve cord (VNC) part of the fly's CNS serves as a sensory processing and motor output center just like our spinal cord. Over the last decade, the VNC has become a fruitful platform to understand neural circuits responsible for motor behavior such as walking and flying. The missing resource was the complete connectome of the VNC neurons. This study provides this needed resource. The authors documented their approaches on how to generate the data from tissue preparation to computer-assisted reconstruction in a simple manner and left the in-depth analysis of the network features of the connecting neurons to two other well-written companion articles.
Strengths:<br /> Unlike many other previously published EM datasets, the authors presented a ready-to-view connectome dataset of the adult fly VNC. Readers, without needing permission, can access the dataset to find their neurons of interest and determine their synaptic partners with a few clicks. The authors also share their novel approaches in a detailed manner for others to reproduce similar EM volumes for other tissues.
Weaknesses:
The reconstruction completion, around 50%, might be considered a weakness. However, the data appear to have ~ %50 completion across all different neuropils suggesting that sampling is homogenous and does not induce bias. Nevertheless, a higher percentage will give a more complete picture.
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bmcdevbiol.biomedcentral.com bmcdevbiol.biomedcentral.com
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BDSC#31501
DOI: 10.1186/s12861-020-00217-1
Resource: RRID:BDSC_31501
Curator: @abever99
SciCrunch record: RRID:BDSC_31501
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Reviewer #1 (Public Review):
This is an important and very well conducted study providing novel evidence on the role of zinc homeostasis for the control of infection with the intracellular bacterium S. typhimurium also disentangling the underlying mechanisms and providing clear evidence on the importance of spatio-temporal distribution of (free) zinc within the cell.
Comments:
It would be important to provide more information on the genotype of mice. It is rather unlikely that C57Bl6 mice survive up to two weeks after i.p. injection of 1x10E5 bacteria.
To be sure that macrophages Slc30A1 fl/fl LysMcre mice really have an impaired clearance of bacteria it would be important to rule out an effect of Slc30A1 deletion of bacterial phagocytosis and containment (f.e. evaluation of bacterial numbers after 30 min of infection).
Does the addition of zinc to macrophages negatively affect iNOS transcription as previously observed for the divalent metal iron and is a similar mechanism also employed (CEBPß/NF-IL6 modulation) (Dlaska M et al. J Immunol 1999)?
How does Zinc or TPEN supplementation to bacteria in LB medium affect the log growth of Salmonella?
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Reviewer #1 (Public Review):
Sertonin is an important neurotransmitter and it synaptic concentration is controlled by re-uptake by the sodium-coupled serotonin transporter SERT. In this paper, some 6000 mutations of SERT were made and tested for surface expression and uptake of a serotonin analogue APP+. The SERT mutants were analysed and compared to the SERT structure and dynamics based on MD simulations. The authors have concluded that mutations located on surface exposed regions are tolerated whilst those involved in packing and structural integrity are not. Gain-of-function mutations map onto regions that in most cases favour opening of a solvent-exposed intracellular vestibule. Closure of the intracellular gate is thought to be rate-limiting to the transport cycle, and thus the evolutionary-based screen is consistent with the clustering of gain-of-function mutations.
Strengths:<br /> This paper using a large unbiased data-set to probe the evolution of the serotonin transporter SERT for the substrate APP+. They have been able to compare both localisation and transport data, which is an interesting data-set. Using MD simulations they are further able to provide some rationale basis for the gain-of-function mutants.
Weaknesses:<br /> They can only detect surface expression of myc-tagged SERT based on conjugation with a fluorescent anti-myc antibody. As such, they cannot distinguish between SERT mutants that abolish expression vs. those that are no longer trafficking to the plasma membrane. This is a downside, as it would have been interesting to know the fraction of SERT mutations disrupt trafficking. Indeed, the relationship between misfolding and targeting is poorly understood beyond the calnexin- calreticulin cycle. Furthermore, there seems to be a gap between the large-scale mutagenesis data and the MD simulations in which the main mechanistic conclusions seem to be based on (carried out in a separate publication). Thus, overall while the mutation data-set is impressive its not clear how this aids to our mechanistic understanding of SERT.
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Reviewer #1 (Public Review):
The authors report a high-quality genome assembly for a member of Xenacoelomorpha, a taxon that is at the center of the last remaining great controversies in animal evolution. The taxon and the species in question have "jumped around" the animal tree of life over the past 25 years, and seemed to have found their place as a sister-group to all remaining bilaterians. This hypothesis posits that the earliest split within Bilateria includes Xenacoelomorpha on the one hand and a clade known as Nephrozoa (Protostomia + Deuterostomia) on the other, and is thus referred to as the Nephrozoa hypothesis. Nephrozoa is supported by phylogenomic evidence, by a number of synapomorphic morphological characters in the Nephrozoa (namely, the presence of nephridia) and lack of some key bilaterian characters in Xenacoelomorpha, and by the presence of unique miRNAs in Nephrozoa.
The Nephrozoa hypothesis has been challenged several times by the authors' groups who alternatively suggest placing Xenacoelomorpha within Deuterostomia as a sister group to a clade known as Ambulacraria. This hypothesis (the Xenambulacraria hypothesis) is supported by alternative phylogenomic datasets and by the shared presence of a number of unique molecular signatures. In this contribution, the authors aim to strengthen their case by providing full genome data for Xenoturbella bocki.<br /> The actual sequencing and analysis are technically and methodologically excellent. Some of the analyses were done several years ago using approaches that may now seem obsolete, but there is no reason not to include them. As a detailed report of a newly sequenced genome, the manuscript meets the highest standards.
The authors emphasize a number of key findings. One is the fact that the genome is not as simple as one might expect from a "basal" taxon, and is on par with other bilaterian genomes and even more complex than the genome of secondarily simplified bilaterians. There is an implicit expectation here that the sister group to all Bilateria would represent the primitive state. This is of course not true, and the authors are aware of this, but it sometimes feels as though they are using this implicit assumption as a straw dog argument to say that since the genome is not as simple as expected, X. bocki must be nested within Bilateria. The authors get around this by acknowledging that their finding is consistent with a "weak version of the Nephrozoa hypothesis", which is essentially the Nephrozoa phylogenetic hypothesis without implicit assumptions of simplicity.
Another finding is a refutation of the miRNA data supporting Nephrozoa. This is an important finding although it is somewhat flogging a dead horse, since there is already a fair amount of skepticism about the validity of the miRNA data (now over 20 years old) for higher-level phylogenetics.
The finding that the authors feel is most important is gene presence-absence data that recovers a topology in which X. bocki is sister to Abulacraria. The problem is that the same tree does not support the monophyly of Xenacoelomorpha. This may be an artifact of fast evolving acoel genomes, as the authors suggest, but it still raises questions about the robustness of the data.
In sum, the authors' results and analyses leave an open window for the Xenambulacraria hypothesis, but do not refute the Nephrozoa hypothesis. The manuscript is a valuable contribution to the debate but does not go a significant way towards its resolution.<br /> The manuscript has gone through several rounds of review and revision on a preprint server and is thus fairly clear of typos, inconsistencies and lack of clarity. The authors are honest and open in their interpretation of the results and their strengths.
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Reviewer #1 (Public Review):
Rubin et al. study chondrocyte columns in the prenatal and postnatal growth plate in 3D for the first time, using a novel analysis pipeline in which Confetti clones in the murine growth plate are analysed morphometrically. Prenatal chondrocytes were found not to be organised in columns parallel to the main orientation of the long bone, but rather, prenatal chondrocytes were commonly organised perpendicular to the main direction of growth. In the postnatal (P40) growth plate there was a diverse arrangement of columns, but more of the columns were vertically aligned
I enjoyed reading the work and the analysis is rigorous. However, I think that it is not valid to state that columns do not form in the embryo. The data only supports the finding that strictly vertical columns do not form in the embryo, as the cells are still organised into columns, albeit with a range of orientations. I do not like the term "typically" aligned, as how can we know what is "typical" when orientation has never before been assessed in 3D... And the authors' data demonstrates that it is certainly not "typical" for chondrocyte to organise into vertical columns prenatally.
It would be very interesting to delve deeper into the reason for the change in orientation of columns between pre- and post-natal. For example, does more circumferential growth happen prenatally as compared to postnatally? Is the rate of circumferential vs longitudinal growth different between prenatal and postnatal, and could the change in column orientation be responsible for a (possible) shift in the balance between longitudinal vs circumferential growth before vs after birth? The first sentence of the Discussion refers to the role of chondrocyte columns in driving bone elongation, but aren't they also involved in driving bone morphology?
I feel describing the activity of the cells as "mis-rotations" which implies the orientations are not intentional. It is likely not accidental or mistaken that the chondrocytes align in the ways they do- the diaphysis is largely for longitudinal growth while the epiphyses, and lateral expansion of the joint is also important. I find the data in Figure 4 fascinating, especially the variation in orientations between the regions of the growth plate (from proximal to distal), with the most lateral orientation at the most proximal and distal ends- it would be nice to see more discussion of these variations and what they may be contributing to.
The abstract focuses solely on the analysis of columns prenatally and would benefit from the inclusion of the data from the postnatal growth plate and from the chondrocyte rotations.
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Reviewer #1 (Public Review):
This manuscript presents, for the first time, the utilization of PRV viral transneuronal tracing to elucidate the central coding and control mechanisms governing sympathetic nervous system (SNS) efferent signals to bone. This groundbreaking work not only holds promising research prospects but also establishes a robust foundation for understanding the neural regulation of bone metabolism.
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Reviewer #1 (Public Review):
Garcia-Saldivar and colleagues present a manuscript investigating connections between diffusion-weighted imaging (DWI) parameters and paced finger tapping measures. A cohort of human participants (n=32) performed a paced finger tapping task with a synchronization-continuation paradigm, in which they were required to listen to a paced metronome, begin tapping in synchrony with it, and then continue tapping at the same rate without it. Both auditory and visual metronomes were used, at a range of intervals. All subjects received structural scans measuring DWI, with an emphasis on superficial and deep white matter structures. This latter analysis was the most innovative, as it allowed the authors to examine microstructural effects in short-range cortical connections.
Behaviorally, the authors replicated some well-known effects in paced finger tapping, with better performance for auditory over visual rhythms, negative lag-1 autocorrelations, and best performance at a range of ~1.5Hz. For the DWI analyses, a large number of correlations were observed across a wide variety of connections with various brain regions. The most salient effects observed were a connection between asynchrony, only for the auditory condition, and connections between the right auditory and motor systems, around the duration of peak performance, as well as a "chronotopic" organization across parts of the corpus callosum, most notably in areas linking motor regions between hemispheres.
Overall, this paper provides a critical missing link between measures of structural connectivity and rhythmic tapping abilities, pointing to some interesting possibilities for how tapping synchronization (at least for auditory intervals) is carried out. Negative aspects of the paper come from the largely exploratory aspects of the analysis, as well as potential biases from the low sample size.
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Reviewer #1 (Public Review):
The manuscript demonstrates an analysis of the synaptic organization within the motor thalamus, emphasizing the interplay between the ventrolateral (VL) and ventroanterior (VA) nuclei and their respective inputs. The primary aim is to unravel the complexities of synaptic interactions among the motor cortex's layer 5 (M1L5), the cerebellum (Cb), and the basal ganglia output nuclei (GPi and SNr), which converge upon the VA/VL nuclei of the motor thalamus. This examination is executed using a combination of anatomical tracing, optogenetics, and electrophysiological recordings in mouse brain slices, which together yield novel insights into the motor control circuitry.
The study uncovers that contrary to traditional models that presumed segregation, some motor thalamic neurons simultaneously integrate inputs from the cerebellum and basal ganglia. Furthermore, a subset of these neurons also receive convergent inputs from M1L5 and basal ganglia, underscoring the complexity of these synaptic networks. Notably, the study reveals that both M1L5 and Cb inputs exhibit driver-type synaptic properties, suggesting a significant impact on thalamic relay neurons.
The functional implications of this synaptic convergence suggest a complex gating mechanism by the inhibitory outputs of the basal ganglia, which could modulate information flow within the motor thalamus. This modulation is significant not only for transthalamic information processing but also for the integration of cerebellar inputs to the motor cortex. The study also highlights direct projections from M1L5 to the motor thalamus, indicating a potential direct influence on thalamic activity, in addition to the known indirect influence through the cortico-basal ganglia-thalamo-cortical loop.
The manuscript suggests that the traditional understanding of motor thalamic connectivity requires reconsideration, and it emphasizes the necessity of further investigation to understand fully the functional implications of this synaptic convergence. Future research may focus on more direct demonstrations of triple-input convergence and its behavioral consequences, as well as cross-species comparative studies to enhance the findings' applicability.
While the study provides valuable contributions to our knowledge of the motor thalamus, illuminating the intricate synaptic architecture of the motor thalamus and setting the stage for future explorations that will deepen our comprehension of motor control and thalamic function.
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Reviewer #1 (Public Review):
This manuscript presents an exciting new method for separating insulin secretory granules using insulator-based dielectrophoresis (iDEP) of immunolabeled vesicles. The method has the advantage of being able to separate vesicles by subtle biophysical differences that do not need to be known by the experimenter, and hence could in principle be used to separate any type of organelle in an unbiased way. Any individual organelle ("particle") will have a characteristic ratio of electrokinetic to dielectrophoretic mobilities (EKMr) that will determine where it migrates in the presence of an electric field. Particles with different EKMr will migrate differently and thus can be separated. The present manuscript is primarily a methods paper to show the feasibility of the iDEP technique applied to insulin vesicles. Experiments are performed on cultured cells in low or high glucose, with the conclusion that there are several distinct subpopulations of insulin vesicles in both conditions, but that the distributions in the two conditions are different. As it is already known that glucose induces release of mature insulin vesicles and stimulates new vesicle biosynthesis and maturation, this finding is not necessarily new, but is intended as a proof of principle experiment to show that the technique works. This is a promising new technology based on solid theory that has the possibility to transform the study of insulin vesicle subpopulations, itself an emerging field. The technique development is a major strength of the paper. Also, cellular fractionation and iDEP experiments are performed well, and it is clear that the distribution of vesicle populations is different in the low and high glucose conditions. However, more work is needed to characterize the vesicle populations being separated, leaving open the possibility that the separated populations are not only insulin vesicles, but might consist of other compartments as well. It is also unclear whether the populations might represent immature and mature vesicles, distinct pools of mature vesicles such as the readily releasable pool and the reserve pool, or vesicles of different age. Without a better characterization of these populations, it is not possible to assess how well the iDEP technique is doing what is claimed.
Major comments:
(1) There is no attempt to relate the separated populations of vesicles to known subpopulations of insulin vesicles such as immature and mature vesicles, or the more recently characterized Syt9 and Syt7 vesicle subpopulations that differ in protein and lipid composition (Kreutzberger et al. 2020). Given that it is unclear exactly what populations of vesicles will be immunolabeled (see point #2 below), it is also possible that some of the "subpopulations" are other compartments being separated in addition to insulin vesicles. It will be important to examine other markers on these separated populations or to perform EM to show that they look like insulin vesicles.
(2) An antibody to synaptotagmin V is used to immunolabel vesicles, but there has been confusion between synaptotagmins V and IX in the literature and it isn't clear what exactly is being recognized by this antibody (this reviewer actually thinks it is Syt 9). If it is indeed recognizing Syt 9, it might already be labeling a restricted population of insulin vesicles (Kreutzberger et al. 2020). The specificity of this antibody should be clarified. Furthermore, Figure 2 is not convincing at showing that this synaptotagmin antibody specifically labels insulin vesicles nor is there convincing colocalization of this synaptotagmin antibody with insulin vesicles. In the image shown, several cells show very weak or no staining of both insulin and the synaptotagmin. The highlighted cell appears to show insulin mainly in a perinuclear structure (probably the Golgi) rather than in mature vesicles (which should be punctate), and insulin is not particularly well-colocalized with the synaptotagmin. Other cells in the image appear to have even less colocalization of insulin and synaptotagmin, and there is no quantification of colocalization. It seems possible that this antibody is recognizing other compartments in the cell, which would change the interpretation of the populations measured in the iDEP experiments. It would also be good to perform synaptotagmin staining under glucose-stimulating conditions, in case this alters the localization.
(3) The EKMr values of the vesicle populations between the low and high glucose conditions don't seem to precisely match. It is unclear if this just a technical limitation in comparing between experiments or instead suggests that glucose stimulation does not just change the proportion of vesicles in the subpopulations (i.e. the relative fluorescent intensities measured), but rather the nature of the subpopulations (i.e. they have distinct biophysical characteristics). This again gets to the issue of what these vesicle subpopulations represent. If glucose stimulation is simply converting immature to mature vesicles, one might expect it to change the proportion of vesicles, but not the biophysical properties of each subpopulation.
(4) The title of the paper promises "isolation" of insulin vesicles, but the manuscript only presents separation and no isolation of the separated populations. Isolation of the separated populations is important to be able to better define what these populations are (see point #1 above). Isolation is also critical if this is to be a valuable technique in the future. Yet the paper is unclear on whether it is actually technically feasible to isolate the populations separated by iDEP. In line 367, it states "this method provides a mechanism for the isolation and concentration of fractions which show the largest difference between the two population patterns for further bioanalysis (imaging, proteomics, lipidomics, etc.)." However, in line 361 it says "developing the capability to port the collected individual boluses will enable downstream analyses such as mass spectrometry or electron microscopy," suggesting that true isolation of these populations is not yet feasible. This should be clarified.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The manuscript describes a model based on 5-state cellular automata of development of an infection. The model is motivated and qualitatively justified by time-resolved measurements of expression levels of viral, interferon-producing, and antiviral genes. The model is set up in such a way that the crucial difference in outcomes (infection spreading vs. confinement) depends on the initial fraction of special virus-sensing cells. Those cells (denoted as 'type a') cannot be infected and do not support the propagation of infection, but rather inhibit it in a somewhat autocatalytic way. Presumably, such feedback makes the transition between two outcomes very sharp: a minor variation in concentration of 'a' cells results in qualitative change from one outcome to another. As in any percolation-like system, the transition between propagation and inhibition of infection goes through a critical state with all its attributes, including a power-law distribution of the cluster size (corresponding to the fraction of infected cells) with a fairly universal exponent and a cutoff at the upper limit of this distribution.
Strengths:
The proposed model suggests a well-justified explanation for the frequently observed yet puzzling diversity of outcomes of viral infections such as COVID.
Weaknesses:
None.
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Reviewer #1 (Public Review):
This paper can be seen as an extension of a recent study by two of the same authors [1]. In the previous paper, the authors considered two variants of the Moran process, labelled Model A and Model B, and examined differences between the evolutionary dynamics of these two models. They further described the site frequency spectra, expected allele counts, and expected singleton counts of these models, building on analytical results from prior studies, and used numerical simulations to investigate the models' evolutionary dynamics. Finally, they compared the site frequency spectra of the two models (using numerical simulations) to spectra derived from a small breast cancer data set (two sets of three samples).
In the new paper, the authors consider the same two Moran process variants (Model A and Model B) and some related branching processes. As before, they compare the site frequency spectra and various summary statistics of these models, but here they present only numerical simulations (except that some prior analytical results are summarized in Appendix A, which are never referred to in the main text and seem unconnected to the study). They then compare the site frequency spectra of these models (again using numerical simulations) to those derived from the same breast cancer samples as before and thus infer some evolutionary parameters.
The first main conclusion is that the critical branching process and the Moran process models behave similarly and generate similar site frequency spectra. This finding is unsurprising (indeed, the authors acknowledge that the result "has been expected"). For a reasonably large population size, the population size in the critical branching process has been shown to vary relatively little over time and the model is thus essentially a continuous time Moran process (see, for example, Equation 8.55 in ref 2). Nor is it surprising that the authors see stronger similarities when they select only the subset of branching process replicates in which the final population size is particularly close to the initial population size (this is because, in these replicates, the population size likely varies even less than usual).
The second main conclusion is that, although "the mutational SFS alone is not adequate" to quantify the strength of selection, "All fitted values for the selective disadvantage of passenger mutations are nonzero, supporting the view that they exert deleterious selection during tumorigenesis". Although the question of whether mildly deleterious mutations play an important role in cancer evolution is of considerable interest, it's debatable whether the results presented here help resolve the issue.
Many prominent researchers have called into question whether cancer evolutionary parameters can be reliably inferred from site frequency spectra (e.g., [3-7]), even using sophisticated statistical methods. The statistical approach used here (though not named as such in the paper) is a crude kind of approximate Bayesian computation. To improve the accuracy of the results, it would have been better to have set reasonably vague priors for the uncertain mutation rates, rather than fixing them arbitrarily. It would also have been better to have chosen a likelihood function explicitly based on an analysis of the sampling and error distributions, rather than just summing the absolute logged deviations. It is well known that "Checking the model is crucial to statistical analysis" and "A good Bayesian analysis, therefore, should include at least some check of the adequacy of the fit of the model to the data and the plausibility of the model for the purposes for which the model will be used" [8]. The authors' failure to describe any attempt to validate or check their model, using simulated data or otherwise, casts doubt on the reliability of their inferences.
Putting aside the potential biassing effects of sampling error, measurement error, and the limitations of the authors' statistical method, it is well established that both population growth and spatial structure profoundly alter the shape of site frequency spectra in ways that can mimic the effects of selection (e.g. [9-11]). Indeed, Figures 3, 4 and 5 show that the critical and super-critical branching processes generate markedly different site frequency spectra. It follows that if the population dynamics and spatial structure of the mathematical model used for inference don't match those of the biological process that produced the data then any inferred evolutionary parameter values will be unreliable. Breast cancer has two indisputable ecological features that shape its evolutionary dynamics: the cell population expands by many orders of magnitude from a single cell, and the population is spatially structured. In the authors' mathematical model, the population size is initially 100 cells and either remains constant or varies little, and there is no spatial structure. These profound mismatches between model and data cast further doubt on what is supposed to be the paper's most important biological finding.
In this paper the authors offer no justification for their decision to model breast cancer as a non-growing, non-spatial cell population. Nor do they engage with the extensive recent literature on the challenges of inferring evolutionary parameters from cancer site frequency spectra (they cite none of the many relevant papers listed at https://www.sottorivalab.org/neutral-evolution.html). Their 2022 paper [1] claims that, "it sometimes makes sense to consider cancer growth in the framework of constant-population models. Our models correspond to the situation in which a constant population of N "healthy" stem cells is gradually replaced by a growing clone of transformed cells with increasing fitness." No evidence was presented to support this hypothesis regarding breast cancer progression. On the other hand, a wealth of evidence supports the consensus view that, in breast cancer and other human solid tumours, the number of cells with unlimited proliferative potential is several orders of magnitude greater than 100 and grows over time (e.g. [12]).
Analytic expressions for the site frequency spectra with neutral mutations are already known. It is well known that the site frequency spectrum of an exponentially growing population has a tail following a power law S_k ~ k^(-2) [13, 14]. Similarly, it is known that for the critical branching process or the Moran process, the site frequency spectrum at equilibrium is S_k ~ k^(-1) [13, 15]. Especially noteworthy yet uncited studies that use those results about site frequency spectra to make inferences based on sequencing data include ref 16, in which selection is inferred, and ref 17, in which evolutionary parameters of constant populations (healthy cell populations) are inferred.
Although the paper is well written, the figures are ineffective in communicating the results. As others have put it, "A figure is meant to express an idea or introduce some facts or a result that would be too long (or nearly impossible) to explain only with words" and "If your figure is able to convey a striking message at first glance, chances are increased that your article will draw more attention from the community" [18]. On the contrary, Figures 3, 4, 5 and 6 are bewilderingly complicated, crowded, and repetitive. These figures comprise no fewer than fifty-six plots, each containing numerous curves or histograms, spread across four pages. To compare the results of different scenarios, the reader is presumably expected to put these figures side by side and try to spot the differences, hampered by inconsistent axis ranges, absence of axis labels, absence of titles, absence of legends, and unreliable captions ("cyan" seems to refer to pale blue, and "orange" to something closer to red). For example, the only notable difference between Figures 3 and 4 is in the shape of a single green curve in panel I. In the main text of a published paper, one would expect fewer, more carefully curated figures drawing attention to salient features, so that the reader can infer the main results with minimal effort. The rest can be put in supplementary figures.
In summary, this paper adds somewhat to our understanding of some standard mathematical models; whether it tells us anything new about cancer is open to debate.
References<br /> (1) Kurpas, Monika K., and Marek Kimmel. "Modes of selection in tumors as reflected by two mathematical models and site frequency spectra." Frontiers in Ecology and Evolution 10 (2022): 889438.<br /> (2) Bailey, Norman TJ. The elements of stochastic processes with applications to the natural sciences. John Wiley & Sons, 1964.<br /> (3) Tarabichi, Maxime, et al. "Neutral tumor evolution?." Nature Genetics 50.12 (2018): 1630-1633.<br /> (4) McDonald, Thomas O., Shaon Chakrabarti, and Franziska Michor. "Currently available bulk sequencing data do not necessarily support a model of neutral tumor evolution." Nature Genetics 50.12 (2018): 1620-1623.<br /> (5) Balaparya, Abdul, and Subhajyoti De. "Revisiting signatures of neutral tumor evolution in the light of complexity of cancer genomic data." Nature Genetics 50.12 (2018): 1626-1628.<br /> (6) Noorbakhsh, Javad, and Jeffrey H. Chuang. "Uncertainties in tumor allele frequencies limit power to infer evolutionary pressures." Nature Genetics 49.9 (2017): 1288-1289.<br /> (7) Bozic, Ivana, Chay Paterson, and Bartlomiej Waclaw. "On measuring selection in cancer from subclonal mutation frequencies." PLoS Computational Biology 15.9 (2019): e1007368.<br /> (8) Neher, Richard A., and Oskar Hallatschek. "Genealogies of rapidly adapting populations." Proceedings of the National Academy of Sciences 110.2 (2013): 437-442.<br /> (9) Gelman, Andrew, et al. Bayesian data analysis (Third Edition). Chapman and Hall/CRC, 2014.<br /> (10) Fusco, Diana, et al. "Excess of mutational jackpot events in expanding populations revealed by spatial Luria-Delbrück experiments." Nature Communications 7.1 (2016): 12760.<br /> (11) Noble, Robert, et al. "Spatial structure governs the mode of tumour evolution." Nature Ecology & Evolution 6.2 (2022): 207-217.<br /> (12) Lawson, Devon A., et al. "Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells." Nature 526.7571 (2015): 131-135.<br /> (13) Gunnarsson, Einar B., Leder, Kevin, and Foo Jasmine. "Exact site frequency spectra of neutrally evolving tumors: A transition between power laws reveals a signature of cell viability" Theoretical Population Biology 142 (2021) 67-90<br /> (14) Durrett, Richard "Branching Process Models of Cancer" Springer (2015)<br /> (15) Durrett, Richard "Probability Models for DNA Sequence Evolution" Springer Science & Business media (2008)<br /> (16) Williams, Mark J. et al. "Quantification of subclonal selection in cancer from bulk sequencing data." Nature Genetics 50 (6). 895-903 (2018)<br /> (17) Moeller, Marius E. et al. "Measures of genetic diversification in somatic tissues at bulk and single-cell resolution" eLife (2024) 12:RP89780<br /> (18) Rougier, Nicolas P., Michael Droettboom, and Philip E. Bourne. "Ten simple rules for better figures." PLoS Computational Biology 10.9 (2014): e1003833.
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Reviewer #1 (Public Review):
Summary:
This study offers a new perspective. ACTL7A and ACTL7B play roles in epigenetic regulation in spermiogenesis. Actin-like 7 A (ACTL7A) is essential for acrosome formation, fertilization, and early embryo development. ACTL7A variants cause acrosome detachment responsible for male infertility and early embryonic arrest. It has been reported that ACTL7A is localized on the acrosome in mouse sperms (Boëda et al., 2011). Previous studies have identified ACTL7A mutations (c.1118G>A:p.R373H; c.1204G>A:p.G402S, c.1117C>T:p.R373C), All these variants were located in the actin domain and were predicted to be pathogenic, affecting the number of hydrogen bonds or the arrangement of nearby protein structures (Wang et al., 2023; Xin et al., 2020; Zhao et al., 2023; Zhou et al., 2023). This work used AI to model the role of ACTL7A/B in the nucleosome remodeling complex and proposed a testis-specific conformation of SCRAP complex. This is different from previous studies.
Strengths:
This study provides a new perspective to reveal the additional roles of these proteins.
Weaknesses:
The results section contains a substantial background description. However, the results and discussion sections require streamlining. There is a lack of mutual support for data between the sections, and direct data to support the authors' conclusions are missing.
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offountainpenstypewriters.blogspot.com offountainpenstypewriters.blogspot.com
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Reviewer #2 (Public Review):
Summary:
The goals of this study were to develop a genetic approach that would specifically and comprehensively target axo-axonic cells (AACs) throughout the brain and then to describe the patterns and characteristics of the targeted AACs in multiple, selected brain regions. The investigators have been successful in providing the most complete description of the regional distribution of putative (pAACs) throughout the brain to date. The supporting evidence is convincing, and the findings should serve as a guide for more detailed studies of AACs within each brain region and lead to new insights into their connectivity and functional organization of this important group of GABAergic interneurons.
Strengths:
The study has numerous strengths. A major strength is the development of a unique intersectional genetic strategy that uses cell lineage (Nkx2.1) and molecular (Unc5b or Pthlh) markers to identify AACs specifically and, apparently, nearly completely throughout the mouse brain. While AACs have been described previously in the cerebral cortex, hippocampus and amygdala, there has been no specific genetic marker that selectively identifies all AACs in these regions.
Importantly, the current genetic strategy labels pAACs in additional brain regions, including the claustrum-insular complex, extended amygdala, and several olfactory centers in which AACs have not been previously recognized. In general, the findings provide support for the specificity of the methods for targeting AACs and include several examples of labeling near markers of axon initial segments, providing validation of their AAC identity.
The descriptions and numerous low magnification images of the brain provide a roadmap for subsequent, detailed studies of AACs in numerous brain regions. The overview and summaries of the findings in the Abstract, Introduction and Discussion are particularly clear and helpful in placing the extensive regional descriptions of AACs in context.
Weaknesses:
Considering the unique and striking characteristics of AACs, it would have been ideal to include a clear, high resolution confocal image of an AAC from the Unc5b;Nkx2.1 mouse that would display the beauty of these cells with their numerous cartridges of axon terminals, emanating from a single AAC. While several cells are illustrated, the processes are often obscured by other labeling or the background created by the blue Dapi labeling. A high-resolution image of an isolated cell would not only support the identity of the cells as AACs but also demonstrate the potential advantages of their labeling for more detailed anatomical and neurophysiological studies. High magnification views of the axon terminals adjacent to AnkG-labeled axon initial segments are included and provide strong support for the identity of the cells. However, they cannot convey the extensiveness and patterns of the axonal arborizations of these cells.
The intersectional genetic methods included use of the lineage marker Nkx2.1 with either Unc5b or Pthlh as the molecular marker. As described, the mice with intersectional targeting of Nkx2.1 and Unc5b appear to show the most specific brain-wide labeling for AACs, and the majority of the descriptions are from these mice. The targeting with Nkx2.1 and Pthlh is less convincing and there appears to be a disconnect between the descriptions and the images. While the descriptions emphasize that the labeling is very similar in the two types of mice, the images suggest distinct differences, including labeling of non AACs in striatum and layer 4 of the cortex in the Pthlh;Nkx2.1 mouse, as described in the manuscript. In addition, the Pthlh;Nkx2.1 mouse has higher cell targeting in some regions and fewer labeled cells in others. Perhaps it would be more accurate to present the Pthlh;Nkx2.1 mouse as differing from the Unc5b;Nkx2.1 mouse, but useful for AAC labeling in select regions and under some conditions, such as following tamoxifen administration at specific ages. As currently presented, the inclusion of the Pthlh;Nkx2.1 detracts from the otherwise convincing argument that the Unc5b;Nkx2.1 mouse provides a specific and comprehensive way to identify AACs.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this study, Rosenblum et al introduce a novel and automatic way of calculating sleep cycles from human EEG. Previous results have shown that the slope of the non-oscillatory component of the power spectrum (called the aperiodic or fractal component) changes with the sleep stage. Building on this, the authors present an algorithm that extracts the continuous-time fluctuations in the fractal slope and propose that peaks in this variable can be used to identify sleep cycle limits. Cycles defined in this way are termed "fractal cycles". The main focus of the article is a comparison of fractal and classical, manually defined sleep cycles in numerous datasets.
Strengths:
The manuscript amply illustrates through examples the strong overlap between fractal and classical cycle identification. Accordingly, a high percentage (81%) can be matched one-to-one between methods and sleep cycle duration is well correlated (around R = 0.5). Moreover, the methods track certain global changes in sleep structure in different populations: shorter cycles in children and longer cycles in patients medicated with REM-suppressing anti-depressants. Finally, a major strength of the results is that they show similar agreement between fractal and classical sleep cycle length in 5 different data sets, showing that it is robust to changes in recording settings and methods.
These results suggest that the fractal cycle methodology could provide a valuable new method to study sleep architecture and avoid the time-consuming steps of manual cycle identification. Moreover, it has the potential to be applied to animal studies which rarely deal with sleep cycle structure.
Weaknesses:
The match between fractal and classical cycles is not one-to-one. For example, the fractal method identifies a correlation between age and cycle duration in adults that is not apparent with the classical method. This raises the question as to whether differences are due to one method being more reliable than another or whether they are also identifying different underlying biological differences. It is not clear for example whether the agreement between the two methods is better or worse than between two human scorers, which generally serve as a gold standard to validate novel methods. The authors provide some insight into differences between the methods that could account for differences in results. However, given that the fractal method is automatic it would be important to clearly identify criteria for recordings in which it will produce similar results to the classical method.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This paper shows that E. coli exhibits a chemotactic response to potassium by measuring both the motor response (using a bead assay) and the intracellular signaling response (CheY phosporylation level via FRET) to step changes in potassium concentration. They find increase in potassium concentration induces a considerable attractant response, with amplitude comparable to aspartate, and cells can quickly adapt (and generally over-adapt). The authors propose that the mechanism for potassium response is through modifying intracellular pH; they find both that potassium modifies pH and other pH modifiers induce similar attractant responses. It is also shown, using Tar- and Tsr-only mutants, that these two chemoreceptors respond to potassium differently. Tsr has a standard attractant response, while Tar has a biphasic response (repellent-like then attractant-like). Finally, the authors use computer simulations to study the swimming response of cells to a periodic potassium signal secreted from a biofilm and find a phase delay that depends on the period of oscillation.
Strengths:
The finding that E. coli can sense and adapt to potassium signals and the connection to intracellular pH is quite interesting and this work should stimulate future experimental and theoretical studies regarding the microscopic mechanisms governing this response. The evidence (from both the bead assay and FRET) that potassium induces an attractant response is convincing, as is the proposed mechanism involving modification of intracellular pH. The updated manuscript controls for the impact of pH on the fluorescent protein brightness that can bias the measured FRET signal. After correction the response amplitude and sharpness (hill coefficient) are comparable to conventional chemoattractants (e.g. aspartate), indicating the general mechanisms underlying the response may be similar. The authors suggest that the biphasic response of Tar mutants may be due to pH influencing the activity of other enzymes (CheA, CheR or CheB), which will be an interesting direction for future study.
Weaknesses:
The measured response may be biased by adaptation, especially for weak potassium signals. For other attractant stimuli, the response typically shows a low plateau before it recovers (adapts). In the case of potassium, the FRET signal does not have an obvious plateau following the stimuli of small potassium concentrations, perhaps due to the faster adaptation compared to other chemoattractants. It is possible cells have already partially adapted when the response reaches its minimum, so the measured response may be a slight underestimate of the true response. Mutants without adaptation enzymes appear to be sensitive to potassium only at much larger concentrations, where the pH significantly disrupts the FRET signal; more accurate measurements would require the development of new mutants and/or measurement techniques.
Note added after the second revision: The authors made a reasonable argument regarding the effects of adaptation, which were estimated to be small.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this paper, the authors evaluate the utility of brain-age derived metrics for predicting cognitive decline by performing a 'commonality' analysis in a downstream regression that enables the different contribution of different predictors to be assessed. The main conclusion is that brain-age derived metrics do not explain much additional variation in cognition over and above what is already explained by age. The authors propose to use a regression model trained to predict cognition ('brain cognition') as an alternative suited to applications of cognitive decline. While this is less accurate overall than brain age, it explains more unique variance in the downstream regression.
Comments on revised version:
I thank the authors for the revision of the manuscript and for being more explicit about the inherent conceptual limitations of Brain Age / Brain Cognition. I have no further comments.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this work the authors propose a new regulatory role for one the most abundant circRNAs, circHIPK3. They demonstrate that circHIPK3 interacts with an RNA binding protein (IGF2BP2), sequestering it away from its target mRNAs. This interaction is shown to regulates the expression of hundreds of genes that share a specific sequence motif (11-mer motif) in their untranslated regions (3'-UTR), identical to one present in circHIPK3 where IGF2BP2 binds. The study further focuses on the specific case of STAT3 gene, whose mRNA product is found to be downregulated upon circHIPK3 depletion. This suggests that circHIPK3 sequesters IGF2BP2, preventing it from binding to and destabilizing STAT3 mRNA. The study presents evidence supporting this mechanism and discusses its potential role in tumor cell progression. These findings contribute to the growing complexity of understanding cancer regulation and highlight the intricate interplay between circRNAs and protein-coding genes in tumorigenesis.
Strengths:
The authors show mechanistic insight into a proposed novel "sponging" function of circHIPK3 which is not mediated by sequestering miRNAs but rather a specific RNA binding protein (IGF2BP2). They address the stoichiometry of the molecules involved in the interaction, which is a critical aspect that is frequently overlooked in this type of studies. They provide both genome-wide analysis and a specific case (STAT3) which is relevant for cancer progression. Overall, the authors have significantly improved their manuscript in their revised version.
Weaknesses:
There are seemingly contradictory effects of circHIPK3 and STAT3 depletion in cancer progression. However, the authors have addressed these issues in their revised manuscript, incorporating potential reasons that might explain such complexity.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This paper describes RNA-sensing guide RNAs for controlled activation of CRISPR modification. This works by having an extended guide RNA with a sequence that folds back onto the targeting sequence such that the guide RNA cannot hybridise to its genomic target. The CRISPR is "activated" by the introduction of another RNA, referred to as a trigger, that competes with this "back folding" to make the guide RNA available for genome targeting. The authors first confirm the efficacy of the approach using several RNA triggers and a GFP reporter that is activated by dCas9 fused to transcriptional activators. A major potential application of this technique is the activation of CRISPR in response to endogenous biomarkers. As these will typically be longer than the first generation triggers employed by the authors they test some extended triggers, which also work though not always to the same extent. They then introduce MODesign which may enable the design of bespoke or improved triggers. After that, they determine that the mode of activation by the RNA trigger involves cleavage of the RNA complexes. Finally, they test the potential for their system to work in a developmental setting - specifically zebrafish embryos. There is some encouraging evidence, though the effects appear more subtle than those originally obtained in cell culture.
Overall, the potential of a CRISPR system that can be activated upon sensing an RNA is high and there are a myriad of opportunities and applications for it. This paper represents a reasonable starting point having developed such a system in principle.<br /> The weakness of the study is that it does not demonstrate that the system can be used in a completely natural setting. This would require an endogenous transcript as the RNA trigger with a clear readout. The authors now acknowledge this limitation in their revised manuscript. Future studies and experiments should focus on these aspects in order for the system to be employed to its full and intended potential.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In the presence of predators, animals display attenuated foraging responses and increased defensive behaviors that serve to protect them from potential predatory attacks. Previous studies have shown that the basolateral nucleus of the amygdala (BLA) and the periaqueductal gray matter (PAG) are necessary for the acquisition and expression of conditioned fear responses. However, it remains unclear how BLA and PAG neurons respond to predatory threats when animals are foraging for food. To address this question, Kim and colleagues conducted in vivo electrophysiological recordings from BLA and PAG neurons and assessed approach-avoidance responses while rats search for food in the presence of a robotic predator.
The authors observed that rats exhibited a significant increase in the latency to obtain the food pellets and a reduction in the pellet success rate when the predator robot was activated. A subpopulation of PAG neurons showing increased firing rate in response to the robot activation didn't change their activity in response to food pellet retrieval during the pre- or post-robot sessions. Optogenetic stimulation of PAG neurons increased the latency to procure the food pellet in a frequency- and intensity-dependent manner, similar to what was observed during the robot test. Combining optogenetics with single-unit recordings, the authors demonstrated that photoactivation of PAG neurons increased the firing rate of 10% of BLA cells. A subsequent behavioral test in 3 of these same rats demonstrated that BLA neurons responsive to PAG stimulation displayed higher firing rates to the robot than BLA neurons nonresponsive to PAG stimulation. Next, because the PAG does not project monosynaptically to the BLA, the authors used a combination of retrograde and anterograde neural tracing to identify possible regions that could convey robot-related information from PAG to the BLA. They observed that neurons in specific areas of the paraventricular nucleus of the thalamus (PVT) that are innervated by PAG fibers contained neurons that were retrogradely labeled by the injection of CTB in the BLA. In addition, PVT neurons showed increased expression of the neural activity marker cFos after the robot test, suggesting that PVT may be a mediator of PAG signals to the BLA.
Overall, the idea that the PAG interacts with the BLA via the midline thalamus during a predator vs. foraging test is new and quite interesting. The authors have used appropriated tools to address their questions.
In this revised version of the manuscript, the authors have made important modifications in the text, inserted new data analyses, and incorporated additional references, as recommended by the reviewers. These modifications have significantly improved the quality of the manuscript.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This work describes a new protein factor required for filamentous phage assembly. The protein PSB15 binds to the packaging signal of the ssDNA, Trx and cardiolipin. A mechanism how the phage DNA is targeted to the assembly site in the bacterial inner membrane is discussed.
Strengths:
The work describes a clever way to detect factors required for phage propagation by looking at the plaque size of pseudorevertants that arise after infection of a phage with a directed mutation in the packaging signal. This led to the detection of a phage protein expressed from ORF9, the PSB15.
The authors convincingly show that PSB15 is expressed in infected cells and can complement a phage with a mutated orf9.
Weaknesses:
Given the fact that the phage LF-UK is not well explored, many open questions should be mentioned in the introduction. For the study, it is important to know if the phageLF-UK has a mimick or homolog of gV and gXI, and if not, whether PSB15 could take their role.
I am not convinced of the proposition of their term "checkpoint". The truth is that the authors do not know the real purpose of PSB15. I do not see an advantage for a checkpoint that only adds an additional step to enter the phage assembly site. There must be a biochemical reason for the action of PSB15. Looking at Figure 7, the step from "Release" to "Loading" is just adding many unknowns, e.g. how to transfer the DNA, how to dispose of PSB15 and Trx? Also, in the previous step are three question marks that do not add any solid information.
The in vivo study of subcellular localization is very questionable. Why is there a single fluorescent dot if there are thousands of PSB15 molecules expressed in the cell? I have my doubts that the conclusions the authors make here are correct and meaningful. The movies do not add anything significant.
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Joint Public Review:
Summary
This manuscript explores the transcriptomic identities of olfactory ensheathing cells (OECs), glial cells that support life-long axonal growth in olfactory neurons, as they relate to spinal cord injury repair. The authors show that transplantation of cultured, immunopurified rodent OECs at a spinal cord injury site can promote injury-bridging axonal regrowth. They then characterize these OECs using single-cell RNA sequencing, identifying five subtypes and proposing functional roles that include regeneration, wound healing, and cell-cell communication. They identify one progenitor OEC subpopulation and also report several other functionally relevant findings, notably, that OEC marker genes contain mixtures of other glial cell type markers (such as for Schwann cells and astrocytes), and that these cultured OECs produce and secrete Reelin, a regrowth-promoting protein that has been disputed as a gene product of OECs.
This manuscript offers an extensive, cell-level characterization of OECs, supporting their potential therapeutic value for spinal cord injury and suggesting potential underlying repair mechanisms. The authors use various approaches to validate their findings, providing interesting images that show the overlap between sprouting axons and transplanted OECs, and showing that OEC marker genes identified using single-cell RNA sequencing are present in vivo, in both olfactory bulb tissue and spinal cord after OEC transplantation.
Despite the breadth of information presented, however, further quantification of results and explanation of experimental approaches would be needed to support some of the authors' claims. Additionally, a more thorough discussion is needed to contextualize their findings relative to previous work.
(1) Important quantification is lacking for the data presented. For example, multiple figures include immunohistochemistry or immunocytochemistry data (Figures 1, 5, 6), but they are presented without accompanying measures like fractions of cells labeled or comparisons against controls. As a result, for axons projecting via OEC bridges in Figure 1, it is unclear how common these bridges are in the presence or absence of OECs. For Figure 6., it is unclear whether cells having an alternative OEC morphology coincide with progenitor OEC subtype marker genes to a statistically significant degree. Similar quantification is missing in other types of data such as Western blot images (Fig. 9) and OEC marker gene data (for which p-values are not reported; Table S2).
The addition of quantitative measures and, where appropriate, statistical comparisons with p-values or other significance measures, would be important for supporting the authors' claims and more rigorously conveying the results.
(2) Some aspects of the experimental design that are relevant to the interpretation of the results are not explained. For example, OECs appear to be collected from only female rats, but the potential implications of this factor are not discussed.
Additionally, it is unclear from the manuscript to what degree immunopurified cells are OECs as opposed to other cell types. The antibody used to retain OECs, nerve growth factor receptor p75 (Ngfr-p75), can also be expressed by non-OEC olfactory bulb cell types including astrocytes [1-3]. The possible inclusion of Ngfr-p75-positive but non-OEC cell types in the OEC culture is not sufficiently addressed. Such non-OEC cell types are also not distinguished in the analysis of single-cell RNA sequencing data (only microglia, fibroblasts, and OECs are identified; Figure 2). Thus, it is currently unclear whether results related to the OEC subtype may have been impacted by these experimental factors.
(3) The introduction, while well written, does not discuss studies showing no significant effect of OEC implantation after spinal cord injury. The discussion also fails to sufficiently acknowledge this variability in the efficacy of OEC implantation. This omission amplifies bias in the text, suggesting that OECs have significant effects that are not fully reflected in the literature. The introduction would need to be expanded to properly address the nuance suggested by the literature regarding the benefits of OECs after spinal cord injury. Additionally, in the discussion, relating the current study to previous work would help clarify how varying observations may relate to experimental or biological factors.
(a) Cragnolini, A.B. et al., Glia, (2009), doi: 10.1002/glia.20857.<br /> (b) Vickland H. et al., Brain Res., (1991), doi: 10.1016/0006-8993(91)91659-O.<br /> (c) Ung K. et al., Nat Commun., (2021), doi: 10.1038/s41467-021-25444-3.
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Reviewer #1 (Public Review):
Summary:
It seems as if the main point of the paper is about the new data related to rat fish although your title is describing it as extant cartilaginous fishes and you bounce around between the little skate and ratfish. So here's an opportunity for you to adjust the title to emphasize ratfish is given the fact that leader you describe how this is your significant new data contribution. Either way, the organization of the paper can be adjusted so that the reader can follow along the same order for all sections so that it's very clear for comparative purposes of new data and what they mean. My opinion is that I want to read, for each subheading in the results, about the the ratfish first because this is your most interesting novel data. Then I want to know any confirmation about morphology in little skate. And then I want to know about any gaps you fill with the cat shark. (It is ok if you keep the order of "skate, ratfish, then shark, but I think it undersells the new data).
Strengths:
The imagery and new data availability for ratfish are valuable and may help to determine new phylogenetically informative characters for understanding the evolution of cartilaginous fishes. You also allude to the fossil record.
Opportunities:
I am concerned about the statement of ratfish paedomorphism because stage 32 and 33 were not statistically significantly different from one another (figure and prior sentences). So, these ratfish TMDs overlap the range of both 32 and 33. I think you need more specimens and stages to state this definitely based on TMD. What else leads you to think these are paedomorphic? Right now they are different, but it's unclear why. You need more outgroups.
Your headings for the results subsection and figures are nice snapshots of your interpretations of the results and I think they would be better repurposed in your abstract, which needs more depth.
Historical literature is more abundant than what you've listed. Your first sentence describes a long fascination and only goes back to 1990. But there are authors that have had this fascination for centuries and so I think you'll benefit from looking back. Especially because several of them have looked into histology and development of these fishes.
I agree that in the past 15 years or so a lot more work has been done because it can be done using newer technologies and I don't think your list is exhaustive. You need to expand this list and history which will help with your ultimate comparative analysis without you needed to sample too many new data yourself.
I'd like to see modifications to figure 7 so that you can add more continuity between the characters, illustrated in figure 7 and the body of the text. Generally Holocephalans are the outgroup to elasmobranchs - right now they are presented as sister taxa with no ability to indicate derivation. Why isn't the catshark included in this diagram?
In the last paragraph of the introduction, you say that "the data argue" and I admit, I am confused. Whose data? Is this a prediction or results or summary of other people's work? Either way, could be clarified to emphasize the contribution you are about to present.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The main observation that the sperm from CRISP proteins 1 and 3 KO lines are post-fertilization less developmentally competent is convincing. However, the molecular characterization of the mechanism that leads to these defects and the temporal appearance of the defects requires additional studies.
Strengths:
The generation of these double mutant mice is valuable for the field. Moreover, the fact that the double mutant line of Crisp 1 and 3 is phenotypically different from the Crisp 1 and 4 line suggests different functions of these epididymis proteins. The methods used to demonstrate that developmental defects are largely due to post-fertilization defects are also a considerable strength. The initial characterization of these sperm has altered intracellular Ca2+ levels, and increased rates of DNA fragmentation are valuable.
Weaknesses:
The study is mechanistically incomplete because there is no direct demonstration that the absence of these proteins alters the epididymal environment and fluid, wherein during the passage through the epididymis the sperm become affected. Also, a direct demonstration of how the proteins in question cause or lead to DNA damage and increased Ca2+ requires further characterization.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Using a cross-modal sensory selection task in head-fixed mice, the authors attempted to characterize how different rules reconfigured representations of sensory stimuli and behavioral reports in sensory (S1, S2) and premotor cortical areas (medial motor cortex or MM, and ALM). They used silicon probe recordings during behavior, a combination of single-cell and population-level analyses of neural data, and optogenetic inhibition during the task.
Strengths:
A major strength of the manuscript was the clarity of the writing and motivation for experiments and analyses. The behavioral paradigm is somewhat simple but well-designed and well-controlled. The neural analyses were sophisticated, clearly presented, and generally supported the authors' interpretations. The statistics are clearly reported and easy to interpret. In general, my view is that the authors achieved their aims. They found that different rules affected preparatory activity in premotor areas, but not sensory areas, consistent with dynamical systems perspectives in the field that hold that initial conditions are important for determining trial-based dynamics.
I think this is a well-performed, well-written and interesting study that shows differences in rule representations in sensory and premotor areas, and finds that rules reconfigure preparatory activity in motor cortex to support flexible behavior.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The classical pro/antisaccade task has become a valuable diagnostic tool in neurology and psychiatry (Antoniades et al., 2013, Vision Res). Although it is well-established that antisaccades require substantially longer latencies than prosaccades, the exact attentional mechanisms underlying these differences are not yet fully elucidated. This study investigates the separate influences of exogenous and endogenous attention on saccade generation. These two mechanisms are often confounded in classical pro/antisaccade tasks. In the current study, the authors build on their previous work using an urgent choice task (Salinas et al., 2019, eLife) to time-resolve the influences of exogenous and endogenous factors on saccade execution. The key contribution of the current study is to show that, when controlling for exogenous capture, antisaccades continue to require longer processing times. This longer processing time may be explained by a coupling between endogenous attention and saccade motor plans.
Strengths:
In the classical pro/antisaccade task the direction of exogenous capture (caused by the presentation of the cue) is typically congruent with the direction of prosaccades and incongruent with antisaccades. A key strength of the current study is the introduction of different experimental conditions that control for the effects of exogenous capture on saccade generation. In particular, Experiments 3 and 4 provide strong evidence for two independent (exogenous and endogenous) mechanisms that guide saccadic choices, acting at different times. Differences in timing for pro and antisaccades during the endogenous phase were consistent and independent of whether the exogenous capture biased early saccades toward the correct prosaccade direction or toward the correct antisaccade directions.
As in previous studies by the same group (Salinas et al., 2019, eLife; Goldstein et al., 2023, eLife), the detailed analysis of the time course of goal-directed saccades allowed the authors to determine the exact, additional time of 30 ms that is necessary to generate a correct antisaccade versus prosaccade.
Overall, the manuscript is very well written, and the data are presented clearly.
Weaknesses:
The main research question could be defined more clearly. In the abstract and at some points throughout the manuscript, the authors indicate that the main purpose of the study was to assess whether the allocation of endogenous attention requires saccade planning [e.g., ll.3-5 or ll.247-248]. While the data show a coupling between endogenous attention and saccades, they do not point to a specific direction of this coupling (i.e., whether endogenous attention is necessary to successfully execute a saccade plan or whether a saccade plan necessarily accompanies endogenous attention).
Some of the analyses were performed only on subgroups of the participants. The reporting of these subgroup analyses is transparent and data from all participants are reported in the supplementary figures. Still, these subgroup analyses may make the data appear more consistent, compared to when data is considered across all participants. For instance, the exogenous capture in Experiments 1 and 2 appears much weaker in Figure 2 (subgroup) than Figure S3 (all participants). Moreover, because different subgroups were used for different analyses, it is often difficult to follow and evaluate the results. For instance, the tachometric curves in Figure 2 (see also Figure 3 and 4) show no motor bias towards the cue (i.e., performance was at ~50% for rPTs <75 ms). I assume that the subsequent analyses of the motor bias were based on a very different subgroup. In fact, based on Figure S2, it seems that the motor bias was predominantly seen in the unreliable participants. Therefore, I often found the figures that were based on data across all participants (Figures 7 and S3) more informative to evaluate the overall pattern of results.
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
This manuscript illustrates the power of "combined" research, incorporating a range of tools, both old and new to answer a question. This thorough approach identifies a novel target in a well-established signalling pathway and characterises a new player in Drosophila CNS development.
Largely, the experiments are carried out with precision, meeting the aims of the project, and setting new targets for future research in the field. It was particularly refreshing to see the use of multi-omics data integration and Targeted DamID (TaDa) findings to triage scRNA-seq data. Some of the TaDa methodology was unorthodox, however, this does not affect the main finding of the study. The authors (in the revised manuscript) have appropriately justified their TaDa approaches and mentioned the caveats in the main text.
Their discovery of Spar as a neuropeptide precursor downstream of Alk is novel, as well as its ability to regulate activity and circadian clock function in the fly. Spar was just one of the downstream factors identified from this study, therefore, the potential impact goes beyond this one Alk downstream effector.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Olszyński and colleagues present data showing variability from canonical "aversive calls", typically described as long 22 kHz calls rodents emit in aversive situations. Similarly long but higher-frequency (44 kHz) calls are presented as a distinct call type, including analyses both of their acoustic properties and animals' responses to hearing playback of these calls. While this work adds an intriguing and important reminder, namely that animal behavior is often more variable and complex than perhaps we would like it to be, there is some caution warranted in the interpretation of these data.
The exclusive use of males is a major concern lacking adequate justification and should be disclosed in the title and abstract to ensure readers are aware of this limitation. With several reported sex differences in rat vocal behaviors this means caution should be exercised when generalizing from these findings. The occurrence of an estrus cycle in typical female rats is not justification for their exclusion. Note also that male rodents experience great variability in hormonal states as well, distinguishing between individuals and within individuals across time. The study of endocrinological influences on behavior can be separated from the study of said behavior itself, across all sexes. Similarly, concerns about needing to increase the number of animals when including all sexes are usually unwarranted (see Shansky [2019] and Phillips et al. [2023]).
Regarding the analysis where calls were sorted using DBSCAN based on peak frequency and duration, my comment on the originally reviewed version stands. It seems that the calls are sorted by an (unbiased) algorithm into categories based on their frequency and duration, and because 44kHz calls differ by definition on frequency and duration the fact that the algorithm sorts them as a distinct category is not evidence that they are "new calls [that] form a separate, distinct group". I appreciate that the authors have softened their language regarding the novelty and distinctness of these calls, but the manuscript contains several instances where claims of novelty and specificity (e.g. the subtitle on line 193) is emphasized beyond what the data justifies.
The behavioral response to call playback is intriguing, although again more in line with the hypothesis that these are not a distinct type of call but merely represent expected variation in vocalization parameters. Across the board animals respond rather similarly to hearing 22 kHz calls as they do to hearing 44 kHz calls, with occasional shifts of 44 kHz call responses to an intermediate between appetitive and aversive calls. This does raise interesting questions about how, ethologically, animals may interpret such variation and integrate this interpretation in their responses. However, the categorical approach employed here does not address these questions fully.
I appreciate the amendment in discussing the idea of arousal being the key determinant for the increased emission of 44kHz, and the addition of other factors. Some of the items in this list, such as annoyance/anger and disgust/boredom, don't really seem to fit the data. I'm not sure I find the idea that rats become annoyed or disgusted during fear conditioning to be a particularly compelling argument. As such the list appears to be a collection of emotion-related words, with unclear potential associations with the 44kHz calls.
Later in the Discussion the authors argue that the 44kHz aversive calls signal an increased intensity of a negative valence emotional state. It is not clear how the presented arguments actually support this. For example, what does the elongation of fear conditioning to 10 trials have to do with increased negative emotionality? Is there data supporting this relationship between duration and emotion, outside anthropomorphism? Each of the 6 arguments presented seems quite distant from being able to support this conclusion.
In sum, rather than describing the 44kHz long calls as a new call type, it may be more accurate to say that sometimes aversive calls can occur at frequencies above 22 kHz. Individual and situational variability in vocalization parameters seems to be expected, much more so than all members of a species strictly adhering to extremely non-variable behavioral outputs.
[Editors' note: The reviewer agrees that the additional analysis has ruled out the possibility that the calls are due to fatigue.]
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors assess the accuracy of short variant calling (SNPs and indels) in bacterial genomes using Oxford Nanopore reads generated on R10.4 flow cells from a very similar genome (99.5% ANI), examining the impact of variant caller choice (three traditional variant callers: bcftools, freebayes, and longshot, and three deep learning based variant callers: clair3, deep variant, and nano caller), base calling model (fast, hac and sup) and read depth (using both simplex and duplex reads).
Strengths:
Given the stated goal (analysis of variant calling for reads drawn from genomes very similar to the reference), the analysis is largely complete and results are compelling. The authors make the code and data used in their analysis available for re-use using current best practices (a computational workflow and data archived in INSDC databases or Zenodo as appropriate).
Weaknesses:
While the medaka variant caller is now deprecated for diploid calling, it is still widely used for haploid variant calling and should at least be mentioned (even if the mention is only to explain its exclusion from the analysis).
Appraisal:
The experiments the authors engaged in are well structured and the results are convincing. I expect that these results will be incorporated into "best practice" bacterial variant calling workflows in the future.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Winged seeds or ovules from the Devonian are crucial to understanding the origin and early evolutionary history of wind dispersal strategy. Based on exceptionally well-preserved fossil specimens, the present manuscript documented a new fossil plant taxon (new genus and new species) from the Famennian Series of Upper Devonian in eastern China and demonstrated that three-winged seeds are more adapted to wind dispersal than one-, two- and four-winged seeds by using mathematical analysis.
Strengths:
The manuscript is well organised and well presented, with superb illustrations. The methods used in the manuscript are appropriate.
Weaknesses:
I would only like to suggest moving the "Mathematical analysis of wind dispersal of ovules with 1-4 wings" section from the supplementary information to the main text, leaving the supplementary figures as supplementary materials.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
By combining an analysis of the evolutionary age of the genes expressed in male germ cells, a study of genes associated with spermatocyte protein-protein interaction networks and functional experiments in Drosophila, Brattig-Correia and colleagues provide evidence for an ancient origin of the genetic program underlying metazoan spermatogenesis. This leads to identifying a relatively small core set of functional interactions between deeply conserved gene expression regulators, whose impairment is then shown to be associated with cases of human male infertility.
Strengths:
In my opinion, the work is important for three different reasons. First, it shows that, even though reproductive genes can evolve rapidly and male germ cells display a significant level of transcriptional noise, it is still possible to obtain convincing evidence that a conserved core of functionally interacting genes lies at the basis of the male germ transcriptome. Second, it reports an experimental strategy that could also be applied to gene networks involved in different biological problems. Third, the authors make a compelling case that, due to its effects on human spermatogenesis, disruption of the male germ cell orthoBackbone can be exploited to identify new genetic causes of infertility.
Weaknesses:
The main strength of the general approach followed by the authors is, inevitably, also a weakness. This is because a study rooted in comparative biology is unlikely to identify newly emerged genes that may adopt key roles in processes such as species-specific gamete recognition. Additionally, using a TPM >1 threshold for protein-coding transcripts may exclude genes, such as those encoding proteins required for gamete fusion, which are thought to be expressed at a very low level. Although these considerations raise the possibility that the chosen approach may miss information that, depending on the species, could be potentially highly functionally important, this by no means reduces its value in identifying genes belonging to the conserved genetic program of spermatogenesis.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The manuscript by Anbarcia et al. re-evaluates the function of the enigmatic Rete Ovarii (RO), a structure that forms in close association with the mammalian ovary. The RO has generally been considered a functionless structure in the adult ovary. This manuscript follows up on a previous study from the lab that analyzed ovarian morphogenesis using high-resolution microscopy (McKey et al., 2022). The present study adds finer details to RO development and possible function by (1) identifying new markers for OR sub-regions (e.g. GFR1a labels the connecting rete) suggesting that the sub-regions are functionally distinct, (2) showing that the OR sub-regions are connected by a luminal system that allows transport of material from the extra-ovarian rete (EOR) to the inter-ovarian rete (IOG), (3) identifies proteins that are secreted into the OR lumen and that may regulate ovarian homeostasis, and finally, (4) better defines how the vasculature, nervous, and immune system integrates with the OR.
Strengths:
The data is beautifully presented and convincing. They show that the RO is composed of three distinct domains that have unique gene expression signatures and thus likely are functionally distinct.
Weaknesses:
It is not always clear what the novel findings are that this manuscript is presenting. It appears to be largely similar to the analysis done by McKey et al. (2022) but with more time points and molecular markers. The novelty of the present study's findings needs to be better articulated.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Summary:
Idiopathic scoliosis (IS) is a common spinal deformity. Various studies have linked genes to IS, but underlying mechanisms are unclear such that we still lack understanding of the causes of IS. The current manuscript analyzes IS patient populations and identifies EPHA4 as a novel associated gene, finding three rare variants in EPHA4 from three patients (one disrupting splicing and two missense variants) as well as a large deletion (encompassing EPHA4) in a Waardenburg syndrome patient with scoliosis. EPHA4 is a member of the Eph receptor family. Drawing on data from zebrafish experiments, the authors argue that EPHA4 loss of function disrupts the central pattern generator (CPG) function necessary for motor coordination.
Strengths:
The main strength of this manuscript is the human genetic data, which provides convincing evidence linking EPHA4 variants to IS. The loss of function experiments in zebrafish strongly support the conclusion that EPHA4 variants that reduce function lead to IS.
Weaknesses:
The conclusion that disruption of CPG function causes spinal curves in the zebrafish model is not well supported. The authors' final model is that a disrupted CPG leads to asymmetric mechanical loading on the spine and, over time, the development of curves. This is a reasonable idea, but currently not strongly backed up by data in the manuscript. Potentially, the impaired larval movements simply coincide with, but do not cause, juvenile-onset scoliosis. Support for the authors' conclusion would require independent methods of disrupting CPG function and determining if this is accompanied by spine curvature. At a minimum, the language of the manuscript could be toned down, with the CPG defects put forward as a potential explanation for scoliosis in the discussion rather than as something this manuscript has "shown". An additional weakness of the manuscript is that the zebrafish genetic tools are not sufficiently validated to provide full confidence in the data and conclusions.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost importance and a reproducible setup essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.
Strengths:
It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.
Also, the comparison between manual and software analysis is appreciated.
Weaknesses:
The authors stress that it can be done in BBB models, but I would argue that it is much more broadly applicable. This is not necessarily a weakness of the study but more an opportunity to strengthen the method. So I would encourage the authors to rewrite some parts and make it more broadly applicable.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The study seeks to establish accurate computational models to explore the role of hydrodynamic interactions on energy savings and spatial patterns in fish schools. Specifically, the authors consider a system of (one degree-of-freedom) flapping airfoils that passively position themselves with respect to the streamwise direction, while oscillating at the same frequency and amplitude, with a given phase lag and at a constant cross-stream distance. By parametrically varying the phase lag and the cross-stream distance, they systematically explore the stability and energy costs of emergent configurations. Computational findings are leveraged to distill insights into universal relationships and clarify the role of the wake of the leading foil.
Strengths:
(1) The use of multiple computational models (computational fluid dynamics, CFD, for full Navier-Stokes equations and computationally efficient inviscid vortex sheet, VS, model) offers an extra degree of reliability of the observed findings and backing to the use of simplified models for future research in more complex settings.
(2) The systematic assessment of the stability and energy savings in multiple configurations of pairs and larger ensembles of flapping foils is an important addition to the literature.
(3) The discovery of a linear phase-distance relationship in the formation attained by pairs of flapping foils is a significant contribution, which helps compare different experimental observations in the literature.
(4) The observation of a critical size effect for in-line formations of larger, above which cohesion and energetic benefits are lost at once, is a new discovery in the field.
Weaknesses:
(1) The extent to which observations on one-degree-of-freedom flapping foils could translate to real fish schools is presently unclear so some of the conclusions on live fish schools are likely to be overstated and would benefit from some more biological framing.
(2) The analysis of non-reciprocal coupling is not as novel as the rest of the study and potentially not as convincing due to the chosen linear metric of interaction (that is, the flow agreement).
Overall, this is a rigorous effort on a critical topic: findings of the research can offer important insight into the hydrodynamics of fish schooling, stimulating interdisciplinary research at the interface of computational fluid mechanics and biology.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This is an interesting study by Xu et al showing the effects of infection with the Treponema pallidum virus (which causes syphilis disease) on neuronal development using iPSC-derived human brain organoids as a model and single-cell RNA sequencing. This work provides an important insight into the impact of the virus on human development, bridging the gap between the phenomena observed in studies using animal models as well as non-invasive human studies showing developmental abnormalities in fetuses infected with the virus in utero through maternal vertical transmission.
Using single-cell RNAseq in combination with qPCR and immunofluorescence techniques, the authors show that T. pallidum infected organoids are smaller in size, in particular during later growth stages, contain a larger number of undifferentiated neuronal lineage cells, and exhibit decreased numbers of specific neuronal subcluster, which the authors have identified as undifferentiated hindbrain neurons.
The study is an important first step in understanding how T. pallidum affects human neuronal development and provides important insight into the potential mechanisms that underlie the neurodevelopmental abnormalities observed in infected human fetuses.
Strengths:
(1) The study is well written, and the data quality is good for the most part.
(2) The study provides an important first step in utilizing human brain organoids to study the impact of T. pallidum infection on neuronal development.
(3) The study's conclusions may provide important insight to other researchers focused on studying how viral infections impact neuronal development.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The revised manuscript "Diffusive lensing as a mechanism of intracellular transport and compartmentalization" is very similar to the original manuscript. The main difference between the revised and the original manuscript is that the authors have removed the reference to viscosity gradient and instead talk of diffusivity gradient. With this change the manuscript the analysis and claims in the manuscript are much more aligned. The manuscript, as the original version, explores the role of spatially varying diffusion constant in three scenarios:
(i) Spatial localization of non-particles<br /> (ii) Clustering in presence of inter-particle interactions<br /> (iii) Moment analysis for non-interacting particles in space with discrete patches of inhomogeneous diffusivity.
Since the manuscript has not changed much the strengths and weaknesses, in my opinion, remain similar to that of the original manuscript.
Strengths: The implications of a heterogeneous environment on phase separation and reaction kinetics in cells are under-explored. This makes the general theme of this manuscript relevant and interesting.
Weaknesses: The central part of the paper "diffusive lensing", i.e., particles localizing in the region of low diffusion constant is not new. Some of the papers authors cite already show that. The parts on phase separation and frap analysis that could provide new results are not rigorous enough for a theory paper.
I reiterate some of my comments from the original version that are valid for the revised version as well.
My main criticism was not to say that some convention should be used or some not. But instead, the main point was to say that just because there is spatial diffusion constant that does not mean there will be a spatial gradient of particles. From the authors response to my comments, it is clear that they understand the subtilties around it and are aware of the relevant papers. However, a reader not familiar with this discussion may work under the impression that if there if there is a spatialy varying diffusion constant in cell there will be an accumulation of particles in the region of low diffusivity but that may not always be the case. Moreover, localisation of particles in the region of low diffusivity has been reported in many different context. Some of the papers that the author cite already show that. For example, in Rupprecht et al. 2018 non-isothermal interpretation is applied to the dynamics of objects inside cells.
Given that the central result is not new. The paper could still be of general interest to the biophysics community if the follow up sections (ii) Clustering in presence of inter-particle interactions and (iii) Moment analysis for non-interacting particles in space with discrete patches of inhomogeneous diffusivity were analysed rigorously.
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Joint Public Review:
This manuscript by Yue et al. aims to understand the molecular mechanisms underlying the better reproductive outcomes of Tibetans at high altitude by characterizing the transcriptome and histology of full-term placenta of Tibetans and compare them to those Han Chinese at high elevations.
The approach is innovative, and the data collected are valuable for testing hypotheses regarding the contribution of the placenta to better reproductive success of populations that adapted to hypoxia. The authors identified hundreds of differentially expressed genes (DEGs) between Tibetans and Han, including the EPAS1 gene that harbors the strongest signals of genetic adaptation. The authors also found that such differential expression is more prevalent and pronounced in the placentas of male fetuses than those of female fetuses, which is particularly interesting, as it echoes with the more severe reduction in birth weight of male neonates at high elevation observed by the same group of researchers (He et al., 2022).
Comments on latest version:
The revised manuscript has incorporated the suggested changes and weakened conclusions regarding natural selection. Limitations of the study are also clearly stated in the Discussion section.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This study is one in a series of excellent papers by the Forstmann group focusing on the ability of fMRI to reliably detect activity in small subcortical nuclei - in this case, specifically those purportedly involved in the hyper- and indirect inhibitory basal ganglia pathways. I have been very fond of this work for a long time, beginning with the demonstration of De Hollander, Forstmann et al. (HBM 2017) of the fact that 3T fMRI imaging (as well as many 7T imaging sequences) do not afford sufficient signal to noise ratio to reliably image these small subcortical nuclei. This work has done a lot to reshape my view of seminal past studies of subcortical activity during inhibitory control, including some that have several thousand citations.
Comments on revised version:
This is my second review of this article, now entitled "Multi-study fMRI outlooks on subcortical BOLD responses in the stop-signal paradigm" by Isherwood and colleagues.
The authors have been very responsive to the initial round of reviews.
I still think it would be helpful to see a combined investigation of the available 7T data, just to really drive the point home that even with the best parameters and a multi-study sample size, fMRI cannot detect any increases in BOLD activity on successful stop compared to go trials. However, I agree with the authors that these "sub samples still lack the temporal resolution seemingly required for looking at the processes in the SST."
As such, I don't have any more feedback.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this work, the authors investigate an important question - under what circumstances should a recurrent neural network optimised to produce motor control signals receive preparatory input before the initiation of a movement, even though it is possible to use inputs to drive activity just-in-time for movement?
This question is important because many studies across animal models have show that preparatory activity is widespread in neural populations close to motor output (e.g. motor cortex / M1), but it isn't clear under what circumstances this preparation is advantageous for performance, especially since preparation could cause unwanted motor output during a delay.
They show that networks optimised under reasonable constraints (speed, accuracy, lack of pre-movement) will use input to seed the state of the network before movement, and that these inputs reduce the need for ongoing input during the movement. By examining many different parameters in simplified models they identify a strong connection between the structure of the network and the amount of preparation that is optimal for control - namely, that preparation has the most value when nullspaces are highly observable relative to the readout dimension and when the controllability of readout dimensions is low. They conclude by showing that their model predictions are consistent with the observation in monkey motor cortex that even when a sequence of two movements is known in advance, preparatory activity only arises shortly before movement initiation.
Overall, this study provides valuable theoretical insight into the role of preparation in neural populations that generate motor output, and by treating input to motor cortex as a signal that is optimised directly this work is able to sidestep many of the problematic questions relating to estimating the potential inputs to motor cortex.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Dasguta et al. have dissected the role of Sema7a in fine tuning of a sensory microcircuit in the posterior lateral line organ of zebrafish. They attempt to also outline the different roles of a secreted verses membrane-bound form of Sema7a in this process. Using genetic perturbations and axonal network analysis, the authors show that loss of both Sema7a isoforms causes abnormal axon terminal structure with more bare terminals and fewer loops in contact with presynaptic sensory hair cells. Further, they show that loss of Sema7a causes decreased number and size of both the pre- and post-synapse. Finally, they show that overexpression of the secreted form of Sema7a specifically can elicit axon terminal outgrowth to an ectopic Sema7a expressing cell. Together, the analysis of Sema7a loss of function and overexpression on axon arbor structure is fairly thorough and revealed a novel role for Sema7a in axon terminal structure.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
People with Parkinson's disease often experience a variety of nonmotor symptoms, the biological bases of which remain poorly understood. Johansson et al began to study potential roles of the dorsal raphe nucleus (DRN) degeneration in the pathophysiology of neuropsychiatric symptoms in PD.
Strengths:
Boi et al validated a transgenic reporter mouse line that can reliably label dopaminergic neurons in the DRN. This brain region shows severe neurodegeneration and has been proposed to contribute to the manifestation of neuropsychiatric symptoms in PD. Using this mouse line (and others), Boi and colleagues characterized electrophysiological and morphological phenotypes of dopaminergic and serotoninergic neurons in the raphe nucleus. This study involved very careful topographical registration of recorded neurons to brain slices for post hoc immunohistochemical validation of cell identity, making it an elegant and thorough piece of work.
Of relevance to PD pathophysiology, the authors evaluated the physiological and morphological changes of DRN serotoninergic and dopaminergic neurons after a partial loss of nigrostriatal dopamine neurons, which serves as a mouse model of early parkinsonian pathology. Moreover, the authors identified a series of physiological and morphological changes of subtypes of DRN neurons that depend on nigral dopaminergic neurodegeneration, LC noradrenergic neurodegeneration, or both. Indeed this work highlights the importance of LC noradrenergic degeneration in PD pathophysiology.
Overall, this is a well-designed study with high significance to the Parkinson's research field.
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
Summary:
Tian et al. aimed to assess differences in biological motion (BM) perception between children with and without ADHD, as well as relationships to indices of social functioning and possible predictors of BM perception (including demographics, reasoning ability and inattention). In their study, children with ADHD showed poorer performance relative to typically developing children in three tasks measuring local, global, and general BM perception. The authors further observed that across the whole sample, performance in all three BM tasks was negatively correlated with scores on the social responsiveness scale (SRS), whereas within groups a significant relationship to SRS scores was only observed in the ADHD group and for the local BM task. Local and global BM perception showed a dissociation in that global BM processing was predicted by age, while local BM perception was not. Finally, general (local & global combined) BM processing was predicted by age and global BM processing, while reasoning ability mediated the effect of inattention on BM processing.
Strengths:
Overall, the manuscript is presented in a clear fashion and methods and materials are presented with sufficient detail so the study could be reproduced by independent researchers. The study uses an innovative, albeit not novel, paradigm to investigate two independent processes underlying BM perception. The results are novel and have the potential to have wide-reaching impact on multiple fields.
Weaknesses:
The manuscript has improved in clarity and conceptual and methodological considerations in response to the last review. However, the reported results still provide incomplete support for the claims the authors make in the paper.
In relation to other reviewers' earlier comments, the model notation used is still not consistent and model results are reported incompletely, which make it difficult to gain a full picture of the data and how they support the authors' secondary claims. For instance, across the models in the supplementary materials, ß coefficients are only reported selectively which makes it difficult to assess the model as a whole. Furthermore, different terms (task 1, task 2 vs. BM-Local, BM-global) are used to refer to the same levels of a variable, and it is unclear which levels of a dummy variable correspond to which task, making it overall very difficult to comprehend the modelling procedure.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors use innovative CRISPRi method to uncover regulators of cell density and volume in neutrophils. The results show that cells require NHE activity during chemoattractant-driven cell migration. Before migration occurs, cells also undergo a rapid cell volume increase. These results indicate that water flux, driven by ion channels, appears to play a central role in neutrophil migration. The paper is very well written and clear. The revised version has addressed all of my questions.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This study presents a novel application of the inverted encoding (i.e., decoding) approach to detect the correlates of crossmodal integration in the human EEG (electrophysiological) signal. The method is successfully applied to data from a group of 41 participants, performing a spatial localization task on auditory, visual, and audio-visual events. The analyses clearly show a behavioural superiority for audio-visual localization. Like previous studies, the results when using traditional univariate ERP analyses were inconclusive, showing once more the need for alternative, more sophisticated approaches. Instead, the principal approach of this study, harnessing the multivariate nature of the signal, captured clear signs of super-additive responses, considered by many as the hallmark of multisensory integration. Unfortunately, the manuscript lacks many important details in the descriptions of the methodology and analytical pipeline. Although some of these details can eventually be retrieved from the scripts that accompany this paper, the main text should be self-contained and sufficient to gain a clear understanding of what was done. (A list of some of these is included in the comments to the authors). Nevertheless, I believe the main weakness of this work is that the positive results obtained and reported in the results section are conditioned upon eye movements. When artifacts due to eye movements are removed, then the outcomes are no longer significant.
Therefore, whether the authors finally achieved the aims and showed that this method of analysis is truly a reliable way to assess crossmodal integration, does not stand on firm ground. The worst-case scenario is that the results are entirely accounted for by patterns of eye movements in the different conditions. In the best-case scenario, the method might truly work, but further experiments (and/or analyses) would be required to confirm the claims in a conclusive fashion.
If finally successful, this approach could bring important advances in the many fields where multisensory integration has been shown to play a role, by providing a way to bring much-needed coherence across levels of analysis, from behaviour to single-cell electrophysiology. To achieve this, one would have to make sure that the pattern of super-additive effects, the standard self-imposed by the authors as a proxy for multisensory integration, shows up reliably regardless of eye movement or artifact corrections. One first step toward this goal would be, perhaps, to facilitate the understanding of results in context by reporting both the uncorrected and corrected analyses in the main results section. Second, one could try to support the argument given in the discussion, pointing out the origin of the super-additive effects in posterior electrode sites, by also modelling frontal electrode clusters and showing they aren't informative as to the effect of interest.
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Joint Public Review:
Mahapatra and Takahashi report on the physiological consequences of pharmacologically blocking either clathrin and dynamin function during compensatory endocytosis or of the cortical actin scaffold both in the calyx of Held synapse and hippocampal boutons in acute slice preparations.
Although many aspects of these pharmacological interventions have been studied in detail during the past decades, this is a comprehensive and comparative study, which reveals some interesting differences between a fast synapse (Calyx of Held) tuned to reliably transmit at several 100 Hz and a more slow hippocampal CA1 synapse. In particular the authors find that acute disturbance of the synaptic actin network leads to a marked frequency-dependent enhancement of synaptic depression in the Calyx, but not in the hippocampal synapse. This striking difference between both preparations is the most interesting finding.
Comments on latest version:
The authors have done a great job revising the paper and only minor revisions are suggested to the Discussion of the paper.
Two quite relevant and recent papers should be cited and briefly discussed because they relate directly to Pitstop2 effects and actin-myosin-scaffold proteins in the calyx of Held synapse.
One is: Paksoy A et al, (2022) "Effects of the clathrin inhibitor Pitstop-2 on synaptic vesicle recycling at a central synapse in vivo." Front. Synaptic Neurosci. 14:1056308. doi: 10.3389/fnsyn.2022.1056308. This paper shows with EM that changes caused by PitStop2 perturbation of "clathrin function suggest that clathrin plays a role in SV recycling from both, the plasma membrane and large endosomes, under physiological activity patterns, in vivo."
Second: A role for actin-myosin and MLCK in short-term plasticity has been shown by Srinivasan G., et al. (2008) "The Pool of Fast Releasing Vesicles Is Augmented by Myosin Light Chain Kinase Inhibition at the Calyx of Held Synapse." J Neurophysiol 99: 1810-1824, 2008. The data here suggests that MLCK plays a crucial role in determining the size of the pool of synaptic vesicles that undergo fast release but not the Pr of the synapse. In other words, MLCK inhibition augments super-priming of vesicles at the calyx of Held synapse.
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Reviewer #1 (Public Review):
Summary:
The authors aim to test the sensory recruitment theory of visual memory, which assumes that visual sensory areas are recruited for working memory and that these sensory areas represent visual memories in a similar fashion to how perceptual inputs are represented. To test the overlap between working memory (WM) and perception, the authors use coarse stimulus (aperture) biases that are known to account for (some) orientation decoding in visual cortex (i.e., stimulus energy is higher for parts of an image where a grating orientation is perpendicular to an aperture edge, and stimulus energy drives decoding). Specifically, the authors show gratings (with a given "carrier" orientation) behind two different apertures: One is a radial modulator (with maximal energy aligned with the carrier orientation) and the other an angular modulator (with maximal energy orthogonal to the carrier orientation). When subject detect contrast changes in these stimuli (the perceptual task), orientation decoding only works when training and testing within each modulator, but not across modulators, showing the impact of stimulus energy on decoding performance. Instead, when subjects remember the orientation over a 12s delay, orientation decoding works irrespective of the modulator used. The authors conclude that representations during WM are therefore not "sensory-like", given that they are immune to aperture biases. This invalidates the sensory recruitment hypothesis, or at least the part assuming that when sensory areas that are recruited during WM, they are recruited in a manner that resembles how these areas are used during perception.
Strengths:
Duan and Curtis very convincingly show that aperture effects that are present during perception, do not appear to be present during the working memory delay. Especially when the debate about "why can we decode orientations from human visual cortex" was in full swing, many may have quietly assumed this to be true (e.g., "the memory delay has no stimuli, and ergo no stimulus aperture effects"), but it is definitely not self-evident and nobody ever thought to test it directly until now. In addition to the clear absence of aperture effects during the delay, Duan and Curtis also show that when stimulus energy aligns with the carrier orientation, cross-generalization between perception and memory does work (which could explain why perception-to-memory cross decoding also works). All in all, this is a clever manipulation, and I'm glad someone did it, and did it well.
Weaknesses:
There seems to be a major possible confound that prohibits strong conclusions about "abstractions" into "line-like" representation, which is spatial attention. What if subjects simply attend the end points of the carrier grating, or attend to the edge of the screen where the carrier orientation "intersects" in order to do the task? This may also result in reconstructions that have higher bold at areas close to the stimulus/screen edges along the carrier orientation. The question then would be if this is truly an "abstracted representation", or if subjects are merely using spatial attention to do the task.
Alternatively (and this reaches back to the "fine vs coarse" debate), another argument could be that during memory, what we are decoding is indeed fine-scale inhomogenous sampling of orientation preferences across many voxels. This is clearly not the most convincing argument, as the spatial reconstructions (e.g., Figure 3A and C) show higher BOLD for voxels with receptive fields that are aligned to the remembered orientation (which is in itself a form of coarse scale bias), but could still play a role.
To conclude that the spatial reconstruction from the data indeed comes from a line-like representation, you'd need to generate modeled reconstructions of all possible stimuli and representations. Yes, Figure 4 shows that a line results in a modeled spatial map that resembles the WM data, but many other stimuli might too, and some may better match the data. For example, the alternative hypothesis (attention to grating endpoints) may very well lead to a very comparable model output to the one from a line. But testing this would not suffice, as there may be an inherent inverse problem (with multiple stimuli that can lead to the same visual field model).
The main conclusion, and title of the paper, that visual working memories are abstractions of percepts, is therefore not supported. Subjects could be using spatial attention, for example. Furthermore, even if it is true that gratings are abstracted into lines, this form of abstraction would not generalize to any non-spatial feature (e.g., color cannot become a line, contrast cannot become a line, etc.), which means it has limited explanatory power.
Additional context:
The working memory and perception tasks are rather different. In this case, the perception task does not require the subject to process the carrier orientation (which is largely occluded, and possibly not that obvious without paying attention to it), but attention is paid to contrast. In this scenario, stimulus energy may dominate the signal. In the WM task, subjects have to work out what orientation is shown to do the task. Given that the sensory stimulus in both tasks is brief (1.5s during memory encoding, and 2.5s total in the perceptual task), it would be interesting to look at decoding (and reconstructions) for the WM stimulus epoch. If abstraction (into a line) happens in working memory, then this perceptual part of the task should still be susceptible to aperture biases. It allows the authors to show that it is indeed during memory (and not merely the task or attentional state of the subject) that abstraction occurs.
What's also interesting is what happens in the passive perceptual condition, and the fact that spatial reconstructions for areas beyond V1 and V2 (i.e., V3, V3AB, and IPS0-1) align with (implied) grating endpoints, even when an angular modulator is used (Figure 3C). Are these areas also "abstracting" the stimulus (in a line-like format)?
Review after revision:
(1) It's nice of the authors to simulate how a dot stimulus affects the image computable model, but this does not entirely address my concern about attention to endpoints. The assumption that attention can be used in the same manner as a physical stimulus to calculate stimulus energy is questionable. (also, why would a dot at 15º lead to high stimulus energy tangential to that orientation?). This simulation also does not at all address my concern about model mimicry (many possible inputs can lead to a line-like output).
(2) It's also nice that the authors agree that much more work needs to be done, and these results may not generalize to all forms of memory. Given this agreement, and until that "more work" is done, I strongly believe we should refrain from making hyperbolic claims that might preemptively imply all visual working memories are abstractions of percepts. Time (and much more work) will likely show things to be much more subtle and complex.
The work presented in this paper is cool, but it uses a specific case: spatial stimuli (gratings) with the task to remember orientation. This limits possible conclusions for several reasons (1) These results are specific to EVC, as visual maps are a prerequisite meaning that these results will not hold up in other, non-retinotopic areas. (2) The fact that subjects are "focusing" along the main stimulus axis (attention or not) can simply be a strategy employed by the majority of (but not all) subjects - a strategy that may not be necessary to do the task, and therefore not a canonical method of Abstraction. It may be a "shared preferred strategy" or something. (3) If subjects had to (for example) remember contrast, and not orientation, results may have been entirely different (I would hypothesize there is no line-like abstraction in this case). Vice versa, if the perceptual task would have been on orientation (instead of contrast), the authors admit that "participants would reformat the grating into a line-like representation to make the judgments" (quote from author's response under "Additional context"). Thus, the results may be entirely about the task/ cognitive state, and not about how perceptual information is abstracted into memory.
Instead of unveiling *the* working memory Abstraction, this work (very nicely) shows a specific instance of possible abstraction. A more correct (but admittedly, less "sexy") conclusion may be "Visual working memories of orientation can be abstracted into a line in early visual cortex". As it stands, the authors still do not acknowledge any of the alternatives that myself (see above) and the other reviewers have put forth, nor do they acknowledge recent work by Chunharas et al. (2023, BioRxiv), that directly applies principles of efficient coding to address the exact same question of working memory abstraction. The link between a "line-like" representation and efficient coding implied by the authors (in their response) is merely tentative to me, but it would be great if the authors could explain this further.
These were, and remain, the major weaknesses in the original submission, that in my view have not been adequately addressed by the authors, as many overly broad conclusions about abstractions are currently still present in the manuscript (in for example the title).
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Reviewer #1 (Public Review):
In this study, Gonzalez Alam et al. report a series of functional MRI results about the neural processing from the visual cortex to high-order regions in the default-mode network (DMN), compiling evidence from task-based functional MRI, resting-state connectivity, and diffusion-weighted imaging. Their participants were first trained to learn the association between objects and rooms/buildings in a virtual reality experiment; after the training was completed, in the task-based MRI experiment, participants viewed the objects from the earlier training session and judged if the objects were in the semantic category (semantic task) or if they were previously shown in the same spatial context (spatial context task). Based on the task data, the authors utilised resting-state data from their previous studies, visual localiser data also from previous studies, as well as structural connectivity data from the Human Connectome Project, to perform various seed-based connectivity analysis. They found that the semantic task causes more activation of various regions involved in object perception while the spatial context task causes more activation in various regions for place perception, respectively. They further showed that those object perception regions are more connected with the frontotemporal subnetwork of the DMN while those place perception regions are more connected with the medial-temporal subnetwork of the DMN. Based on these results, the authors argue that there are two main pathways connecting the visual system to high-level regions in the DMN, one linking object perception regions (e.g., LOC) leading to semantic regions (e.g., IFG, pMTG), the other linking place perception regions (e.g., parahippocampal gyri) to the entorhinal cortex and hippocampus.
Below I provide my takes on (1) the significance of the findings and the strength of evidence, (2) my guidance for readers regarding how to interpret the data, as well as several caveats that apply to their results, and finally (3) my suggestions for the authors.
(1) Significance of the results and strength of the evidence
I would like to praise the authors for, first of all, trying to associate visual processing with high-order regions in the DMN. While many vision scientists focus specifically on the macroscale organisation of the visual cortex, relatively few efforts are made to unravel how neural processing in the visual system goes on to engage representations in regions higher up in the hierarchy (a nice precedent study that looks at this issue is by Konkle and Caramazza, 2017). We all know that visual processing goes beyond the visual cortex, potentially further into the DMN, but there's no direct evidence. So, in this regard, the authors made a nice try to look at this issue.
Having said this, the authors' characterisation of the organisation of the visual cortex (object perception/semantics vs. place perception/spatial contexts) does not go beyond what has been known for many decades by vision neuroscience. Specifically, over the past two decades, numerous proposals have been put forward to explain the macroscale organisation of the visual system, particularly the ventrolateral occipitotemporal cortex. A lateral-medial division has been reliably found in numerous studies. For example, some researchers found that the visual cortex is organised along the separation of foveal vision (lateral) vs. peripheral vision (medial), while others found that it is structured according to faces (lateral) vs. places (medial). Such a bipartite division is also found in animate (lateral) vs. inanimate (medial), small objects (lateral) vs. big objects (medial), as well as various cytoarchitectonic and connectomic differences between the medial side and the lateral side of the visual cortex. Some more recent studies even demonstrate a tripartite division (small objects, animals, big objects; see Konkle and Caramazza, 2013). So, in terms of their characterisation of the visual cortex, I think Gonzalez Alam et al. do not add any novel evidence to what the community of neuroscience has already known.
However, the authors' effort to link visual processing with various regions of the DMN is certainly novel, and their attempt to gather converging evidence with different methodologies is commendable. The authors are able to show that, in an independent sample of resting-state data, object-related regions are more connected with semantic regions in the DMN while place-related regions are more connected with navigation-related regions in the DMN, respectively. Such patterns reveal a consistent spatial overlap with their Kanwisher-type face/house localiser data and also concur with the HCP white-matter tractography data. Overall, I think the two pathways explanation that the authors seek to argue is backed by converging evidence. The lack of travelling wave type of analysis to show the spatiotemporal dynamics across the cortex from the visual cortex to high-level regions is disappointing though because I was expecting this type of analysis would provide the most convincing evidence of a 'pathway' going from one point to another. Dynamic caudal modelling or Granger causality may also buttress the authors' claim of pathway because many readers, like me, would feel that there is not enough evidence to convincingly prove the existence of a 'pathway'.
(2) Guidance to the readers about interpretation of the data
The organisation of the visual cortex and the organisation of the DMN historically have been studied in parallel with little crosstalk between different communities of researchers. Thus, the work by Gonzalez Alam et al. has made a nice attempt to look at how visual processing goes beyond the realm of the visual cortex and continues into different subregions of the DMN.
While the authors of this study have utilised multiple methods to obtain converging evidence, there are several important caveats in the interpretation of their results:
(1) While the authors choose to use the term 'pathway' to call the inter-dependence between a set of visual regions and default-mode regions, their results have not convincingly demonstrated a definitive route of neural processing or travelling. Instead, the findings reveal a set of DMN regions are functionally more connected with object-related regions compared to place-related regions. The results are very much dependent on masking and thresholding, and the patterns can change drastically if different masks or thresholds are used.
(2) Ideally, if the authors could demonstrate the dynamics between the visual cortex and DMN in the primary task data, it would be very convincing evidence for characterising the journey from the visual cortex to DMN. Instead, the current connectivity results are derived from a separate set of resting state data. While the advantage of the authors' approach is that they are able to verify certain visual regions are more connected with certain DMN regions even under a task-free situation, it falls short of explaining how these regions dynamically interact to convert vision into semantic/spatial decision.
(3) There are several results that are difficult to interpret, such as their psychophysiological interactions (PPI), representational similarity analysis, and gradient analysis. For example, typically for PPI analysis, researchers interrogate the whole brain to look for PPI connectivity. Their use of targeted ROI is unusual, and their use of spatially extensive clusters that encompass fairly large cortical zones in both occipital and temporal lobes as the PPI seeds is also an unusual approach. As for the gradient analysis, the argument that the semantic task is higher on Gradient 1 than the spatial task based on the statistics of p-value = 0.027 is not a very convincing claim (unhelpfully, the figure on the top just shows quite a few blue 'spatial dots' on the hetero-modal end which can make readers wonder if the spatial context task is really closer to the unimodal end or it is simply the authors' statistical luck that they get a p-value under 0.05). While it is statistically significant, it is weak evidence (and it is not pertinent to the main points the authors try to make).
(3) My suggestion for the authors
There are several conceptual-level suggestions that I would like to offer to the authors:
(1) If the pathway explanation is the key argument that you wish to convey to the readers, an effective connectivity type of analysis, such as Granger causality or dynamic caudal modelling, would be helpful in revealing there is a starting point and end point in the pathway as well as revealing the directionality of neural processing. While both of these methods have their issues (e.g., Granger causality is not suitable for haemodynamic data, DCM's selection of seeds is susceptible to bias, etc), they can help you get started to test if the path during task performance does exist. Alternatively, travelling wave type of analysis (such as the results by Raut et al. 2021 published in Science Advances) can also be useful to support your claims of the pathway.
(2) I think the thresholding for resting state data needs to be explained - by the look of Figure 2E and 3E, it looks like whole-brain un-thresholded results, and then you went on to compute the conjunction between these un-thresholded maps with network templates of the visual system and DMN. This does not seem statistically acceptable, and I wonder if the conjunction that you found would disappear and reappear if you used different thresholds. Thus, for example, if the left IFG cluster (which you have shown to be connected with the visual object regions) would disappear when you apply a conventional threshold, this means that you need to seriously consider the robustness of the pathway that you seek to claim... it may be just a wild goose that you are chasing.
(3) There are several analyses that are hard to interpret and you can consider only reporting them in the supplementary materials, such as the PPI results and representational similarity analysis, as none of these are convincing. These analyses do not seem to add much value to make your argument more convincing and may elicit more methodological critiques, such as statistical issues, the set-up of your representational theory matrix, and so on.
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Reviewer #1 (Public Review):
The study by Chikermane and colleagues investigates the functional, structural, and dopaminergic network substrates of cortical beta oscillations (13-30 Hz). The major strength of the work lies in the methodology taken by the authors, namely a multimodal lesion network mapping. First, using invasive electrophysiological recordings from healthy cortical territories of epileptic patients they identify regions with the highest beta power. Next, they leverage open-access MRI data and PET atlases and use the identified high-beta regions as seeds to find (1) the whole-brain functional and structural maps of regions that form the putative underlying network of high-beta regions and (2) the spatial distribution of dopaminergic receptors that show correlation with nodal connectivity of the identified networks. These steps are achieved by generating aggregate functional, structural, and dopaminergic network maps using lead-DBS toolbox, and by contrasting the results with those obtained from high-alpha regions.
The main findings are:<br /> (1) Beta power is strongest across frontal, cingulate, and insular regions in invasive electrophysiological data, and these regions map onto a shared functional and structural network.<br /> (2) The shared functional and structural networks show significant positive correlations with dopamine receptors across the cortex and basal ganglia (which is not the case for alpha, where correlations are found with GABA).
Nevertheless, a few clarifications regarding the choice of high-power electrodes and distributions of functional connectivity maps (i.e., strength and sign across cortex and sub-cortex) can help with understanding the results.
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Reviewer #1 (Public Review):
Vision is a highly active process. Humans move their eyes 3-4 times per second to sample information with high visual acuity from our environment, and where eye movements are directed is critical to our understanding of active vision. Here, the authors propose that the cost of making a saccade contributes critically to saccade selection (i.e., whether and where to move the eyes). The authors build on their own recent work that the effort (as measured by pupil size) that comes with planning and generating an eye movement varies with saccade direction. To do this, the authors first measured pupil size for different saccade directions for each participant. They then correlated the variations in pupil size obtained in the mapping task with the saccade decision in a free-choice task. The authors observed a striking correlation: pupil size in the mapping task predicted the decision of where to move the eyes in the free choice task. In this study, the authors provide a number of additional insightful analyses (e.g., based on saccade curvature, and saccade latency) and experiments that further support their claim that the decision to move the eyes is influenced by the effort to move the eyes in a particular direction. One experiment showed that the same influence of assumed saccade costs on saccade selection is observed during visual search in natural scenes. Moreover, increasing the cognitive load by adding an auditory counting task reduced the number of saccades, and in particular reduced the costly saccades. In sum, these experiments form a nice package that convincingly establishes the association between pupil size and saccade selection.
In my opinion, the causal structure underlying the observed results is not so clear. While the relationship between pupil size and saccade selection is compelling, it is not clear that saccade-related effort (i.e., the cost of a saccade) really drives saccade selection. Given the correlational nature of this relationship, there are other alternatives that could explain the finding. For example, saccade latency and the variance in landing positions also vary across saccade directions. This can be interpreted for instance that there are variations in oculomotor noise across saccade directions, and maybe the oculomotor system seeks to minimize that noise in a free-choice task. In fact, given such a correlational result, many other alternative mechanisms are possible. While I think the authors' approach of systematically exploring what we can learn about saccade selection using pupil size is interesting, it would be important to know what exactly pupil size can add that was not previously known by simply analyzing saccade latency. For example, saccade latency anisotropies across saccade directions are well known, and the authors also show here that saccade costs are related to saccade latency. An important question would be to compare how pupil size and saccade latency uniquely contribute to saccade selection. That is, the authors could apply the exact same logic to their analysis by first determining how saccade latencies (or variations in saccade landing positions; see Greenwood et al., 2017 PNAS) vary across saccade directions and how this saccade latency map explains saccade selection in subsequent tasks. Is it more advantageous to use one or the other saccade metric, and how well does a saccade latency map correlate with a pupil size map?
In addition to eye-movement-related anisotropies across the visual field, there are of course many studies reporting visual field anisotropies (see Himmelberg, Winawer & Carrasco, 2023, Trends in Neuroscience for a review). It would be interesting to understand how the authors think about visual field anisotropies in the context of their own study. Do they think that their results are (in)dependent on such visual field variations (see Greenwood et al., 2017, PNAS; Ohl, Kroell, & Rolfs, 2024, JEP:Gen for a similar discussion)?
Finally, the authors conclude that their results "suggests that the eye-movement system and other cognitive operations consume similar resources that are flexibly allocated among each other as cognitive demand changes. The authors should speculate what these similar resources could mean? What are the specific operations of the auditory task that overlap in terms of resources with the eye movement system?
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Reviewer #1 (Public Review):
In the manuscript "A microglia clonal inflammatory disorder in Alzheimer's Disease", Vicario et al. provide a compelling study elucidating a potential contribution of somatic mutations within the microglia population of the CNS that accelerates microglia activation and disease-associated gene signatures in Alzheimer's disease. Here they especially identified an "enrichment" of pathological SNVs in microglia, but not the peripheral blood, that are associated with clonal proliferative disorders and neurological diseases in a subset of patients with AD. Convincingly, they identified P-SNVs in microglia of AD patients located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in human microglia-like cells in vitro.
Overall, this study provides clear and detailed evidence from an AD patient cohort pointing to a potential contribution of microglia-specific somatic mutations to disease onset and/or progression in a subset of patients with Alzheimer's disease.
Strengths:<br /> As outlined above, the study identified P-SNVs in microglia of AD patients associated with clonal proliferative disorders, but also gave an in-depth analysis of re-occurring P-SNVs located within the ring domain of CBL, a negative regulator of MAPK signaling. They further provide mechanistic insights into how these variants result in MAPK over-activation and subsequently in a pro-inflammatory phenotype in HEK cells, BV2 cells, MAC cells, and human microglia-like cells in vitro.
Great care was taken here to validate their hypotheses at each step, as well as to identify the limitations of the possible conclusions. For example, they highlight that the pathway proposed to be affected may be an explanation for a subset of AD patients, and emphasize that it is yet unclear whether this accumulation of pathological SNVs is a cause or consequence of disease progression
The study clearly supports an enrichment of P-SNVs in several genes associated with clonal proliferative disorders in microglia and nicely separates this from SNVs associated with clonal hematopoiesis in the peripheral blood found in AD patients and controls.
The authors further acknowledged that several age-matched control patients were diagnosed with cancer or tumor-associated diseases and carefully dissected the occurring SNVs in these patients are not associated with the P-SNVs identified in the microglial compartment of the AD cohort.
Weaknesses:
Even though the study is overall very convincing, several points could help to connect the seen somatic variants in microglia more with a potential role in disease progression. The connection of P-SNVs in the genes chosen from neurological disorders was not further highlighted by the authors.
The authors show in snRNA-seq data that a disease-associated microglia state seems to be enriched in patients with somatic variants in the CBL ring domain, however, this analysis could be deepened. For example, how this knowledge may translate to patient benefits when the relevant cell populations appear concentrated in a single patient sample (Figure 5; AD52) is unclear; increasing the analyzed patient pool for Figure 5 and showcasing the presence of this microglia state of interest in a few more patients with driving mutations for CBL or other MAPK pathway associated mutations would lend their hypotheses further credibility.
A potential connection between P-SNVs in microglia and disease pathology and symptoms was not further explored by the authors.
A recent preprint (Huang et al., 2024) connected the occurrence of somatic variants in genes associated with clonal hematopoiesis in microglia in a large cohort of AD patients, this study is not further discussed or compared to the data in this manuscript.
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Reviewer #1 (Public Review):
Summary:
The study of human intelligence has been the focus of cognitive neuroscience research, and finding some objective behavioral or neural indicators of intelligence has been an ongoing problem for scientists for many years. Melnick et al, 2013 found for the first time that the phenomenon of spatial suppression in motion perception predicts an individual's IQ score. This is because IQ is likely associated with the ability to suppress irrelevant information. In this study, a high-resolution MRS approach was used to test this theory. In this paper, the phenomenon of spatial suppression in motion perception was found to be correlated with the visuo-spatial subtest of gF, while both variables were also correlated with the GABA concentration of MT+ in the human brain. In addition, there was no significant relationship with the excitatory transmitter Glu. At the same time, SI was also associated with MT+ and several frontal cortex FCs.
Strengths:
(1) 7T high-resolution MRS is used.
(2) This study combines the behavioral tests, MRS, and fMRI.
Weaknesses:
(1) In the intro, it seems to me that the multiple-demand (MD) regions are the key in this study. However, I didn't see any results associated with the MD regions. Did I miss something??
(2) How was the sample size determined? Is it sufficient??
(3) In Schallmo elife 2018, there was no correlation between GABA concentration and SI. How can we justify the different results different here?
(4) Basically this study contains the data of SI, BDT, GABA in MT+ and V1, Glu in MT+ and V1-all 6 measurements. There should be 6x5/2 = 15 pairwise correlations. However, not all of these results are included in Figure 1 and supplementary 1-3. I understand that it is not necessary to include all figures. But I suggest reporting all values in one Table.
(5) In Melnick (2013), the IQ scores were measured by the full set of WAIS-III, including all subtests. However, this study only used the visual spatial domain of gF. I wonder why only the visuo-spatial subtest was used not the full WAIS-III?
(6) In the functional connectivity part, there is no explanation as to why only the left MT+ was set to the seed region. What is the problem with the right MT+?
(7) In Melnick (2013), the authors also reported the correlation between IQ and absolute duration thresholds of small and large stimuli. Please include these analyses as well.
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Reviewer #1 (Public Review):
Summary:
This is a fine paper that serves the purpose to show that the use of light sheet imaging may be used to provide whole brain imaging of axonal projections. The data provided suggest that at this point the technique provides lower resolution than with other techniques. Nonetheless, the technique does provide useful, if not novel, information about particular brain systems.
Strengths:
The manuscript is well written. In the introduction a clear description of the functional organization of the barrel cortex is provided provides the context for applying the use of specific Cre-driver lines to map the projections of the main cortical projection types using whole brain neuroanatomical tracing techniques. The results provided are also well written, with sufficient detail describing the specifics of how techniques were used to obtain relevant data. Appropriate controls were done, including the identification of whisker fields for viral injections and determination of the laminar pattern of Cre expression. The mapping of the data provides a good way to visualize low resolution patterns of projections.
Weaknesses:
(1) The results provided are, as stated in the discussion, "largely in agreement with previously reported studies of the major projection targets". However it must be stated that the study does not "extend current knowledge through the high sensitivity for detecting sparse axons, the high specificity of labeling of genetically defined classes of neurons and the brain wide analysis for assigning axons to detailed brain regions" which have all been published in numerous other studies. ( the allen connectivity project and related papers, along with others). If anything the labeling of axons obtained with light sheet imaging in this study does not provide as detailed mapping obtained with other techniques. Some detail is provided of how the raw images are processed to resolve labeled axons, but the images shown in the figures do not demonstrate how well individual axons may be resolved, of particular interest would be to see labeling in terminal areas such as other cortical areas, striatum and thalamus. As presented the light sheet imaging appears to be rather low resolution compared to the many studies that have used viral tracing to look at cortical projections from genetically identified cortical neurons.<br /> (2) Amongst the limitations of this study is the inability to resolve axons of passage and terminal fields. This has been done in other studies with viral constructs labeling synaptophysin. This should be mentioned.<br /> (3) There is no quantitative analysis of differences between the genetically defined neurons projecting to the striatum, what is the relative area innervated by, density of terminals, other measures.<br /> (4) Figure 5 is an example of the type of large sets of data that can be generated with whole brain mapping and registration to the Allen CCF that provides information of questionable value. Ordering the 50 plus structures by the density of labeling does not provide much in terms of relative input to different types of areas. There are multiple subregions for different functional types ( ie, different visual areas and different motor subregions are scattered not grouped together. Makes it difficult to understand any organizing principles.<br /> (5) The GENSAT Cre driver lines used must have the specific line name used, not just the gene name as the GENSAT BAC-Cre lines had multiple lines for each gene and often with very different expression patterns. Rbp4_KL100, Tlx3_PL56, Sim1_KJ18, Ntsr1_ GN220.
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Reviewer #1 (Public Review):
Abbasi et al. assess in this MEG study the directed connectivity of both cortical and subcortical regions during continuous speech production and perception. The authors observed bidirectional connectivity patterns between speech-related cortical areas as well as subcortical areas in production and perception. Interestingly, they found in speaking low-frequency connectivity from subcortical (the right cerebellum) to cortical (left superior temporal) areas, while connectivity from the cortical to subcortical areas was in the high frequencies. In listening a similar cortico-subcortical connectivity pattern was observed for the low frequencies, but the reversed connectivity in the higher frequencies was absent.
The work by Abbasi and colleagues addresses a relevant, novel topic, namely understanding the brain dynamics between speaking and listening. This is important because traditionally production and perception of speech and language are investigated in a modality-specific manner. To have a more complete understanding of the neurobiology underlying these different speech behaviors, it is key to also understand their similarities and differences. Furthermore, to do so, the authors utilize state-of-the-art directed connectivity analyses on MEG measurements, providing a quite detailed profile of cortical and subcortical interactions for the production and perception of speech. Importantly, and perhaps most interesting in my opinion, is that the authors find evidence for frequency-specific directed connectivity, which is (partially) different between speaking and listening. This could suggest that both speech behaviors rely (to some extent) on similar cortico-cortical and cortico-subcortical networks, but different frequency-specific dynamics.
These elements mentioned above (investigation of both production and perception, both cortico-cortical and cortico-subcortical connectivity is considered, and observing frequency-specific connectivity profiles within and between speech behaviors), make for important novel contributions to the field. Notwithstanding these strengths, I find that they are especially centered on methodology and functional anatomical description, but that precise theoretical contributions for neurobiological and cognitive models of speech are less transparent. This is in part because the study compares speech production and perception in general, but no psychophysical or psycholinguistic manipulations are considered. I also have some critical questions about the design which may pose some confounds in interpreting the data, especially with regard to comparing production and perception.
(1) While the cortico-cortical and cortico-subcortical connectivity profiles highlighted in this study and the depth of the analyses are impressive, what these data mean for models of speech processing remains on the surface. This is in part due, I believe, to the fact that the authors have decided to explore speaking and listening in general, without targeting specific manipulations that help elucidate which aspects of speech processing are relevant for the particular connectivity profiles they have uncovered. For example, the frequency-specific directed connectivity is it driven by low-level psychophysical attributes of the speech or by more cognitive linguistic properties? Does it relate to the monitoring of speech, timing information, and updating of sensory predictions? Without manipulations trying to target one or several of these components, as some of the referenced work has done (e.g., Floegel et al., 2020; Stockert et al., 2021; Todorović et al., 2023), it is difficult to draw concrete conclusions as to which representations and/or processes of speech are reflected by the connectivity profiles. An additional disadvantage of not having manipulations within each speech behavior is that it makes the comparison between listening and speaking harder. That is, speaking and listening have marked input-output differences which likely will dominate any comparison between them. These physically driven differences (or similarities for that matter; see below) can be strongly reduced by instead exploring the same manipulations/variables between speaking and listening. If possible (if not to consider for future work), it may be interesting to score psychophysical (e.g., acoustic properties) or psycholinguistic (e.g., lexical frequency) information of the speech and see whether and how the frequency-specific connectivity profiles are affected by it.
(2) Recent studies comparing the production and perception of language may be relevant to the current study and add some theoretical weight since their data and interpretations for the comparisons between production and perception fit quite well with the observations in the current work. These studies highlight that language processes between production and perception, specifically lexical and phonetic processing (Fairs et al., 2021), and syntactic processing (Giglio et al., 2024), may rely on the same neural representations, but are differentiated in their (temporal) dynamics upon those shared representations. This is relevant because it dispenses with the classical notion in neurobiological models of language where production and perception rely on (partially) dissociable networks (e.g., Price, 2010). Rather those data suggest shared networks where different language behaviors are dissociated in their dynamics. The speech results in this study nicely fit and extend those studies and their theoretical implications.
(3) The authors align the frequency-selective connectivity between the right cerebellum and left temporal speech areas with recent studies demonstrating a role for the right cerebellum for the internal modelling in speech production and monitoring (e.g., Stockert et al., 2021; Todorović et al., 2023). This link is indeed interesting, but it does seem relevant to point out that at a more specific scale, it does not concern the exact same regions between those studies and the current study. That is, in the current study the frequency-specific connectivity with temporal regions concerns lobule VI in the right cerebellum, while in the referenced work it concerns Crus I/II. The distinction seems relevant since Crus I/II has been linked to the internal modelling of more cognitive behavior, while lobule VI seems more motor-related and/or contextual-related (e.g., D'Mello et al., 2020; Runnqvist et al., 2021; Runnqvist, 2023).
(4) On the methodological side, my main concern is that for the listening condition, the authors have chosen to play back the speech produced by the participants in the production condition. Both the fixed order as well as hearing one's own speech as listening condition may produce confounds in data interpretation, especially with regard to the comparison between speech production and perception. Could order effects impact the observed connectivity profiles, and how would this impact the comparison between speaking and listening? In particular, I am thinking of repetition effects present in the listening condition as well as prediction, which will be much more elevated for the listening condition than the speaking condition. The fact that it also concerns their own voice furthermore adds to the possible predictability confound (e.g., Heinks-Maldonado et al., 2005). In addition, listening to one's speech which just before has been articulated may, potentially strategically even, enhance inner speech and "mouthing" in the participants, hereby thus engaging the production mechanism. Similarly, during production, the participants already hear their own voice (which serves as input in the subsequent listening condition). Taken together, both similarities or differences between speaking and listening connectivity may have been due to or influenced by these order effects, and the fact that the different speech behaviors are to some extent present in both conditions.
(5) The ability of the authors to analyze the spatiotemporal dynamics during continuous speech is a potentially important feat of this study, given that one of the reasons that speech production is much less investigated compared to perception concerns motor and movement artifacts due to articulation (e.g., Strijkers et al., 2010). Two questions did spring to mind when reading the authors' articulation artifact correction procedure: If I understood correctly, the approach comes from Abbasi et al. (2021) and is based on signal space projection (SSP) as used for eye movement corrections, which the authors successfully applied to speech production. However, in that study, it concerned the repeated production of three syllables, while here it concerns continuous speech of full words embedded in discourse. The articulation and muscular variance will be much higher in the current study compared to three syllables (or compared to eye movements which produce much more stable movement potentials compared to an entire discourse). Given this, I can imagine that corrections of the signal in the speaking condition were likely substantial and one may wonder (1) how much signal relevant to speech production behavior is lost?; (2) similar corrections are not necessary for perception, so how would this marked difference in signal processing affect the comparability between the modalities?
References:<br /> - Abbasi, O., Steingräber, N., & Gross, J. (2021). Correcting MEG artifacts caused by overt speech. Frontiers in Neuroscience, 15, 682419.<br /> - D'Mello, A. M., Gabrieli, J. D., & Nee, D. E. (2020). Evidence for hierarchical cognitive control in the human cerebellum. Current Biology, 30(10), 1881-1892.<br /> - Fairs, A., Michelas, A., Dufour, S., & Strijkers, K. (2021). The same ultra-rapid parallel brain dynamics underpin the production and perception of speech. Cerebral Cortex Communications, 2(3), tgab040.<br /> - Floegel, M., Fuchs, S., & Kell, C. A. (2020). Differential contributions of the two cerebral hemispheres to temporal and spectral speech feedback control. Nature Communications, 11(1), 2839.<br /> - Giglio, L., Ostarek, M., Sharoh, D., & Hagoort, P. (2024). Diverging neural dynamics for syntactic structure building in naturalistic speaking and listening. Proceedings of the National Academy of Sciences, 121(11), e2310766121.<br /> - Heinks‐Maldonado, T. H., Mathalon, D. H., Gray, M., & Ford, J. M. (2005). Fine‐tuning of auditory cortex during speech production. Psychophysiology, 42(2), 180-190.<br /> - Price, C. J. (2010). The anatomy of language: a review of 100 fMRI studies published in 2009. Annals of the new York Academy of Sciences, 1191(1), 62-88.<br /> - Runnqvist, E., Chanoine, V., Strijkers, K., Pattamadilok, C., Bonnard, M., Nazarian, B., ... & Alario, F. X. (2021). Cerebellar and cortical correlates of internal and external speech error monitoring. Cerebral Cortex Communications, 2(2), tgab038.<br /> - Runnqvist, E. (2023). Self-monitoring: The neurocognitive basis of error monitoring in language production. In Language production (pp. 168-190). Routledge.<br /> - Stockert, A., Schwartze, M., Poeppel, D., Anwander, A., & Kotz, S. A. (2021). Temporo-cerebellar connectivity underlies timing constraints in audition. Elife, 10, e67303.<br /> - Strijkers, K., Costa, A., & Thierry, G. (2010). Tracking lexical access in speech production: electrophysiological correlates of word frequency and cognate effects. Cerebral cortex, 20(4), 912-928.<br /> - Todorović, S., Anton, J. L., Sein, J., Nazarian, B., Chanoine, V., Rauchbauer, B., ... & Runnqvist, E. (2023). Cortico-cerebellar monitoring of speech sequence production. Neurobiology of Language, 1-21.
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Reviewer #1 (Public Review):
Summary:
This paper reports the finding that less fat accumulates in C. elegans that are feeding on Comamonas aquatica DA1877 (DA) vs the standard lab diet of Escherichia coli OP50 (OP50). While these bacteria are likely to be different in many ways, the authors found that fat accumulation phenotype depends on the vitamin B12 content of the bacterial diet and the involvement of B12 in the methionine cycle, affecting SAMS-1 and phosphatidylcholine (PC) synthesis. They report that low PC levels activate SREBP-1 (SBP-1 in C. elegans) and that an important target of SBP-1 is the delta 9 desaturase FAT-7. Finally, they describe a role for ASM-3, an acid sphingomyelinase, in influencing PC synthesis and fat accumulation in the worm.
Strengths:
This is a comprehensive story about how a dietary change affects fat accumulation in C. elegans. Their experimental evidence is convincing. The most novel aspect of this paper is that the coelomecyte expression of asm-3 contributes to PC/TAG homeostasis in C. elegans, which most likely occurs through the production of phosphocholine by the enzymatic breakdown of sphingomyelin by ASM-3. The phosphocholine will provide precursors for phosphatidylcholine (PC) synthesis, contributing to the PC synthesis pathway.
Weaknesses:
In the way the story is presented, the authors tend to imply that they discovered the pathways of B12, PC, SBP-1, and FAT-7, ignoring some important studies describing the relationship between PC synthesis and TAG accumulation in both the mammalian lipid metabolism field (liver) as well as in C. elegans. Many previous studies with similar results are not cited appropriately. Thus, the pathways reported in the paper are not new, and in this sense, the work is mostly confirmatory.
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www.biorxiv.org www.biorxiv.org
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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 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.
Notes on revision: The authors provided responses to some of my critiques. I think the central discrepancy between the lack of a phenotype in Pou2f3 and Trpm5 KO mice compared to the stronger phenotype in the Chat-Cre / Gng13 KO mice remains unresolved and will require future work to provide a clear model. This may or may not ultimately involve tuft cell heterogeneity.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The revised manuscript is much improved. As stated previously, it is on an interesting and important topic and provides many new potentially important findings. The manuscript contains a large amount of high-quality data. In the revised manuscript, the authors have done a nice job addressing the concerns raised in the previous review. They have refined their conclusions and the evidence provided supports conclusions drawn. Likewise, the writing and low of the manuscript is much improved.
Strengths:
The manuscript contains a large amount of high-quality data that is used to draw interesting and important conclusions.
Weaknesses:
There are still some issues with grammar and word usage, but these should be easily corrected with some additional minor editing. Other than some minor editing, my only real question/concern is whether the data presented shows that SCC3 is directly involved in gene regulation. It may well be that changes in chromatin structure caused by mutations in SCC3 and the axial element protein containing genes examined indirectly affect transcript levels for the genes examined.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Interactions known to be important for melanosome transport include exon F and the globular tail domain (GTD) of MyoVa with Mlph. Motivated by a discrepancy between in vitro and cell culture results regarding necessary interactions for MyoVa to be recruited to the melanosome, the authors used a series of pull-down and pelleting assays experiments to identify an additional interaction that occurs between exon G of MyoVa and Mlph. This interaction is independent of and synergistic with the interaction of Mlph with exon F. However, the interaction of the actin-binding domain of Mlph can occur either with exon G or with the actin filament, but not both simultaneously. These data lead to a modified recruitment model where both exon F and exon G enhance binding of Mlph to auto-inhibited MyoVa, and then via an unidentified switch (PKA?) the actin-binding domain of Mlph dissociates from MyoVa and interacts with the actin filament to enhance MyoVa processivity.
The only weakness noted is that the authors could have had a more complete story if they pursued whether PKA phosphorylation/dephosphorylation of Mlph is indeed the switch for the actin-binding domain of Mlph to interact with exon G versus the actin filament.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
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 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 has not been explored.
Strengths:
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. The results are clear and support the conclusions.
Weaknesses:
(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.
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Reviewer #1 (Public Review):
The comments below are from my review of the first submission of this article. I would now like to thank the authors for their hard work in responding to my comments. I am happy with the changes they have made, in particular the inclusion of further experimental evidence in Figures 2 and 4. I have no further comments to make.
In 'Systems analysis of miR-199a/b-5p and multiple miR-199a/b-5p targets during chondrogenesis', Patel et al. present a variety of analyses using different methodologies to investigate the importance of two miRNAs in regulating gene expression in a cellular model of cartilage development. They first re-analysed existing data to identify these miRNAs as one of the most dynamic across a chondrogenesis development timecourse. Next, they manipulated the expression of these miRNAs and showed that this affected the expression of various marker genes as expected. An RNA-seq experiment on these manipulations identified putative mRNA targets of the miRNAs which were also supported by bioinformatics predictions. These top hits were validated experimentally and, finally, a kinetic model was developed to demonstrate the relationship between the miRNAs and mRNAs studied throughout the paper.
I am convinced that the novel relationships reported here between miR-199a/b-5p and target genes FZD6, ITGA3 and CAV1 are likely to be genuine. It is important for researchers working on this system and related diseases to know all the miRNA/mRNA relationships but, as the authors have already published work studying the most dynamic miRNA (miR-140-5p) in this biological system I was not convinced that this study of the second miRNA in their list provided a conceptual advance on their previous work.
I was also concerned with the lack of reporting of details of the manipulation experiments. The authors state that they have over-expressed miR-199a-5p (Figure 2A) and knocked down miR-199b-5p (Figure 2B) but they should have reported their proof that these experiments had worked as predicted, e.g. showing the qRT-PCR change in miRNA expression. Similarly, I was concerned that one miRNA was over-expressed while the other was knocked down - why did the authors not attempt to manipulate both miRNAs in both directions? Were they unable to achieve a significant change in miRNA expression or did these experiments not confirm the results reported in the manuscript?
I had a number of issues with the way in which some of the data is presented. Table 1 only reported whether a specific pathway was significant or not for a given differential expression analysis but this concealed the extent of this enrichment or the level of statistical significance reported. Could it be redrawn to more similarly match the format of Figure 3A? The various shades of grey in Figure 2 and Figure 4 made it impossible to discriminate between treatments and therefore identify whether these data supported the conclusions made in the text. It also appeared that the same results were reported in Figure 3B and 3C and, indeed, Figure 3B was not referred to in the main text. Perhaps this figure could be made more concise by removing one of these two sets of panels?
Overall, while I think that this is an interesting and valuable paper, I think its findings are relatively limited to those interested in the role of miRNAs in this specific biomedical context.
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Reviewer #1 (Public Review):
The inferior colliculus (IC) is the central auditory system's major hub. It integrates ascending brainstem signals to provide acoustic information to the auditory thalamus. The superficial layers of the IC ("shell" IC regions as defined in the current manuscript) also receive a massive descending projection from the auditory cortex. This auditory cortico-collicular pathway has long fascinated the hearing field, as it may provide a route to funnel "high-level" cortical signals and impart behavioral salience upon an otherwise behaviorally agnostic midbrain circuit.
Accordingly, IC neurons can respond differently to the same sound depending on whether animals engage in a behavioral task (Ryan and Miller 1977; Ryan et al., 1984; Slee & David, 2015; Saderi et al., 2021; De Franceschi & Barkat, 2021). Many studies also report a rich variety of non-auditory responses in the IC, far beyond the simple acoustic responses one expects to find in a "low-level" region (Sakurai, 1990; Metzger et al., 2006; Porter et al., 2007). A tacit assumption is that the behaviorally relevant activity of IC neurons is inherited from the auditory cortico-collicular pathway. However, this assumption has never been tested, owing to two main limitations of past studies:
(1) Prior studies could not confirm if data were obtained from IC neurons that receive monosynaptic input from the auditory cortex.
(2) Many studies have tested how auditory cortical inactivation impacts IC neuron activity; the consequence of cortical silencing is sometimes quite modest. However, all prior inactivation studies were conducted in anesthetized or passively listening animals. These conditions may not fully engage the auditory cortico-collicular pathway. Moreover, the extent of cortical inactivation in prior studies was sometimes ambiguous, which complicates interpreting modest or negative results.
Here, the authors' goal is to directly test if the auditory cortex is necessary for behaviorally relevant activity in IC neurons. They conclude that surprisingly, task relevant activity in cortico-recipient IC neuron persists in absence of auditory cortico-collicular transmission. To this end, a major strength of the paper is that the authors combine a sound-detection behavior with clever approaches that unambiguously overcome the limitations of past studies.
First the authors inject a transsynaptic virus into the auditory cortex, thereby expressing a genetically encoded calcium indicator in the auditory cortex's postsynaptic targets in the IC. This powerful approach enables 2-photon Ca2+ imaging from IC neurons that unambiguously receive monosynaptic input from auditory cortex. Thus, any effect of cortical silencing should be maximally observable in this neuronal population. Second, they abrogate auditory cortico-collicular transmission using lesions of auditory cortex. This "sledgehammer" approach is arguably the most direct test of whether cortico-recipient IC neurons will continue to encode task-relevant information in absence of descending feedback. Indeed, their method circumvents the known limitations of more modern optogenetic or chemogenetic silencing, e.g. variable efficacy.
The authors have revised their manuscript and adequately addressed the major concerns. Although more in depth analyses of these rich datasets are definitely possible, the current results nevertheless stand on their own. Indeed, the work serves as a beacon to move away from the idea that cortico-collicular projections function primarily to impart behavioral relevance upon auditory midbrain neurons. This knowledge inspires a search for alternative explanations as to the role of auditory cortico-collicular synapses in behavior.
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Reviewer #1 (Public Review):
Summary:<br /> This is an excellent study by a superb investigator who discovered and is championing the field of migrasomes. This study contains a hidden "gem" - the induction of migrasomes by hypotonicity and how that happens. In summary, an outstanding fundamental phenomenon (migrasomes) en route to becoming transitionally highly significant.
Strengths:
Innovative approach at several levels. Migrasomes - discovered by Dr Yu's group - are an outstanding biological phenomenon of fundamental interest and now of potentially practical value.
Weaknesses:
I feel that the overemphasis on practical aspects (vaccine), however important, eclipses some of the fundamental aspects that may be just as important and actually more interesting. If this can be expanded, the study would be outstanding.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this paper, Wang et al show that differentiated peridermal cells of the zebrafish epidermis extend cytoneme-like protrusions toward the less differentiated, intermediate layer below. They present evidence that expression of a dominant-negative cdc42, inhibits cytoneme formation and leads to elevated expression of a marker of undifferentiated keratinocytes, krtt1c19e, in the periderm layer. Data is presented suggesting the involvement of Delta-Notch signaling in keratinocyte differentiation. Finally, changes in expression of the inflammatory cytokine IL-17 and its receptors is shown to affect cytoneme number and periderm structure in a manner similar to Notch and cdc42 perturbations.
Strengths:
Overall, the idea that differentiated cells signal to underlying undifferentiated cells via membrane protrusions in skin keratinocytes is interesting and novel, and it is clear that periderm cells send out thin membrane protrusions that contain a Notch ligand. Further, perturbations that affect cytoneme number, Notch signaling, and IL-17 expression clearly lead to changes in periderm structure and gene expression.
Weaknesses:
More work is needed to determine whether the effects on keratinocyte differentiation are due to a loss of cytonemes themselves, or to broader effects of inhibiting cdc42. Moreover, more evidence is needed to support the claim that periderm cytonemes deliver Delta ligands to induce Notch signaling below. Without these aspects of the study being solidified, understanding how IL-17 affects these processes seems premature.
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Reviewer #1 (Public Review):
Summary:
Juvenile Hormone (JH) plays a key role in insect development and physiology. Although the intracellular receptor for JH was identified long ago, a number of studies have shown that part of JH functions should be fulfilled through binding to an unknown membrane receptor, which was proposed to belong to the RTK family. In this study, the authors screened all RTKs from the H. armigera genome for their ability to mediate responses to JH III treatment both in cultured cells and in developing animals. They also present convincing evidence that CAD96CA and FGFR1 directly bind JH III, and that their role might be conserved in other insect species.
Strengths:
Altogether, the experimental approach is very complete and elegant, providing evidence for the role of CAD96CA and FGFR1 in JH signalling using different techniques and in different contexts. I believe that this work will open new perspectives to study the role of JH and better understand what is the contribution of signalling through membrane receptors for JH-dependent developmental processes.
Weaknesses:
I don't see major weaknesses in this study. However, I think that the manuscript would benefit from further information or discussion regarding the relationship between the two newly identified receptors. Experiments (especially in HEK-293T cells) suggest that CAD96CA and FGFR1 are sufficient on their own to transduce JH signalling. However, they are also necessary since loss-of-function conditions for each of them are sufficient to trigger strong effects (while the other is supposed to be still present).
In addition, despite showing different expression patterns, the two receptors seem to display similar developmental functions according to loss-of-function phenotypes. It is therefore unclear how to draw a model for membrane receptor-mediated JH signalling that includes both CAD96CA and FGFR1.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Chen and colleagues first compared the cartilage tissues collected from OA and HA patients using histology and immunostaining. Then, a genome-wide DNA methylation analysis was performed, which informed the changes of a novel gene, TNXB. IHC confirmed that TNXB has a lower expression level in HA cartilage than OA. Next, the authors demonstrated that TNXB levels were reduced in HA animal model, and intraarticular injection of AAV carrying TNXB siRNA induced cartilage degradation and promoted chondrocyte apoptosis. Based on KEGG enrichment, histopathological analysis, and western blot, the authors also showed the relationship between TNXB and AKT phosphorylation. Lastly, AKT agonist, specifically SC79 in this study, was shown to partially rescue the changes of in vitro-cultured chondrocytes induced by Tnxb knock-down. Overall, this is an interesting study and provided sufficient data to support their conclusion.
Strengths:
(1) Both human and mouse samples were examined.<br /> (2) The HA model was used.<br /> (3) genome-wide DNA methylation analysis was performed.
Weaknesses:
(1) In some experiments, the selection of the control groups was not ideal.<br /> (2) More details on analyzing methods and information on replicates need to be included.<br /> (3) Discussion can be improved by comparing findings to other relevant studies.<br /> (4) The use of transgenic mice with conditional Tnxb depletion can further define the physiological roles of Tnxb.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this study the authors use an elegant set of single-molecule experiments to assess the transcriptional and post-transcriptional regulation of RecB. The question stems from a previous observation from the same lab, that RecB protein levels are low and not induced under DNA damage. The authors first show that recB transcript levels are low and have a short half-life. They further show that RecB levels are likely regulated via translational control. They provide evidence for low noise in RecB protein levels across cells and show that the translation of the mRNA increases under double-strand break conditions. Authors identify Hfq binding sites in the recBCD operon and show that Hfq regulates the levels of RecB protein without changing the mRNA levels. They suggest that RecB translation is directly controlled by Hfq binding to mRNA, as mutating one of the binding sites has a direct effect on RecB protein levels.
Strengths:
The implication of Hfq in regulation of RecB translation is important and suggests mechanisms of cellular response to DNA damage that are beyond the canonically studied mechanisms (such as transcriptional regulation by LexA). Data are clearly presented and the writing is direct and easy to follow. Overall, the study is well-designed and provides novel insights into the regulation of RecB, that is part of the complex required to process break ends.
Weaknesses:
Some key findings need additional support/ clarifications to strengthen the conclusions. These are suggested to the authors.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
I thank the authors for addressing almost all my comments on the previous version of this manuscript, which studies the representation by gender and name origin of authors from Nature and Springer Nature articles in Nature News.
The representation of author identities is an important step towards equality in science, and the authors found that women are underrepresented in news quotes and mentions with respect to the proportion of women authors.
The research is rigorously conducted. It presents relevant questions and compelling answers. The documentation of the data and methods is thoroughly done, and the authors provide the code and data for reproduction.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The study offers intriguing insights, yet interpretations warrant caution, as the authors themselves acknowledged in their discussion of limitations.
The observed grid-like neural activity might not signify navigating a social landscape but rather a sensory feature space. The study's design had participants associate each face with a pair of bar lengths, with the purported 'navigation' being merely a response to the morphing of bar graph images. Crucially, the task did not necessitate any social cognitive processing to estimate grid-like activity. When making social decisions in a separate task, it is unclear whether participants were actually traversing a social space mentally or simply recalling the bar graphs linked to each face to calculate decision values. Notably, during the trust game, competence and trustworthiness did not equally influence decision-making (as illustrated by Equation 1), implying the possibility that the space represented may be more perceptual than social in nature.
The existence of a universal brain representation for faces within a social context is still debatable. Participants were not required to form a cognitive map of the six faces based on social traits; they could simply remember each face's trait values. While the study suggests that reaction times correlated with the perceived social distances between faces hint at the creation of internal representations, this phenomenon could occur without a true cognitive map of the face relationships. To convincingly argue for such internal representations in the brain, additional multivariate pattern analysis would be necessary to demonstrate that these are not merely the result of perceptual differences in the bar graphs associated with each face.
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
Summary:
The study by He et al. investigates the relationship of an increased susceptibility of diabetes patients towards COVID-19. The paper raises the possibility that hyperglycemia-induced cathepsin L maturation could be one of the driving forces in this pathology, suggesting that an increased activity of CTSL leads to accelerated virus infection rates due to an elevated processing of the SARS-CoV-2 spike protein.
In a clinical case-control study, the team found that severity of corona infections was higher in diabetic patients, and their CTSL levels correlated well with the progression of the disease. They further showed an increase in CTSL activity in long term as well as acute hyperglycemia. SARS-CoV-2 increasingly infected cells that were cultured in serum from diabetic patients, the same was observed using high glucose medium. No effect was observed in the medium with increased concentrations of insulin. CTSL knockout abolished the glucose-dependent increase in infection.
Increased glucose levels did not correlate with an increase in CTSL transcription. Rather He et al. could show that high glucose levels led to CTSL translocation from the ER into the lysosome. It was the glucose-dependent processing of the protease to its active form which promoted infection.
Overall, it is a very complete study starting from a clinical observation and ending on the molecular mechanism. A strength is certainly the wide selection of experiments. The clinical study to investigate the effect of glucose on CTSL concentrations in healthy individuals sets the stage for experiments in cell culture, animal models and human tissue. The effect of CTSL knockout cell lines on glucose-induced SARS-CoV2 infection rates are convincing. Finally, the team used a combination of Western blots and confocal microscopy to identify the underlying molecular mechanisms.
The authors keep the diabetic condition at the center of their study and extend on previous knowledge of glucose-induced CTSL activation and their consequences for Covid19 infections. By doing so, they create a novel connection between CTSL involvement in SARS-CoV2 infections and diabetes. This enables novel, public awareness of the susceptibility of diabetes patients to the disease.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this study, Liu et al. investigate the signaling pathway that triggers sliding motility in the bacterium B. subtilis in response to subinhibitory concentrations of the antibiotic chloramphenicol. The authors used a genetic approach to identify the master regulator CodY playing a regulatory role in this behavior. They used transcriptional and metabolomic profiling to delineate the spatiotemporal separation of the regulatory networks that define distinct metabolic states related to purine metabolism and pyruvate utilization, which are ultimately responsible for the induction of sliding in response to chloramphenicol. Many readers would be interested to read this work showing how extracellular signals modulate microbial physiology and metabolism.
Strengths:
This work presents numerous technical and conceptual strengths. In the opinion of this referee, the most significant conceptual strength of this work is to (once again) provide evidence that antibiotics are not merely produced by bacteria to eliminate competitors. Bacteria have evolved to respond to their presence and activate a range of physiological responses, which are poorly understood. Understanding these responses is critical to fully understand the evolutionary consequences associated with the use of antibiotics. From a technical standpoint, perhaps the most relevant aspect is the robust phenotypic assay developed by the authors to study sliding motility in the presence of chloramphenicol. This robustness enables genetic work using mutants and performing omics assays to characterize the response to chloramphenicol in detail. Additionally, two sets of results stood out and provided important value to this work. One is the comparison established between the sliding induced by chloramphenicol and the sliding generated in the ΔcodY mutant, to determine the genes and the metabolites (using transcriptomics and metabolomics) specifically associated with the response to chloramphenicol without being part of the general Cody-mediated induction of sliding. The second set of results led the authors to identify precise genes of bacterial metabolism (pdhA) responsible for the sliding phenotype in response to chloramphenicol, and conducted genetic experiments to demonstrate that the pdhA mutant does not respond to the presence of chloramphenicol.
Weaknesses:
This work has three main weaknesses, all related to transcriptomic and metabolomic analyses. Firstly, there is the challenge of understanding the essence of the omics results. This section presents an overwhelming array of genes involved in different metabolic pathways, without an obvious thread to tie these hits together. It is easy to get lost in this section. For instance, one cannot be certain if the hits from one particular metabolic pathway are significant enough to figure out to which degree is this pathway responsible for the sliding phenotype. This section contains a huge diversity of genes and pathways and needs to be streamlined. Related to this, the message of the omics experiments highlights a very close relationship between purine and pyruvate metabolism in sliding motility. However, it is unclear how these metabolic pathways may influence sliding or any other specific bacterial behavior. I do not mean to say that it is not possible, just that the connection/mechanism is missing. The third weakness concerns the omics results that sometimes are in conflict. The authors proposed that this may stem from a division of labor and the coexistence of different subpopulations with different metabolisms within the microbial community. While plausible, other possibilities are equally plausible and should be tested in a revised version of the work.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
By identifying a loss of function mutant of IQCH in infertile patient, Ruan et al. shows that IQCH is essential for spermiogenesis by generating a knockout mouse model of IQCH. Similar to infertile patient with mutant of IQCH, Iqch knockout mice are characterized by a cracked flagellar axoneme and abnormal mitochondrial structure. Mechanistically, IQCH regulates the expression of RNA-binding proteins (especially HNRPAB), which are indispensable for spermatogenesis.
Although this manuscript contains a potentially interesting piece of work that delineates a mechanism of IQCH that associates with spermatogenesis, this reviewer feels that a number of issues require clarification and re-evaluation for a better understanding of the role of IQCH in spermatogenesis.
Line 251 - 253, "To elucidate the molecular mechanism by which IQCH regulates male fertility, we performed liquid chromatography tandem mass spectrometry (LC‒MS/MS) analysis using mouse sperm lysates and detected 288 interactors of IQCH (Figure 5-source data 1)."
The reviewer had already raised significant concerns regarding the text above, noting that "LC‒MS/MS analysis using mouse sperm lysates" would not identify interactors of IQCH. However, this issue was not addressed in the revised manuscript. In the Methods section detailing LC-MS/MS, the authors stated that it was conducted on "eluates obtained from IP". However, there was no explanation provided on how IP for LC-MS/MS was performed. Additionally, it was unclear whether LC-MS or LC-MS/MS was utilized. The primary concern is that if LC‒MS/MS was conducted for the IP of IQCH, IQCH itself should have been detected in the results; however, as indicated by Figure 5-source data 1, IQCH was not listed.
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Reviewer #2 (Public Review):
The authors analysed functional MRI recordings of brain activity at rest, using state-of-the-art methods that reveal the diverse ways in which information can be integrated in the brain. In this way, they found brain areas that act as (synergistic) gateways for the 'global workspace', where conscious access to information or cognition would occur, and brain areas that serve as (redundant) broadcasters from the global workspace to the rest of the brain. The results are compelling and are consistent with the already assumed role of several networks and areas within the Global Neuronal Workspace framework. Thus, in a way, this work comes to stress the role of synergy and redundancy as complementary information processing modes, which fulfill different roles in the bigger context of information integration.
In addition, to prove that the identified high-order interactions are relevant to the phenomenon of consciousness, the same analysis was performed in subjects under anesthesia or with disorders of consciousness (DOC), showing that indeed the loss of consciousness is associated with a deficient integration of information within the gateway regions.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this study, the authors obtained multiple, novel and compelling datasets to better understand the relationship between histone H1 and RNA-directed DNA methylation in plants. Most of the authors' claims concerning H1 and RNA polymerase V (Pol V) are backed by convincing and independent lines of evidence. However, Pol V produces noncoding transcripts that act as scaffold RNAs, which AGO4-bound siRNAs recognize in plant chromatin to mediate RNA-directed DNA methylation. Detection of Pol V transcript products at the sites of Pol V redistribution in h1 mutants would significantly enhance the impact of this manuscript. Below I have listed several strengths and a weakness of the manuscript.
Strengths:
- The authors report high-quality NRPE1 ChIP-seq data, allowing them to directly test how and where Pol V occupancy depends on histone H1 function in Arabidopsis.<br /> - nrpe1 mutants generated via CRISPR/Cas9 in the h1 mutant background (nrpe1 h1.1-1 h1.2-1 triple mutants), allow the authors to study the role of Pol V in ectopic DNA methylation in H1-deficient plants.<br /> - Pol V recruitment via ZincFinger-DMS3 expression (a modified version of Pol V's DMS3 recruitment factor) sends Pol V to new genomic loci and thus provides the authors with an innovative dataset for understanding H1 function at these sites.
Weakness:
- The manuscript does not include detection or quantification of Pol V transcripts generated at ectopic sites in the h1 mutant background. Pol V encroachment into heterochromatin in the h1 mutant is indirectly shown by NRPE1-dependent methylation at such ectopic sites.
Previous studies have charted the relationship between H1 function and RNA-directed DNA methylation (RdDM) via analyses of Pol IV-dependent 24 nt siRNAs and factors that recruit Pol IV (Choi et al., 2021 and Papareddy et al., 2020). Harris and colleagues have extended this work and shown that histone H1 function also antagonizes Pol V occupancy in the context of constitutive heterochromatin. The authors thus provide important evidence to show that H1 limits the encroachment of both polymerases Pol IV and Pol V into plant heterochromatin.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The authors design an automated 24-well Barnes maze with 2 orienting cues inside the maze, then model what strategies the mice use to reach the goal location across multiple days of learning. They consider a set of models and conclude that the animals begin with a large proportion of random choices (choices irrespective of the goal location), which over days of experience becomes a combination of spatial choices (choices targeted around the goal location) and serial choices (successive stepwise choices in a given direction). Moreover, the authors show that after the animal has many days of experience in the maze, they still often began each trial with a random choice, followed by spatial or serial choices.
This study is written concisely and the results are presented concisely. The best fit model provides valuable insight into how the animals solve this task, and therefore offers a quantitative foundation upon which tests of neural mechanisms of the components of the behavioral strategy can be performed. These tests will also benefit from the automated nature of the task.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Jiang et al. demonstrated that ablating Neurexins results in alterations to glycinergic transmission and its calcium sensitivity, utilizing a robust experimental system. Specifically, the authors employed rAAV-Cre-EGFP injection around the MNTB in Nrxn1/2/3 triple conditional mice at P0, measuring Glycine receptor-dependent IPSCs from postsynaptic LSO neurons at P13-14. Notably, the authors presented a clear reduction of 60% and 30% in the amplitudes of opto- and electric stimulation-evoked IPSCs, respectively. Additionally, they observed changes in kinetics, alterations in PPR, and sensitivity to lower calcium and the calcium chelator, EGTA, indicating solid evidence for changes in presynaptic properties of glycinergic transmission.
Furthermore, the authors uncovered an unexpected increase in sIPSC frequency without altering amplitude. Although the precise mechanism remains unknown, the authors discussed this complex phenotype by considering various possibilities, including the potential scenario where the augmentation in synapses may result from Nrxn deletion rather than being a causal effect.
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www.biorxiv.org www.biorxiv.org
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Reviewer #3 (Public Review):
This study demonstrated the application of OPM-MEG in neurodevelopment studies of somatosensory beta oscillations and connections with children as young as 2 years old. It provides a new functional neuroimaging method which has high spatial-temporal resolution as well wearable which makes it a new useful tool for studies in young children. They have constructed a 192-channel wearable OPM-MEG system includes field compensation coils which allows free head movement scanning with relatively high ratio of usable trials. Beta band oscillations during somatosensory tasks are well localized and the modulation with age are found in the amplitude, connectivity, and pan-spectral burst probability. It is demonstrated that the wearable OPM-MEG could be used in children as a quite practical and easy to deploy neuroimaging method with performance as good as conventional MEG. With both good spatial (several millimeter) and temporal (milliseconds) resolution, it provides a novel and powerful technology to neurodevelopment research and clinical application not limited to somatosensory areas.
The conclusions of this paper are mostly well supported by data acquired under proper method.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Chartampila et al. describe the effect of early-life choline supplementation on cognitive functions and epileptic activity in a mouse model of Alzheimer's disease. The cognitive abilities were assessed by the novel object recognition test and the novel object location test, performed in the same cohort of mice at 3 months and 6 months of age. Neuronal loss was tested using NeuN immunoreactivity, and neuronal hyperexcitability was examined using FosB and video-EEG recordings, providing multi-level correlations between these different parameters.
Strengths:
The study was designed as a 6-month follow-up, with repeated behavioral and EEG measurements through disease development and multilevel correlations providing valuable and interesting findings on AD progression and the effect of early-life choline supplementation. Moreover, the behavioral data that suggest an adverse effect of low choline in WT mice are interesting and important also beyond the context of AD, highlighting the dramatic effect of diet on the phenotypes of animal models.
Weaknesses:
The readability could be improved.
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
Although the study by Xiaolin Yu et al is largely limited to in vitro data, the results of this study convincingly improve our current understanding of leukocyte migration.
(1) The conclusions of the paper are mostly supported by the data and in the revised manuscript clarification is provided concerning the exact CCL5 forms (without or with a fluorescent label or His-tag) and amounts/concentrations that were used in the individual experiments. This is important since it is known that modification of CCL5 at the N-terminus affects the interactions of CCL5 with the GPCRs CCR1, CCR3 and CCR5 and random labeling using monosuccinimidyl esters (as done by the authors with Cy-3) is targeting lysines. The revised manuscript more clearly indicates for each individual experiment which form is used. However, a discussion on the potential effects of the modifications on CCL5 in the results and discussion sections is still missing.<br /> (2) In general, authors used high concentrations of CCL5 in their experiments. In their reply to the comments they indicate that at lower CCL5 concentrations no LLPS is detected. This is important information since it may indicate the need for chemokine oligomerization for LLPS. This info should be added to the manuscript and comparison with for instance the obligate monomer CCL7 and another chemokine such as CXCL4 that easily forms oligomers may clarify whether LLPS is controlled by oligomerization.<br /> (3) Statistical analyses have been improved in the revised manuscript.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The authors perform RNA-seq on FACS isolated neurons from adult worms at days 1 and 8 of adulthood to profile the gene expression changes that occur with cognitive decline. Supporting data are included indicating that by day 7 of adulthood, learning and memory are reduced, indicating that this timepoint or after represents cognitively aged worms. Neuronal identity genes are reduced in expression within the cognitively aged worms, whereas genes involved in proteostasis, transcription/chromatin, and the stress response are elevated. A number of specific examples are provided, representing markers of specific neuronal subtypes, and correlating expression changes to the erosion of particular functions (e.g. motor neurons, chemosensory neurons, aversive learning neurons, etc).
To investigate whether upregulation of genes in neurons with age is compensatory or deleterious, the authors reduced expression of a set of three significantly upregulated genes and performed behavioral assays in young adults. In each case, reduction of expression improved memory, consistent with a model in which age-associated increases impair neuronal function.
The authors then characterize learning and memory in wild type, daf-2, and daf-2/daf-16 worms with age and find that daf-2 worms have an extended ability to learn for approximately 10 days longer that wild types. This was daf-16 dependent. Memory was extended in daf-2 as well, and strikingly, daf-2;daf-16 had no short term memory even at day 1. Transcriptomic analysis of FACS-sorted neurons was performed on the three groups at day 8. The authors focus their analysis on daf-2 vs. daf-2;daf-16 and present evidence that daf-2 neurons express a stress-resistance gene program. They also find small differences between the N2 and daf-2;daf-16 neurons, which correlate with the observed behavioral differences, though these differences are modest.
The authors tested eight candidate genes that were more highly expressed in daf-2 neurons vs. daf-2;daf-16 and showed that reduction of 2 and 5 of these genes impaired learning and memory, respectively, in daf-2 worms. This finding implicates specific neuronal transcriptional targets of IIS in maintaining cognitive ability in daf-2 with age, which, importantly, are distinct from those in young wild type worms.
Overall, this is a strong study with rigorously performed experiments. The authors achieved their aim of identifying transcriptional changes in neurons that underlie loss of learning and memory in C. elegans, and how cognition is maintained in insulin/IGF-1-like signaling mutants.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors utiilze the model organism C. elegans to interrogate cell non-autonomous signaling between GABAergic neurons and somatic tissues. They demonstrate that RNAi of isp-1 or spg-7 in GABAergic neurons leads to lifespan extension and improved healthspan (by resistance to paraquat or heat stress), which are dependent on the transcription factor daf-16/FOXO3a.
Strengths:
The authors are clear and straightforward in their study. They examine the healthspan of C. elegans at days 3, 6, and 9 to give a wide perspective on how the phenotypes changes with aging. They use two methods to specifically knockdown isp-1 or spg-7 in GABAergic neurons: (1) a previously published rde-1 mutant that has rde-1 and sid-1 restored only in GABAergic neurons and (2) a novel model uses a sid-1 mutant that makes dsRNA of isp-1 or spg-7 in GABAergic neurons. They use multiple methods to examine healthspan. They identified daf-16/FOXO3a as the mechanism of their phenotype and ruled out other transcription factors. The authors do not use FUdR in their studies, which is known to confound experiments.
Weaknesses:
(1) Incomplete validation of GABAergic knockdown. The study relies on the specific knockdown of isp-1 or spg-7 in GABAergic neurons, but in the opinion of this reviewer, the authors do not adequately validate their models to demonstrate GABAergic specificity. For the previously published rde-1 mutant model, a simple validation of specific knockdown of GFP in GFP-labeled GABAergic neurons should be included. They should also show that GFP RNAi would not be effective in knocking down intestinal GFP, for example.
Their second model is poorly explained and not validated and this reviewer could not find similar previously published models of its kind. This model claims that dsRNA of isp-1 was made in the GABAergic neurons of a sid-1 mutant, but no evidence is shown to support this claim. The authors point to changes in phenotypes such as lifespan extension and reduced lipofuscin in the intestines as proof that knockdown is occurring in the GABAergic neurons, but this is indirect evidence. Rigorous validation of this model is needed, especially if it is the first model of its kind.
(2) Lifespan. The control lifespans using the rde-1 mutants are very short-lived and no explanation for this is provided (eg. Figure 1D, E). The authors use two RNAis in their lifespan with daf-16 and isp-1. For their controls, they should use empty vector mixed with isp-1, not only isp-1 RNAi.
(3) Cell non-autonomous effects. The claims that GABAergic mitochondrial dysfunction have effects on somatic tissues is weak. More specific tests on somatic stress resistance are warranted for their claims. Better quality images of intestinal mitochondria are needed. Examining additional tissues, such as muscle, would also strengthen their claims. For example, they could examine muscle mitochondria and determine if muscle strength is improved in their models.
(4) Dependence on daf-16/FOXO3a. The authors show that loss of daf-16 reverses the lifespan and healthspan effects in their model. Next, they show that loss of daf-16 reverses the effects of isp-1 in the intestines and in the germline. However, they only show the daf-16 mutant data and not the positive control (EV and isp-1 alone), which should be included. Furthermore, the phenotypes they examine are only a subset of somatic phenotypes, and this reviewer would be more convinced with the additional controls and with more parameters examined.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review)
Summary:
In this study, Xie and colleagues aimed to explore the function and potential mechanisms of the gut microbiota in a hamster model of severe leptospirosis. The results demonstrated that Leptospira infection was able to cause intestine damage and inflammation. Leptospira infection promoted an expansion of Proteobacteria, increased gut barrier permeability, and elevated LPS levels in the serum. Thus, they proposed an LPS-neutralization therapy which improved the survival rate of moribund hamsters combined with antibody therapy or antibiotic therapy.
Strengths:
The work is well-designed and the story are interesting to me. The gut microbiota is essential for immunity and systemic health. Many life-threatening pathogens, such as SARS-CoV-2 and other gut-damaged infection, have the potential to disrupt the gut microbiota in the later stages of infection, causing some harmful gut microbiota-derived substances to enter the bloodstream. It is emphasized that in addition to exogenous pathogenic pathogens, harmful substances of intestinal origin should also be considered in critically ill patients.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Spikol et al. investigate the roles of two distinct populations of neurons in the nucleus incertus (NI). The authors established two new transgenic lines that label gsc2- and rln3a-expressing neurons. They show that the gsc2+ and rln3a+ NI neurons show divergent projection patterns and project to different parts of the interpeduncular nucleus (IPN), which receive inputs from the habenula (Hb). Furthermore, calcium imaging shows that gsc2 neurons are activated by the optogenetic activation of the dorsal Hb-IPN and respond to aversive electric shock stimuli, while rln3a neurons are highly spontaneously active. The ablation of rln3a neurons, but not gsc2 neurons, alters locomotor activity of zebrafish larvae.
The strength of the paper is their genetic approach that enabled the authors to characterize many different features of the two genetically targeted populations in the NI. These two neuronal populations are anatomically closely apposed and would have been indistinguishable without their genetic tools. Their analyses provide valuable information on the diverse anatomical, physiological and behavioral functions of the different NI subtypes. On the other hand, these pieces of evidence are loosely linked with each other to reach a mechanistic understanding of how the NI works in a circuit. For example, the anatomical study revealed the connections from the NI to the IPN, while the optogenetic mapping experiments investigate the other way around, i.e. the connection from the IPN to the NI.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors define a new metric for visual displays, derived from psychophysical response times, called visual homogeneity (VH). They attempt to show that VH is explanatory of response times across multiple visual tasks. They use fMRI to find visual cortex regions with VH-correlated activity. On this basis, they declare a new visual region in human brain, area VH, whose purpose is to represent VH for the purpose of visual search and symmetry tasks.
Strengths:
The authors present carefully designed experiments, combining multiple types of visual judgments and multiple types of visual stimuli with concurrent fMRI measurements. This is a rich dataset with many possibilities for analysis and interpretation.
Weaknesses:
The datasets presented here should provide a rich basis for analysis. However, in this version of the manuscript, I believe that there are major problems with the logic underlying the authors' new theory of visual homogeneity (VH), with the specific methods they used to calculate VH, and with their interpretation of psychophysical results using these methods. These problems with the coherency of VH as a theoretical construct and metric value make it hard to interpret the fMRI results based on searchlight analysis of neural activity correlated with VH. In addition, the large regions of VH correlations identified in Experiments 1 and 2 vs. Experiments 3 and 4 are barely overlapping. This undermines the claim that VH is a universal quantity, represented in a newly discovered area of visual cortex, that underlies a wide variety of visual tasks and functions.
Maybe I have missed something, or there is some flaw in my logic. But, absent that, I think the authors should radically reconsider their theory, analyses, and interpretations, in light of detailed comments below, in order to make the best use of their extensive and valuable datasets combining behavior and fMRI. I think doing so could lead to a much more coherent and convincing paper, albeit possibly supporting less novel conclusions.
THEORY AND ANALYSIS OF VH
(1) VH is an unnecessary, complex proxy for response time and target-distractor similarity.
VH is defined as a novel visual quality, calculable for both arrays of objects (as studied in Experiments 1-3) and individual objects (as studied in Experiment 4). It is derived from a center-to-distance calculation in a perceptual space. That space in turn is derived from multi-dimensional scaling of response times for target-distractor pairs in an oddball detection task (Experiments 1 and 2) or in a same different task (Experiments 3 and 4). Proximity of objects in the space is inversely proportional to response times for arrays in which they were paired. These response times are higher for more similar objects. Hence, proximity is proportional to similarity. This is visible in Fig. 2B as the close clustering of complex, confusable animal shapes.
VH, i.e. distance-to-center, for target-present arrays is calculated as shown in Fig. 1C, based on a point on the line connecting target and distractors. The authors justify this idea with previous findings that responses to multiple stimuli are an average of responses to the constituent individual stimuli. The distance of the connecting line to the center is inversely proportional to the distance between the two stimuli in the pair, as shown in Fig. 2D. As a result, VH is inversely proportional to distance between the stimuli and thus to stimulus similarity and response times. But this just makes VH a highly derived, unnecessarily complex proxy for target-distractor similarity and response time. The original response times on which the perceptual space is based are far more simple and direct measures of similarity for predicting response times.
(2) The use of VH derived from Experiment 1 to predict response times in Experiment 2 is circular and does not validate the VH theory.
The use of VH, a response time proxy, to predict response times in other, similar tasks, using the same stimuli, is circular. In effect, response times are being used to predict response times across two similar experiments using the same stimuli. Experiment 1 and the target present condition of Experiment 2 involve the same essential task of oddball detection. The results of Experiment 1 are converted into VH values as described above, and these are used to predict response times in experiment 2 (Fig. 2F). Since VH is a derived proxy for response values in Experiment 1, this prediction is circular, and the observed correlation shows only consistency between two oddball detection tasks in two experiments using the same stimuli.
(3) The negative correlation of target-absent response times with VH as it is defined for target-absent arrays, based on distance of a single stimulus from center, is uninterpretable without understanding the effects of center-fitting. Most likely, center-fitting and the different VH metric for target-absent trials produce an inverse correlation of VH with target-distractor similarity.
The construction of the VH perceptual space also involves fitting a "center" point such that distances to center predict response times as closely as possible. The effect of this fitting process on distance-to-center values for individual objects or clusters of objects is unknowable from what is presented here. These effects would depend on the residual errors after fitting response times with the connecting line distances. The center point location and its effects on distance-to-center of single objects and object clusters are not discussed or reported here.
Yet, this uninterpretable distance-to-center of single objects is chosen as the metric for VH of target-absent displays (VHabsent). This is justified by the idea that arrays of a single stimulus will produce an average response equal to one stimulus of the same kind. But it is not logically clear why response strength to a stimulus should be a metric for homogeneity of arrays constructed from that stimulus, or even what homogeneity could mean for a single stimulus from this set. And it is not clear how this VHabsent metric based on single stimuli can be equated to the connecting line VH metric for stimulus pairs, i.e. VHpresent, or how both could be plotted on a single continuum.
It is clear, however, what *should* be correlated with difficulty and response time in the target-absent trials, and that is the complexity of the stimuli and the numerosity of similar distractors in the overall stimulus set. Complexity of the target, similarity with potential distractors, and number of such similar distractors all make ruling out distractor presence more difficult. The correlation seen in Fig. 2G must reflect these kinds of effects, with higher response times for complex animal shapes with lots of similar distractors and lower response times for simpler round shapes with fewer similar distractors.
The example points in Fig. 2G seem to bear this out, with higher response times for the deer stimulus (complex, many close distractors in the Fig. 2B perceptual space) and lower response times for the coffee cup (simple, few close distractors in the perceptual space). While the meaning of the VH scale in Fig. 2G, and its relationship to the scale in Fig. 2F, are unknown, it seems like the Fig. 2G scale has an inverse relationship to stimulus complexity, in contrast to the expected positive relationship for Fig. 2F. This is presumably what creates the observed negative correlation in Fig. 2G.
Taken together, points 1-3 suggest that VHpresent and VHabsent are complex, unnecessary, and disconnected metrics for understanding target detection response times. The standard, simple explanation should stand. Task difficulty and response time in target detection tasks, in both present and absent trials, are positively correlated with target-distractor similarity.
I think my interpretations apply to Experiments 3 and 4 as well, although I find the analysis in Fig. 4 especially hard to understand. The VH space in this case is based on Experiment 3 oddball detection in a stimulus set that included both symmetric and asymmetric objects. But the response times for a very different task in Experiment 4, a symmetric/asymmetric judgment, are plotted against the axes derived from Experiment 3 (Fig. 4F and 4G). It is not clear to me why a measure based on oddball detection that requires no use of symmetry information should be predictive of within-stimulus symmetry detection response times. If it is, that requires a theoretical explanation not provided here.
(4) Contrary to the VH theory, same/different tasks are unlikely to depend on a decision boundary in the middle of a similarity or homogeneity continuum.
The authors interpret the inverse relationship of response times with VHpresent and VHabsent, described above, as evidence for their theory. They hypothesize, in Fig. 1G, that VHpresent and VHabsent occupy a single scale, with maximum VHpresent falling at the same point as minimum VHabsent. This is not borne out by their analysis, since the VHpresent and VHabsent value scales are mainly overlapping, not only in Experiments 1 and 2 but also in Experiments 3 and 4. The authors dismiss this problem by saying that their analyses are a first pass that will require future refinement. Instead, the failure to conform to this basic part of the theory should be a red flag calling for revision of the theory.
The reason for this single scale is that the authors think of target detection as a boundary decision task, along a single scale, with a decision boundary somewhere in the middle, separating present and absent. This model makes sense for decision dimensions or spaces where there are two categories (right/left motion; cats vs. dogs), separated by an inherent boundary (equal left/right motion; training-defined cat/dog boundary). In these cases, there is less information near the boundary, leading to reduced speed/accuracy and producing a pattern like that shown in Fig. 1G.
This logic does not hold for target detection tasks. There is no inherent middle point boundary between target present and target absent. Instead, in both types of trial, maximum information is present when target and distractors are most dissimilar, and minimum information is present when target and distractors are most similar. The point of greatest similarity occurs at then limit of any metric for similarity. Correspondingly, there is no middle point dip in information that would produce greater difficulty and higher response times. Instead, task difficulty and response times increase monotonically with similarity between targets and distractors, for both target present and target absent decisions. Thus, in Figs. 2F and 2G, response times appear to be highest for animals, which share the largest numbers of closely similar distractors.
DEFINITION OF AREA VH USING fMRI
(1) The area VH boundaries from different experiments are nearly completely non-overlapping.
In line with their theory that VH is a single continuum with a decision boundary somewhere in the middle, the authors use fMRI searchlight to find an area whose responses positively correlate with homogeneity, as calculated across all of their target present and target absent arrays. They report VH-correlated activity in regions anterior to LO. However, the VH defined by symmetry Experiments 3 and 4 (VHsymmetry) is substantially anterior to LO, while the VH defined by target detection Experiments 1 and 2 (VHdetection) is almost immediately adjacent to LO. Fig. S13 shows that VHsymmetry and VHdetection are nearly non-overlapping. This is a fundamental problem with the claim of discovering a new area that represents a new quantity that explains response times across multiple visual tasks. In addition, it is hard to understand why VHsymmetry does not show up in a straightforward subtraction between symmetric and asymmetric objects, which should show a clear difference in homogeneity.
(2) It is hard to understand how neural responses can be correlated with both VHpresent and VHabsent.
The main paper results for VHdetection are based on both target-present and target-absent trials, considered together. It is hard to interpret the observed correlations, since the VHpresent and VHabsent metrics are calculated in such different ways and have opposite correlations with target similarity, task difficulty, and response times (see above). It may be that one or the other dominates the observed correlations. It would be clarifying to analyze correlations for target-present and target-absent trials separately, to see if they are both positive and correlated with each other.
(3) Definition of the boundaries and purpose of a new visual area in the brain requires circumspection, abundant and convergent evidence, and careful controls.
Even if the VH metric, as defined and calculated by the authors here, is a meaningful quantity, it is a bold claim that a large cortical area just anterior to LO is devoted to calculating this metric as its major task. Vision involves much more than target detection and symmetry detection. Cortex anterior to LO is bound to perform a much wider range of visual functionalities. If the reported correlations can be clarified and supported, it would be more circumspect to treat them as one byproduct of unknown visual processing in cortex anterior to LO, rather than treating them as the defining purpose for a large area of visual cortex.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This is an interesting report examining activity patterns in mouse ACC and in the OFC neurons projecting to ACC. In addition, the effects of inactivation are examined. In aggregate, the results provide new and interesting information about these two brain areas and they translate motivation into action - a function that it seems intuitively plausible that ACC might perform but, despite this intuition, there have been comparatively few direct tests of the idea and little is known of the specific mechanisms. The study is performed carefully and is written up clearly.
The combination of recording and inactivation/inhibition experiments and the combination of investigation of ACC neurons and of OFC regions projecting to ACC are very impressive.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this paper, the authors introduce a new deep learning-based algorithm for tracking animal poses, especially in minimizing drift effects. The algorithm's performance was validated by comparing it with two other popular algorithms, DeepLabCut and LEAP.
Strengths:
The authors showcased the effectiveness of their new algorithm in a systematic manner, covering individual levels of mice, drosophilas, macaques, and multi-animal poses.
Weaknesses:
(1) The accessibility of this tool for biological research is not clearly addressed, despite its potential usefulness. Researchers in biology often have limited expertise in deep learning training, deployment, and prediction. A detailed, step-by-step user guide is crucial, especially for applications in biological studies.
(2) The proposed algorithm focuses on tracking and is compared with DLC and LEAP, which are more adept at detection rather than tracking.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The mechanisms of how axonal projections find their correct target requires the interplay of signalling pathways, and cell adhesion that act over short and long distances. The current study aims to use the small ventral lateral clock neurons (s-LNvs) of the Drosophila clock circuit as a model to study axon projections. These neurons are born during embryonic stages and are part of the core of the clock circuit in the larval brain. Moreover, these neurons are maintained through metamorphosis and become part of the adult clock circuit. The authors use the axon length by means of anti-Pdf antibody or Pdf>GFP as a read-out for the axonal length. Using ablation of the MB- the overall target region of the s-LNvs, the authors find defects in the projections. Next, by using Dscam mutants or knock-down they observe defects in the projections. Manipulations by the DNs - another group of clock neurons- can induce defects in the s-LNvs axonal form, suggesting an active role of these neurons in the morphology of the s-LNvs.
Strengths:
The use of Drosophila genetics and a specific neural type allows targeted manipulations with high precision.
Proposing a new model for a small group of neurons for axonal projections allows us to explore the mechanism with high precision.
Weaknesses:
It is unclear how far the proposed model can be seen as developmental.
The study of changes in fully differentiated and functioning neurons may affect the interpretation of the findings.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The study investigated the neural circuits underlying social novelty preference in mice. Using viral circuit tracing, chemogenetics, and optogenetics in the vHPC, LS, and VTA, the authors found that vHPC to LS projections may contribute to the salience of social novelty investigations. In addition, the authors identify LS projections to the VTA involved in social novelty and familiar food responses. Finally, via viral tracing, they demonstrate that vHPC-LS neurons may establish direct monosynaptic connections with VTA dopaminergic neurons. The experiments are well-designed, and the conclusions are mostly very clear. The manuscript is well-written and logically organized, and the content will be of interest to specialists in the field and to the broad readership of the journal.
Strengths:
(1) The vHPC has been involved in social memory for novel and familiar conspecifics. Yet, how the vHPC conveys this information to drive motivation for novel social investigations remains unclear. The authors identified a pathway from the vHPC to the LS and eventually the VTA, that may be involved in this process.
(2) Mice became familiar with a novel conspecific by co-housing for 72h. This represents a familiarization session with a longer duration as compared to previous literature. Using this new protocol, the authors found robust social novelty preference when animals were given a choice between a novel and familiar conspecific.
(3) The effects of vHPC-LS inhibition are specific to novel social stimuli. The authors included novel food and novel object control experiments and those were not affected by neuronal manipulations.
(4) For optogenetic studies, the authors applied closed-loop photoinhibition only when the animals investigated either the novel conspecific or the familiar. This optogenetic approach allowed for the investigation of functional manipulations to selective novel or familiar stimuli approaches.
Weaknesses:
(1) The abstract and the overall manuscript pose that the authors identified a novel vHPC-LS-VTA pathway that is necessary for mice to preferentially investigate novel conspecifics. However, the authors assessed the functional manipulations of vHPC-LS and LS-VTA circuits independently and the sentence could be misleading. Therefore, a viral strategy specifically designed to target the vHPC-LS-VTA circuit combined with optogenetic/chemogenetic tools and behavior may be necessary for the statement of this conclusion.
(2) The authors combined males and females in their analysis, as neural circuit manipulation affected novelty discrimination ratios in both sexes. However, supplementary Figure 1 demonstrates the chemogentic inhibition of vHPC-LS circuit may cause stronger effects in male mice as compared to females.
(3) In most experiments, the same animals were used for social novelty preference, for food or object novelty responses but washout periods between experiments are not mentioned in the methods section. In this line, the authors did not mention the time frame between the closed-loop optogenetic experiments that silenced the vHPC-LS only during familiar and then only novel social investigations. When using the same animals tested for social experiments in the same context there may be an effect of context-dependent social behaviors that could affect future outcomes.
(4) All the experiments were performed in a non-cell-type-specific manner. The viral strategies used targeted multiple neuronal subpopulations that could have divergent effects on social novelty preference. This constraint could be added in the discussion section.
(5) The authors' assumptions were all based on experiments of necessity. The authors could use an experiment of sufficiency by targeting for instance the LS-VTA circuit and assess if animals reduce novel social investigations with LS-VTA photostimulation.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this manuscript by Mou and Ji, the authors describe the correlation between firing rates in the ACC with that of CA1 ensembles during observational learning. Their main findings include trajectory selective (observational) responses in ACC, correlations between ACC and CA1 place cells for specific trajectories during observational learning, and correlations between ACC and CA1 place cells that are reactivated during SWRs, specifically during CA1 replays.
Strengths:
The study is well designed, the data presented is very clear and the conclusions are appropriate regarding their results. The study is novel and of high relevance for the understanding of social learning.
Weaknesses:
Lack of physiological characterization of the neurons that could have been included, such as regular firing rates of neurons in different regions (not only constrained to behavioral landmarks) or PSTH during sharp-wave ripples. The first experiment, NMDA blockage, is a bit disconnected from the rest of the results. Perhaps clarifying in the text a bit further that this proves that ACC is necessary for social learning would help.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this manuscript, Zhang et al. report a genetic screen to identify novel transcriptional regulators that could coordinate mitochondrial biogenesis. They performed an RNAi-based modifier screen wherein they systematically knocked down all known transcription factors in the developing Drosophila eye, which was already sensitised and had decreased mitochondrial DNA content. Through this screen, they identify CG1603 as a potential regulator of mitochondrial content. They show that protein levels of mitochondrial proteins like TFAM, SDHA, and other mitochondrial proteins and mtDNA content are downregulated in CG1603 mutants. RNA-Seq and ChIP-Seq further show that CG1603 binds to the promoter regions of several known nuclear-encoded mitochondrial genes and regulates their expression. Finally, they also identified YL-1 as an upstream regulator of CG1603. Overall, it is a very important study as our understanding of the regulation of mitochondrial biogenesis remains limited across metazoans. Most studies have focused on PGC-1α as a master regulator of mitochondrial biogeneis, which seems a context-dependent regulator. Also, PGC-1α mediated regulation could not explain the regulation of 1100 genes that are required for mitochondrial biogenesis. Therefore, identifying a new regulator is crucial for understanding the overall regulation of mitochondrial biogenesis.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors investigate ligand and protein-binding processes in GPCRs (including dimerization) by the multiple walker supervised molecular dynamics method. The paper is interesting and it is very well written.
Strengths:
The authors' method is a powerful tool to gain insight into the structural basis for the pharmacology of G protein-coupled receptors.
Weaknesses:
Cholesterol may play a fundamental role in GPCR dimerization (as cited by the authors, Prasanna et al, "Cholesterol-Dependent Conformational Plasticity in GPCR Dimers"). Yet they do not use cholesterol in their simulations of the dimerization.
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Reviewer #1 (Public Review):
Summary:
This manuscript presents evidence of 'vocal style' in sperm whale vocal clans. Vocal style was defined as specific patterns in the way that rhythmic codas were produced, providing a fine-scale means of comparing coda variations. Vocal style effectively distinguished clans similar to the way in which vocal repertoires are typically employed. For non-identity codas, vocal style was found to be more similar among clans with more geographic overlap. This suggests the presence of social transmission across sympatric clans while maintaining clan vocal identity.
Strengths:
This is a well-executed study that contributes exciting new insights into cultural vocal learning in sperm whales. The methodology is sound and appropriate for the research question, building on previous work and ground-truthing much of their theories. The use of the Dominica dataset to validate their method lends strength to the concept of vocal style and its application more broadly to the Pacific dataset. The results are framed well in the context of previous works and clearly explain what novel insights the results provide to the current understanding of sperm whale vocal clans. The discussion does an overall great job of outlining why horizontal social learning is the best explanation for the results found.
Weaknesses:
The primary issues with the manuscript are in the technical nature of the writing and a lack of clarity at times with certain terminology. For example, several tree figures are presented and 'distance' between trees is key to the results, yet 'distance' is not clearly defined in a way for someone unfamiliar with Markov chains to understand. However, these are issues that can easily be dealt with through minor revisions with a view towards making the manuscript more accessible to a general audience.
I also feel that the discussion could focus a bit more on the broader implications - specifically what the developed methods and results might imply about cultural transmission in other species. This is specifically mentioned in the abstract but not really delved into in detail during the discussion.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The microtubule cytoskeleton is essential for basic cell functions, enabling intracellular transport, and establishment of cell polarity and motility. Microtubule-associated proteins (MAPs) contribute to the regulation of microtubule dynamics and stability - mechanisms that are specifically important for the development and physiological function of neurons. Here, the authors aimed to elucidate the neuronal function of the MAP Hmmr, which they had previously identified in a (yet unpublished) quantitative study of the proteome associated with neuronal microtubules. The authors conduct well-controlled experiments to demonstrate the localization of endogenous as well as exogenous Hmmr on microtubules within the soma as well as all neurites of hippocampal neurons. Functional analysis using gain- and loss-of-function approaches demonstrates that Hmmr levels are crucial for neuronal morphogenesis, as the length of both dendrites and axons decreases upon loss of Hmmr and increases upon Hmmr overexpression. In addition to length alterations, the branching pattern of neurites changes with Hmmr levels. To uncover the mechanism of how Hmmr influences neuronal morphology, the authors follow the lead that Hmmr overexpression induces looped microtubules in the soma, indicative of an increase in microtubule stability. Microtubule acetylation indeed decreases and increases with Hmmr LOF and GOF, respectively. Together with a rescue of nocodazole-induced microtubule destabilization by Hmmr GOF, these results argue that Hmmr regulates microtubule stability. Highlighted by the altered movement of a plus-end-associated protein, Hmmr also has an effect on the dynamic nature of microtubules. The authors present evidence suggesting that the nucleation frequency of neuronal microtubules depends on Hmmr's ability to recruit the microtubule nucleator Tpx2. The authors discuss how branching may be regulated by Hmmr-mediated microtubule dynamics and speculate about the physiological significance of altered neuronal morphogenesis. Together, their work adds novel insight into MAP-mediated regulation of microtubules as a prerequisite for neuronal morphogenesis.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
In this work, the authors provide a valuable transcriptomic resource for the intermediate free-living transmission stage (miracidium larva) of the blood fluke. The single-cell transcriptome inventory is beautifully supplemented with in situ hybridization, providing spatial information and absolute cell numbers for many of the recovered transcriptomic states. The identification of sex-specific transcriptomic states within the populations of stem cells was particularly unexpected. The work comprises a rich resource to complement the biology of this complex system, however falls short in some technical aspects of the bioinformatic analyses of the generated sequence data.
(1) Four sequencing libraries were generated and then merged for analysis, however, the authors fail to document any parameters that would indicate that the clustering does not suffer from any batch effects.
(2) Additionally, the authors switch between analysis platforms without a clear motivation or explanation of what the fundamental differences between these platforms are. While in theory, any biologically robust observation should be recoverable from any permutation of analysis parameters, it has been recently documented that the two popular analysis platforms (Seurat - R and scanPy - python) indeed do things slightly differently and can give different results (https://www.biorxiv.org/content/10.1101/2024.04.04.588111v1). For this reason, I don't think that one can claim that Seurat fails to find clusters resolved by SAM without running a similar pipeline on the cluster alone as was done with SAM/scanPy here. The manuscript itself needs to be checked carefully for misleading statements in this regard.
(3) Similarly, the manuscript contains many statements regarding clusters being 'connected to', or forming a 'bridge' on the UMAP projection. One must be very careful about these types of statements, as the relative position of cells on a reduced-dimension cell map can be misleading (see Chari and Pachter 2023). To support these types of interpretations, the authors should provide evidence of gene expression transitions that support connectivity as well as stability estimates of such connections under different parameter conditions. Otherwise, these descriptors hold little value and should be dropped and the transcriptomic states simply defined as clusters with no reference to their positions on the UMAP.
(4) The underlying support for the clusters as transcriptomically unique identities is not well supported by the dot plots provided. The authors used very permissive parameters to generate marker lists, which hampers the identification of highly specific marker genes. This permissive approach can allow for extensive lists of upregulated genes for input into STRING/GO analyses, this is less useful for evaluating the robustness of the cluster states. Running the Seurat::FindAllMarkers with more stringent parameters would give a more selective set of genes to display and thereby increase the confidence in the reader as to the validity of profiles selected as being transcriptomically unique.
(5) Figure 5B shows a UMAP representation of cell positions with a statement that the clustering disappears. As a visual representation of this phenomenon, the UMAP is a very good tool, however, to make this statement you need to re-cluster your data after the removal of this gene set and demonstrate that the data no longer clusters into A/B and C/D. Also, as a reader, these data beg the question: which genes are removed here? Is there an over-representation of any specific 'types' of genes that could lead to any hypotheses of the function? Perhaps the STRING/GO analyses of this gene set could be informative.
(6) How do the proportions of cell types characterized via in situ here compare to the relative proportions of clusters obtained? It does not correspond to the percentages of the clusters captured (although this should be quantified in a similar manner in order to make this comparison direct: 10,686/20,478 = ~50% vs. 7%), how do you interpret this discrepancy? While this is mentioned in the discussion, there is no sufficient postulation as to why you have an overabundance of the stem cells compared to their presence in the tissue. While it is true that you could have a negative selection of some cell types, for example as stated the size of the penetration glands exceeds both that of the 10x capabilities (40uM), and the 30uM filters used in the protocol, this does not really address why over half of the captured cells represent 'stem cells'. A more realistic interpretation would be biological rather than merely technical. For example, while the composition of the muscle cells and the number of muscle transcriptomes captured are quite congruent at ~20%, the organism is composed of more than 50% of neurons, but only 15% of the transcriptomic states are assigned to neuronal. Could it be that a large fraction of the stem cells are actually neural progenitors? Are there other large inconsistencies between the cluster sizes and the fraction of expected cells? Could you look specifically at early transcription factors that are found in the neurons (or other cell types) within the various stem cell populations to help further refine the precursor/cell type relationships?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The article explores the connection between immunogenic cell death (ICD)-related genes and bladder cancer prognosis, immune infiltration, and response to therapy. The study identifies a risk-scoring model involving four ICD-related genes (CALR, IL1R1, IFNB1, IFNG), showing a correlation between higher risk scores and weaker anti-tumor immune function.
Strengths:
The significance lies in the potential for personalized treatment guidance in bladder cancer. The establishment of a risk-scoring model to predict patient survival is noteworthy.
Weaknesses:
However, the identification of ICD-related genes is somewhat conventional, focusing on known genes regulating cancer immune response. To enhance the significance of the risk-scoring model, it would be better if the authors could validate the model across various cancer types. The strength of evidence appears moderate, but broader applicability would strengthen the findings.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
This is a very interesting study by Kyle Spinler et al., demonstrating the novel role of MSI2-HOXA9 translocation in the development and pathogenesis of blast crisis CML. The authors employed appropriate in vitro and in vivo assays, including a sophisticated transplantation-based model of CML, which is well-established in the field of studying the pathogenesis of CML. Additionally, the authors successfully concluded that the MSI2 RNA binding domain RRM1 has a preferential impact on the growth of blast crisis CML.
The quality of this research article could be significantly enhanced by addressing the following points:
Major:
(1) Do mice with BCR-ABL/MSI2-HOXA9 leukemia have an increased pool of leukemic stem cells (LSC), or do they have an increased propensity to develop blast cells? Is it the number of LSCs that has increased, or is it the function of LSC to give rise to the disease that has increased? It is not clear if the detected differences in Lineage-negative cells (Figure S1D) were detected in vitro in retrovirally transduced cells or were detected in vivo in transplanted mice. If the differences were detected in vitro, could the author confirm the same findings in vivo? This will greatly enhance the understanding of in vivo disease pathogenesis and could directly link the aggressivity of the disease (shortened survival) with an increased stem cell-like population.
(2) The authors suggest that BCR-ABL/MSI2-HOXA9 leads to the development of blast crisis-CML. One of the main characteristics of blast crisis-CML is drug resistance. Is BCR-ABL/MSI2-HOXA9 leukemia resistant to classical CML treatment drugs?
(3) The authors have emphasized the heightened expression of Polrmt in delineating the mitochondrial phenotype of BCR-ABL/MSI2-HOXA9 leukemia cells. However, the regulatory mechanism governing the expression of Polrmt by MSI2-HOXA9 has not been clearly demonstrated by the authors. Unveiling this mechanism would constitute a novel finding and significantly elevate the quality of the research.
(4) Did the authors observe any survival differences between BCR-ABL/NUP98-HOXA9 and BCR-ABL/MSI2-HOXA9?
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this manuscript, the authors investigate the contributions of the long noncoding RNA snhg3 in liver metabolism and MAFLD. The authors conclude that liver-specific loss or overexpression of Snhg3 impacts hepatic lipid content and obesity through epigenetic mechanisms. More specifically, the authors invoke that the nuclear activity of Snhg3 aggravates hepatic steatosis by altering the balance of activating and repressive chromatin marks at the Pparg gene locus. This regulatory circuit is dependent on a transcriptional regulator SNG1.
Strengths:
The authors developed a tissue-specific lncRNA knockout and KI models. This effort is certainly appreciated as few lncRNA knockouts have been generated in the context of metabolism. Furthermore, lncRNA effects can be compensated in a whole organism or show subtle effects in acute versus chronic perturbation, rendering the focus on in vivo function important and highly relevant. In addition, Snhg3 was identified through a screening strategy and as a general rule the authors the authors attempt to follow unbiased approaches to decipher the mechanisms of Snhg3.
Weaknesses:
Despite efforts at generating a liver-specific knockout, the phenotypic characterization is not focused on the key readouts. Notably missing are rigorous lipid flux studies and targeted gene expression/protein measurement that would underpin why the loss of Snhg3 protects from lipid accumulation. Along those lines, claims linking the Snhg3 to MAFLD would be better supported with careful interrogation of markers of fibrosis and advanced liver disease. In other areas, significance is limited since the presented data is either not clear or rigorous enough. Finally, there is an important conceptual limitation to the work since PPARG is not established to play a major role in the liver.
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52.53.155.43 52.53.155.43
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Reviewer #1 (Public Review):
Summary:
In this study, Djebar et al. perform a comprehensive analysis of mutant phenotypes associated with the onset and progression of scoliosis in zebrafish ciliary transition zone mutants rpgrip1l and cep290. They determine that rpgrip1l is required in foxj1a-expressing cells for normal spine development, and that scoliosis is associated with brain ventricle dilations, loss of Reissner fiber polymerization, and the loss of 'tufts' of multi-cilia surrounding the subcommissural organ (the source of Reissner substance). Informed by transcriptomic and proteomic analyses, they identify a neuroinflammatory response in rpgrip1l and cep290 mutants that is associated with astrogliosis and CNS macrophage/microglia recruitment. Furthermore, anti-inflammatory drug treatment reduced scoliosis penetrance and severity in rpgrip1l mutants. Based on their data, the authors propose a feed-forward loop between astrogliosis, induced by perturbed ventricular homeostasis, and immune cell recruitment as a novel pathogenic mechanism of scoliosis in zebrafish ciliary transition zone mutants.
Strengths:
(1) Comprehensive characterization of the causes of scoliosis in ciliary transition zone mutants rpgrip1l and cep290.
(2) Comparison of rpgrip1l mutants pre- and post-scoliosis onset allowed authors to identify specific phenotypes as being correlated with spine curvature, including brain ventricle dilations, loss of Reissner fiber, and loss of cilia in proximity to the sub-commissural organ.
(3) Elegant genetic demonstration that increased urotensin peptide levels do not account for spinal curvature in rpgrip1l mutants.
(4) The identification of astrogliosis and Annexin over-expression in glial cells surrounding diencephalic and rhombencephalic ventricles as being correlated with scoliosis onset and severe curve progression is a very interesting finding, which may ultimately inform pathogenic mechanisms driving spine curvature
Weaknesses:
(1) The fact that cilia loss/dysfunction and Reissner fiber defects cause scoliosis in zebrafish is already well established in the literature, as is the requirement for cilia in foxj1a-expressing cells.
(2) Neuroinflammation has already been identified as the underlying pathogenic mechanism in at least 2 previously published scoliosis models (zebrafish ptk7a and sspo mutants).
(3) Anti-inflammatory drugs like aspirin, NAC, and NACET have also previously been demonstrated to suppress scoliosis onset and severe curve progression in these models.
Therefore, although similar observations in rpgrip1l and cep290 mutants (as reported here) add to a growing body of literature that supports a common biological mechanism underlying spine curvature in zebrafish, the novelty of reported findings is diminished.
(4) Although authors demonstrate that astrogliosis and/or macrophage or microglia cell recruitment are correlated with scoliosis, they do not formally demonstrate that these events are sufficient to drive spine curvature. Thus, the functional consequences of astrogliosis and microglia infiltration remain uncertain.
(5) The authors do not investigate the effect of anti-inflammatory treatments on other phenotypes they have correlated with spinal curve onset (like ventricle dilation, Reissner fiber loss, and multi-cilia loss around the subcommissural organ). This would help to identify causal events in scoliosis.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Strengths:
The manuscript utilizes a previously reported misfolding-prone reporter to assess its behaviour in ER in different cell line models. They make two interesting observations:
(1) Upon prolonged incubation, the reporter accumulates in nuclear aggregates.
(2) The aggregates are cleared during mitosis. They further provide some insight into the role of chaperones and ER stressors in aggregate clearance. These observations provide a starting point for addressing the role of mitosis in aggregate clearance. Needless to say, going ahead understanding the impact of aggregate clearance on cell division will be equally important.
Weaknesses:
The study almost entirely relies on an imaging approach to address the issue of aggregate clearance. A complementary biochemical approach would be more insightful. The intriguing observations pertaining to aggregates in the nucleus and their clearance during mitosis lack mechanistic understanding. The issue pertaining to the functional relevance of aggregation clearance or its lack thereof has not been addressed. Experiments addressing these issues would be a terrific addition to this manuscript.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The manuscript by Boudjema et al. describes the cellular events underlying centriole amplification and apical migration to allow the assembly of hundreds of motile cilia in multi-ciliated cells. For this, they use cell culture models in combination with fixed and live cell imaging using antibody staining and fluorescence from endogenously tagged centriole and deuterostome markers, respectively. The work is largely descriptive and functional analyses are restricted to treatment with the microtubule depolymerizing drug nocodazole. The imaging is state-of-the-art including confocal microscopy, live imaging with optical sectioning and high optical and temporal resolution, as well as super-resolution imaging by ultra-expansion microscopy.
The study does a good job of providing a very detailed description of the dynamics of centrioles and deuterostomes that lead to centriole amplification and apical migration in multiciliated cells. This detailed view was missing in previous work. It also reveals the involvement of microtubules at multiple steps: the formation of a cloud of deuterostome precursors, the nuclear envelope tethering of newly formed centrioles, their separation, and their migration to the apical surface.
It would have been useful to expand the analysis of the role of microtubules by including analyses of the requirement for specific microtubule motors, for a better understanding and additional evidence that microtubule-based transport is involved. A weak point is that there is no visualization of microtubules together with deuterosomes and centrioles at the different steps of centriole amplification and migration, to directly address how these structures may interact with and move along microtubules.
Overall, apart from experimental aspects and since this is largely a descriptive study, the manuscript would benefit from more precise language and a better description of the complex events underlying centriole amplification and movements.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The manuscript by Majhi and colleagues describes the effects of manipulating ROS levels in somatic stem cells of the testis on overall testis architecture, signaling, and function. The conclusions made by the authors are somewhat difficult to judge as the changes to the testis cell types are mostly not apparent in the representative images shown. This is true in examining gstD1-GFP expression and in the analysis of cell types and behaviours (e.g. cell cycle) and cell signaling pathway activity. Thus, the reader is left to try and interpret the quantification of the data to justify the authors' conclusions, but it is often not clear how the quantification was accomplished. For example, it is not clear how CySC vs. GSC quantification is done when the molecular markers used do not define the surface of these cells (plasma membrane) and mark different cellular compartments (Tj is nuclear while Vasa is perinuclear or cytoplasmic). Why the changes reported in quantification are not apparent in the specific example images chosen for the figures is worrisome. I'm much more used to being able to clearly see what the authors are reporting in the images, and then using the quantification to illustrate the range of data observed and demonstrate statistical significance. For this reason, I'm very concerned about the strength and validity of the conclusions. In addition, while many different characteristics of the testis somatic and germline cells are analyzed, a general and consistent view of how ROS affects these cells is not presented. In particular, one of the principle conclusions, that ROS signaling in the CySCs affects ROS signaling in the GSCs, is not well-supported by the data presented.
Specific Comments:
In Figure 1, it is very difficult to identify where CySCs end and GSCs begin without using a cell surface marker for these different cell types. In addition, the methods for quantifying the mitochondrial distribution in GSCs vs. CySCs are very much unclear, and appear to rely on colocalization with molecular markers that are not in the same cellular compartment (Tj-nuclear vs Vasa-perinuclear and cytoplasmic), the reader has no way to determine the validity of the mitochondrial distribution. Similarly, the labeling with gstD1-GFP is also very much unclear - I see little to no GFP signal in either GSCs or CySCs in panels 1G-K. Lastly, while the expression of SOD in CySCs does increase the gstD1-GFP signal in CySCs, the effects on GSCs claimed by the authors are not apparent.
In Figure 2, while the cell composition of the niche region does appear to be different from controls when SOD1 is knocked down in the CySCs, at least in the example images shown in Figures 2A and B, how cell type is quantified in Figures 2E-G is very much unclear in the figure and methods. Are these counts of cells contacting the niche? If so, how was that defined? Or were additional regions away from the niche also counted and, if so, how were these regions defined?
In Figure 3, it is quite interesting that there is an increase in Eya+, differentiating cyst cells in SOD1 knockdown animals, and that these Eya+ cells appear closer to the niche than in controls. However, this seems at odds with the proliferation data presented in Figure 2, since Eya+ somatic cells do not normally divide at all. Are they suggesting that now differentiating cyst cells are proliferative? In addition, it is important for them to show example images of the changes in Socs36E and ptp61F expression.
Overall, the various changes in signaling are quite puzzling-while Jak/Stat signaling from the niche is reduced, hh signaling appears to be increased. Similarly, while the authors conclude that premature differentiation occurs close to the niche, EGF signaling, which occurs from germ cells to cyst cells during differentiation, is decreased. Many times these changes are contradictory, and the authors do not provide a suitable explanation to resolve these contradictions.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors set up a pipeline for automated high-through single-molecule fluorescence imaging (htSMT) in living cells and analysis of molecular dynamics.
Strengths:
htSMT reveals information on the diffusion and bound fraction of molecules, dose-response curves, relative estimates on 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 on the mechanism of action or interaction networks, which can be a starting point for more in-depth analysis. The authors describe their automated imaging and analysis procedures as well as the measures taken to assure data and analysis quality.
Weaknesses:
While elegantly showcasing the power of high-throughput measurements, htSMT relies on a sophisticated robot-based workflow and several microscopes for parallel imaging, thus limiting wide-spread application of htSMT by other scientists.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Detection of early-stage colorectal cancer is of great importance. Laboratory scientists and clinicians have reported different exosomal biomarkers to identify colorectal cancer patients. This is a proof-of-principle study of whether exosomal RNAs, and particularly predicted lncRNAs, potential biomarkers of early-stage colorectal cancer and its precancerous lesions.
Strengths:
The study provides a valuable dataset of the whole-transcriptomic profile of circulating sEVs, including miRNA, mRNA, and lncRNA. This approach adds to the understanding of sEV-RNAs' role in CRC carcinogenesis and facilitates the discovery of potential biomarkers.
The developed 60-gene t-SNE model successfully differentiated T1a stage CRC/AA from normal controls with high specificity and sensitivity, indicating the potential of sEV-RNAs as diagnostic markers for early-stage colorectal lesions.
The study combines RNA-seq, RT-qPCR, and modelling algorithms to select and validate candidate sEV-RNAs, maximising the performance of the developed RNA signature. The comparison of different algorithms and consideration of other factors enhance the robustness of the findings.
Weaknesses:
Validation in larger cohorts would be required to establish as biomarkers, and to demonstrate whether the predicted lncRNAs implicated in these biomarkers are indeed present, and whether they are robustly predictive/prognostic.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors aimed to modify the characteristics of the extracellular matrix (ECM) produced by immortalized mesenchymal stem cells (MSCs) by employing the CRISPR/Cas9 system to knock out specific genes. Initially, they established VEGF-KO cell lines, demonstrating that these cells retained chondrogenic and angiogenic properties. Additionally, lyophilized carriage tissues produced by these cells exhibited retained osteogenic properties.
Subsequently, the authors established RUNX2-KO cell lines, which exhibited reduced COLX expression during chondrogenic differentiation and notably diminished osteogenic properties in vitro. Transplantation of lyophilized carriage tissues produced by RUNX2-KO cell lines into osteochondral defects in rat knee joints resulted in the regeneration of articular cartilage tissues as well as bone tissues, a phenomenon not observed with tissues derived from parental cells. This suggests that gene-edited MSCs represent a valuable cell source for producing ECM with enhanced quality.
Strengths:
The enhanced cartilage regeneration observed with ECM derived from RUNX2-KO cells supports the authors' strategy of creating gene-edited MSCs capable of producing ECM with superior quality. Immortalized cell lines offer a limitless source of off-the-shelf material for tissue regeneration.
Weaknesses:
Most data align with anticipated outcomes, offering limited novelty to advance scientific understanding. Methodologically, the chondrogenic differentiation properties of immortalized MSCs appeared deficient, evidenced by Safranin-O staining of 3D tissues and histological findings lacking robust evidence for endochondral differentiation. This presents a critical limitation, particularly as authors propose the implantation of cartilage tissues for in vivo experiments. Instead, the bulk of data stemmed from type I collagen scaffold with factors produced by MSCs stimulated by TGFβ.
The rationale behind establishing VEGF-KO cell lines remains unclear. What specific outcomes did the authors anticipate from this modification?
Insufficient depth was given to elucidate the disparity in osteogenic properties between those observed in ectopic bone formation and those observed in transplantation into osteochondral defects. While the regeneration of articular cartilage in RUNX2-KO ECM presents intriguing results, the study lacked an exploration into underlying mechanisms, such as histological analyses at earlier time points.
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www.medrxiv.org www.medrxiv.org
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Reviewer #1 (Public Review):
The authors Wilming and colleagues set out to determine the impact of regularity of feeding per se on the efficiency of weight loss. The idea was to determine if individuals who consume 2-3 meals within individualized time frames, as opposed to those who exhibit stochastic feeding patterns throughout the circadian period, will cause weight loss.
The methods are rigorous, and the research is conducted using a two-group, single-center, randomized-controlled, single-blinded study design. The participants were aged between 18 and 65 years old, and a smartphone application was used to determine preferred feeding times, which were then used as defined feeding times for the experimental group. This adds strength to the study since restricting feeding within preferred/personalized feeding windows will improve compliance and study completion. Following a 14-day exploration phase and a 6-week intervention period in a cohort of 100 participants (inclusive of both the controls and the experimental group that completed the study), the authors conclude that when meals are restricted to 45min or less durations (MTVS of 3 or less), this leads to efficient weight loss. Surprisingly, the study excludes the impact of self-reported meal composition on the efficiency of weight loss in the experimental group. In light of this, it is important to follow up on this observation and develop rigorous study designs that will comprehensively assess the impact of changes (sustained) in dietary composition on weight loss. The study also reports interesting effects of regularity of feeding on eating behavior, which appears to be independent of weight loss. Perhaps the most important observation is that personalized interventions that cater to individual circadian needs will likely result in more significant weight loss than when interventions are mismatched with personal circadian structures. One are of concern for the study is its two-group design; however, single-group cross-over designs are tedious to develop, and an adequate 'wash-out' period may be difficult to predict. A second weakness is not considering the different biological variables and racial and ethnic diversity and how that might impact outcomes. In sum, the authors have achieved the aims of the study, which will likely help move the field forward.
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www.researchsquare.com www.researchsquare.com
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Reviewer #2 (Public Review):
The authors solved the crystal structure of CDV H-protein head domain at 3,2 A resolution to better understand the detailed mechanism of membrane fusion triggering. The structure clearly showed that the orientation of the H monomers in the homodimer was similar to that of measles virus H and different from other paramyxoviruses. The authors used the available co-crystal strictures of the closely related measles virus H structures with the SLAM and Nectin4 receptors to map the receptor binding site on CDV H. The authors also confirmed which N-linked sites were glycosylated in the CDV H protein and showed that both wildtype and vaccine strains of CDV H have the same glycosylation pattern. The authors documented that the glycans cover a vast majority of the H surface while leaving the receptor binding site exposed, which may in part explain the long-term success of measles virus and CDV vaccines. Finally, the authors used HS-AFM to visualize the real-time dynamic characteristics of CDV-H under physiological conditions. This analysis indicated that homodimers may dissociate into monomers, which has implications for the model of fusion triggering.
The structural data and analysis were thorough and well-presented. The HS-AFM data, while very exciting, needs to be further validated, perhaps by alternate approaches to further support the authors' model describing the molecular dynamics of fusion triggering.
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Annotators
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Given that KRAS inhibition approaches are a relatively new innovation and that resistance is now being observed to such therapies in patients with NSCLC, investigation of combination therapies is valuable. The manuscript furthers our understanding of combination therapy for KRAS mutant non-small cell lung cancer by providing evidence that combined inhibition of ULK1/2 (and therefore autophagy) and KRAS can inhibit KRAS-mutant lung cancer growth. The manuscript will be of interest to the lung cancer community but also to researchers in other cancer types where KRAS inhibition is relevant.
Strengths:
The manuscript combines cell line, cell line-derived xenograft, and genetically-engineered mouse model data to provide solid evidence for the proposed combination therapy.
The manuscript is well written, and experiments are broadly well performed and presented.
Weaknesses:
With 3-4 mice per group in many experiments, experimental power is a concern and some comparisons (e.g. mono vs combination therapy) seem to be underpowered to detect a difference. Both male and female mice are used in experiments which may increase variability.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this meticulously conducted study, the authors show that Drosophila epidermal cells can modulate escape responses to noxious mechanical stimuli. First, they show that activation of epidermal cells evokes many types of behaviors including escape responses. Subsequently, they demonstrate that most somatosensory neurons are activated by activation of epidermal cells, and that this activation has a prolonged effect on escape behavior. In vivo analyses indicate that epidermal cells are mechanosensitive and require stored-operated calcium channel Orai. Altogether, the authors conclude that epidermal cells are essential for nociceptive sensitivity and sensitization, serving as primary sensory noxious stimuli.
Strengths:
The manuscript is clearly written. The experiments are logical and complementary. They support the authors' main claim that epidermal cells are mechanosensitive and that epidermal mechanically evoked calcium responses require the stored-operated calcium channel Orai. Epidermal cells activate nociceptive sensory neurons as well as other somatosensory neurons in Drosophila larvae, and thereby prolong escape rolling evoked by mechanical noxious stimulation.
Weaknesses:
Core details are missing in the protocols, including the level of LED intensity used, which are necessary for other researchers to reproduce the experiments. For most experiments, the epidermal cells are activated for 60 s, which is long when considering that nocifensive rolling occurs on a timescale of milliseconds. It would be informative to know the shortest duration of epidermal cell activation that is sufficient for observing the behavioral phenotype (prolongation of escape behavior) and activation of sensory neurons.
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Reviewer #1 (Public Review):
Summary:
This study assumes but also demonstrates that auditory rhythm processing is produced by internal oscillating systems and evaluates the properties of internal oscillators across individuals. The authors designed an experiment and performed analyses that address individuals' preferred rate and flexibility, with a special focus on how much past rhythms influence subsequent trials. They find evidence for such historical dependence and show that we adapt less well to new rhythms as we age. Furthermore, the revised version of this manuscript includes evidence for detuning; i.e., a gradual reduction in accuracy as the difference between a participant's preferred rate and stimulus rate increases. Such detuning also correlates with modelled oscillator flexibility measures. Such outcomes increase our credence that an entrainment-based interpretation is indeed warranted. Regardless of mechanism though, this work contributes to our understanding of individual differences in rhythm processing.
Strengths:
The inclusion of two tasks -- a tapping and a listening task -- complement each other methodologically. By analysing both the production and tracking of rhythms, the authors emphasize the importance of the characteristics of the receiver, the external world, and their interplay. The relationship between the two tasks and components within tasks are explored using a range of analyses. The visual presentation of the results is very clear. The age-related changes in flexibility are useful and compelling. The paper includes a discussion of the study assumptions, and it contextualizes itself more explicitly as taking entrainment frameworks as a starting point. Finally, the revised versions show creative additional analyses that increase our credence in an entrainment-based interpretation versus an interpretation of timekeeper other models, increasing the theoretical relevance of this study as compared to previous work.
Weaknesses:
The authors have addressed many of the weaknesses of previous peer review rounds. One final point is that our credence in an entrainment-based interpretation of these results could further increase by not only carefully outlining what is expected under entrainment (as is now done), but to also specify more extensively what predictions emerge from a timekeeper or other model, and how these data do not bear out such predictions.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
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:
The biochemical conclusions are supported by multiple configurations of the experiments. They combine multiple approaches to study a complex problem.
Weaknesses:
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:
The manuscript by Mäkelä et al. presents compelling experimental evidence that the amount of chromosomal DNA can become limiting for the total rate of mRNA transcription and consequently protein production in the model bacterium Escherichia coli. Specifically, the authors demonstrate that upon inhibition of DNA replication the single-cell growth rate continuously decreases, in direct proportion to the concentration of active ribosomes, as measured indirectly by single-particle tracking. The decrease of ribosomal activity with filamentation, in turn, is likely caused by a decrease of the concentration of mRNAs, as suggested by an observed plateau of the total number of active RNA polymerases. These observations are compatible with the hypothesis that DNA limits the total rate of transcription and thus translation. The authors also demonstrate that the decrease of RNAp activity is independent of two candidate stress response pathways, the SOS stress response and the stringent response, as well as an anti-sigma factor previously implicated in variations of RNAp activity upon variations of nutrient sources.
Remarkably, the reduction of growth rate is observed soon after the inhibition of DNA replication, suggesting that the amount of DNA in wild-type cells is tuned to provide just as much substrate for RNA polymerase as needed to saturate most ribosomes with mRNAs. While previous studies of bacterial growth have most often focused on ribosomes and metabolic proteins, this study provides important evidence that chromosomal DNA has a previously underestimated important and potentially rate-limiting role for growth.
Strengths:
This article links the growth of single cells to the amount of DNA, the number of active ribosomes and to the number of RNA polymerases, combining quantitative experiments with theory. The correlations observed during depletion of DNA, notably in M9gluCAA medium, are compelling and point towards a limiting role of DNA for transcription and subsequently for protein production soon after reduction of the amount of DNA in the cell. The article also contains a theoretical model of transcription-translation that contains a Michaelis-Menten type dependency of transcription on DNA availability and is fit to the data. While the model fits well with the continuous reduction of relative growth rate in rich medium (M9gluCAA), the behavior in minimal media without casamino acids is a bit less clear (see comments below).
At a technical level, single-cell growth experiments and single-particle tracking experiments are well described, suggesting that different diffusive states of molecules represent different states of RNAp/ribosome activities, which reflect the reduction of growth. However, I still have a few points about the interpretation of the data and the measured fractions of active ribosomes (see below).
Apart from correlations in DNA-deplete cells, the article also investigates the role of candidate stress response pathways for reduced transcription, demonstrating that neither the SOS nor the stringent response are responsible for the reduced rate of growth. Equally, the anti-sigma factor Rsd recently described for its role in controlling RNA polymerase activity in nutrient-poor growth media, seems also not involved according to mass-spec data. While other (unknown) pathways might still be involved in reducing the number of active RNA polymerases, the proposed hypothesis of the DNA substrate itself being limiting for the total rate of transcription is appealing.
Finally, the authors confirm the reduction of growth in the distant Caulobacter crescentus, which lacks overlapping rounds of replication and could thus have shown a different dependency on DNA concentration.
Weaknesses:
There are a range of points that should be clarified or addressed, either by additional experiments/analyses or by explanations or clear disclaimers.
First, the continuous reduction of growth rate upon arrest of DNA replication initiation observed in rich growth medium (M9gluCAA) is not equally observed in poor media. Instead, the relative growth rate is immediately/quickly reduced by about 10-20% and then maintained for long times, as if the arrest of replication initiation had an immediate effect but would then not lead to saturation of the DNA substrate. In particular, the long plateau of a constant relative growth rate in M9ala is difficult to reconcile with the model fit in Fig 4S2. Is it possible that DNA is not limiting in poor media (at least not for the cell sizes studied here) while replication arrest still elicits a reduction of growth rate in a different way? Might this have something to do with the naturally much higher oscillations of DNA concentration in minimal medium?
The authors argue that DNA becomes limiting in the range of physiological cell sizes, in particular for M9glCAA (Fig. 1BC). It would be helpful to know by how much (fold-change) the DNA concentration is reduced below wild-type (or multi-N) levels at t=0 in Fig 1B and how DNA concentration decays with time or cell area, to get a sense by how many-fold DNA is essentially 'overexpressed/overprovided' in wild-type cells.
Fig. 2: The distribution of diffusion coefficients of RpsB is fit to Gaussians on the log scale. Is this based on a model or on previous work or simply an empirical fit to the data? An exact analytical model for the distribution of diffusion constants can be found in the tool anaDDA by Vink, ..., Hohlbein Biophys J 2020. Alternatively, distributions of displacements are expressed analytically in other tools (e.g., in SpotOn).
The estimated fraction of active ribosomes in wild-type cells shows a very strong reduction with decreasing growth rate (down from 75% to 30%), twice as strong as measured in bulk experiments (Dai et al Nat Microbiology 2016; decrease from 90% to 60% for the same growth rate range) and probably incompatible with measurements of growth rate, ribosome concentrations, and almost constant translation elongation rate in this regime of growth rates. Might the different diffusive fractions of RpsB not represent active/inactive ribosomes? See also the problem of quantification above. The authors should explain and compare their results to previous work.
To measure the reduction of mRNA transcripts in the cell, the authors rely on the fluorescent dye SYTO RNAselect. They argue that 70% of the dye signal represents mRNA. The argument is based on the previously observed reduction of the total signal by 70% upon treatment with rifampicin, an RNA polymerase inhibitor (Bakshi et al 2014). The idea here is presumably that mRNA should undergo rapid degradation upon rif treatment while rRNA or tRNA are stable. However, work from Hamouche et al. RNA (2021) 27:946 demonstrates that rifampicin treatment also leads to a rapid degradation of rRNA. Furthermore, the timescale of fluorescent-signal decay in the paper by Bakshi et al. (half life about 10min) is not compatible with the previously reported rapid decay of mRNA (2-4min) but rather compatible with the slower, still somewhat rapid, decay of rRNA reported by Hamouche et al.. A bulk method to measure total mRNA as in the cited Balakrishnan et al. (Science 2022) would thus be a preferred method to quantify mRNA. Alternatively, the authors could also test whether the mass contribution of total RNA remains constant, which would suggest that rRNA decay does not contribute to signal loss. However, since rRNA dominates total RNA, this measurement requires high accuracy. The authors might thus tone down their conclusions on mRNA concentration changes while still highlighting the compelling data on RNAp diffusion.
The proteomics experiments are a great addition to the single-cell studies, and the correlations between distance from ori and protein abundance is compelling. However, I was missing a different test, the authors might have already done but not put in the manuscript: If DNA is indeed limiting the initiation of transcription, genes that are already highly transcribed in non-perturbed conditions might saturate fastest upon replication inhibition, while genes rarely transcribed should have no problem to accommodate additional RNA polymerases. One might thus want to test, whether the (unperturbed) transcription initiation rate is a predictor of changes in protein composition. This is just a suggestion the authors may also ignore, but since it is an easy analysis, I chose to mention it here.
Related to the proteomics, in l. 380 the authors write that the reduced expression close to the ori might reflect a gene-dosage compensatory mechanism. I don't understand this argument. Can the authors add a sentence to explain their hypothesis?
In Fig. 1E the authors show evidence that growth rate increases with cell length/area. While this is not a main point of the paper it might be cited by others in the future. There are two possible artifacts that could influence this experiment: a) segmentation: an overestimation of the physical length of the cell based on phase-contrast images (e.g., 200 nm would cause a 10% error in the relative rate of 2 um cells, but not of longer cells). b) time-dependent changes of growth rate, e.g., due to change from liquid to solid or other perturbations. To test for the latter, one could measure growth rate as a function of time, restricting the analysis to short or long cells, or measuring growth rate for short/long cells at selected time points. For the former, I recommend comparison of phase-contrast segmentation with FM4-64-stained cell boundaries.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
As a reviewer for this manuscript, I recognize its significant contribution to understanding the immune response to saprophytic Leptospira exposure and its implications for leptospirosis prevention strategies. The study is well-conceived, addressing an innovative hypothesis with potentially high impact. However, to fully realize its contribution to the field, the manuscript would benefit greatly from a more detailed elucidation of immune mechanisms at play, including specific cytokine profiles, antigen specificity of the antibody responses, and long-term immunity. Additionally, expanding on the methodological details, such as immunophenotyping panels, qPCR normalization methods, and the rationale behind animal model choice, would enhance the manuscript's clarity and reproducibility. Implementing functional assays to characterize effector T-cell responses and possibly investigating the microbiota's role could offer novel insights into the protective immunity mechanisms. These revisions would not only bolster the current findings but also provide a more comprehensive understanding of the potential for saprophytic Leptospira exposure in leptospirosis vaccine development. Given these considerations, I believe that after substantial revisions, this manuscript could represent a valuable addition to the literature and potentially inform future research and vaccine strategy development in the field of infectious diseases.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study uses single nucleus multiomics to profile the transcriptome and chromatin accessibility of mouse XX and XY primordial germ cells (PGCs) at three time-points spanning PGC sexual differentiation and entry of XX PGCs into meiosis (embryonic days 11.5-13.5). They find that PGCs can be clustered into sub-populations at each time point, with higher heterogeneity among XX PGCs and more switch-like developmental transitions evident in XY PGCs. In addition, they identify several transcription factors that appear to regulate sex-specific pathways as well as cell-cell communication pathways that may be involved in regulating XX vs XY PGC fate transitions. The findings are important and overall rigorous. The study could be further improved by a better connection to the biological system, including the addition of experiments to validate the 'omics-based findings in vivo and putting the transcriptional heterogeneity of XX PGCs in the context of findings that meiotic entry is spatially asynchronous in the fetal ovary. Overall, this study represents an advance in germ cell regulatory biology and will be a highly used resource in the field of germ cell development.
Strengths:
(1) The multiomics data is mostly rigorously collected and carefully interpreted.
(2) The dataset is extremely valuable and helps to answer many long-standing questions in the field.
(3) In general, the conclusions are well anchored in the biology of the germ line in mammals.
Weaknesses:
(1) The nature of replicates in the data and how they are used in the analysis are not clearly presented in the main text or methods. To interpret the results, it is important to know how replicates were designed and how they were used. Two "technical" replicates are cited but it is not clear what this means.
(2) Transcriptional heterogeneity among XX PGCs is mentioned several times (e.g., lines 321-323) and is a major conclusion of the paper. It has been known for a long time that XX PGCs initiate meiosis in an anterior-to-posterior wave in the fetal ovary starting around E13.5. Some heterogeneity in the XX PGC populations could be explained by spatial position in the ovary without having to invoke novel sub-populations.
(3) There is essentially no validation of any of the conclusions. Heterogeneity in the expression of a given marker could be assessed by immunofluorescence or RNAscope.
(4) The paper sometimes suffers from a problem common to large resource papers, which is that the discussion of specific genes or pathways seems incomplete. An example here is from the analysis of the regulation of the Bnc2 locus, which seems superficial. Relatedly, although many genes and pathways are nominated for important PGC functions, there is no strong major conclusion from the paper overall.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this study, the authors advance their previous findings on the role of the SLAM-SAP signaling pathway in the development and function of multiple innate-like gamma-delta T-cell subsets. Using a high throughput single-cell proteogenomics approach, the authors uncover SAP-dependent developmental checkpoints, and the role of SAP signaling in regulating the diversion of γδ T cells into the αβ T cell developmental pathway. Finally, the authors define TRGV4/TRAV13-4(DV7)-expressing T cells as a novel, SAP-dependent Vγ4 γδT1 subset.
Strengths:
This study furthers our understanding of the importance and complexity of the SLAM-SAP signaling pathway not only in the development of innate-like γδ T cells but also in how it potentially balances the γδ/αβ T cell lineage commitment. Additionally, this study reveals the role of SAP-dependent events in the generation of γδ TCR repertoire.
The conclusions of the study are supported by well-thought-out experiments and compelling data.
Weaknesses:
No major weaknesses in the study were identified.
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www.biorxiv.org www.biorxiv.org
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Reviewer #2 (Public Review):
This work focuses on the biochemical features of the SARS-CoV-2 Nucleocapsid (N) protein, which condenses the large viral RNA genome inside the virus and also plays other roles in the infected cell. The N protein of SARS-CoV-2 and other coronaviruses is known to contain two globular RNA-binding domains, the NTD and CTD, flanked by disordered regions. The central disordered linker is particularly well understood: it contains a long SR-rich region that is extensively phosphorylated in infected cells, followed by a leucine-rich helical segment that was shown previously by these authors to promote N protein oligomerization.
In the current work, the authors analyze 5 million viral sequence variants to assess the conservation of specific amino acids and general sequence features in the major regions of the N protein. This analysis shows that disordered regions are particularly variable but that the general hydrophobic and charge character of these regions are conserved, particularly in the SR and leucine-rich regions of the central linker. The authors then construct a series of N proteins bearing the most prevalent mutations seen in the Delta and Omicron variants, and they subject these mutant proteins to a comprehensive array of biophysical analyses (temperature sensitivity, circular dichroism, oligomerization, RNA binding, and phase separation).
The results include a number of novel findings that are worthy of further exploration. Most notable are the analyses of the previously unstudied P31L mutation of the Omicron variant. The authors use ColabFold and sedimentation analysis to suggest that this mutation promotes self-association of the disordered N-terminal region and stimulates the formation of N protein condensates. Although the affinity of this interaction is low, it seems likely that this mutation enhances viral fitness by promoting N-terminal interactions. The work also addresses the impact of another unstudied mutation, D63G, that is located on the surface of the globular NTD and has no significant effect on the properties analyzed here, raising interesting questions about how this mutation enhances viral fitness. Finally, the paper ends with studies showing that another common mutant, R203K/G204R, disrupts phase separation and might thereby alter N protein function in a way that enhances viral fitness. These provocative results set the stage for in-depth analyses of these mutations in future work.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors were using an innovative technic to study the visual vigilance based on high-acuity vision, the fovea. Combining motion-capture features and visual space around the head, the authors were able to estimate the visual fixation of free-feeding pigeon at any moment. Simulating predator attacks on screens, they showed that 1) pigeons used their fovea to inspect predators cues, 2) the behavioural state (feeding or head-up) influenced the latency to use the fovea and 3) the use of the fovea decrease the latency to escape of both the individual that foveate the predators cues but also the other flock members.
Strengths:
The paper is very interesting, and combines innovative technic well adapted to study the importance of high-acuity vision for spotting a predator, but also of improving the behavioural response (escaping). The results are strong and the models used are well-adapted. This paper is a major contribution to our understanding of the use of visual adaptation in a foraging context when at risk. This is also a major contribution to the understanding of individual interaction in a flock.
Weaknesses:
I have identified only two weaknesses:
(1) The authors often mixed the methods and the results, Which reduces the readability and fluidity of the manuscript. I would recommend the authors to re-structure the manuscript.<br /> (2) In some parts, the authors stated that they reconstructed the visual field of the pigeon, which is not true. They identified the foveal positions, but not the visual fields, which involve different sectors (binocular, monocular or blind). Similarly, they sometimes mix-up the area centralis and the fovea, which are two different visual adaptations.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Plasmacytoid dendritic cells (pDCs) represent a specialized subset of dendritic cells (DCs) known for their role in producing type I interferons (IFN-I) in response to viral infections. It was believed that pDCs originated from common DC progenitors (CDP). However, recent studies by Rodrigues et al. (Nature Immunology, 2018) and Dress et al. (Nature Immunology, 2019) have challenged this perspective, proposing that pDCs predominantly develop from lymphoid progenitors expressing IL-7R and Ly6D. A minor subset of pDCs arising from CDP has also been identified as functionally distinct, exhibiting reduced IFN-I production but a strong capability to activate T-cell responses. On the other hand, clonal lineage tracing experiments, as recently reported by Feng et al. (Immunity, 2022), have demonstrated a shared origin between pDCs and conventional DCs (cDCs), suggesting a contribution of common DC precursors to the pDC lineage.
In this context, Araujo et al. investigated the heterogeneity of pDCs in terms of both development and function. Their findings revealed that approximately 20% of pDCs originate from lymphoid progenitors common to B cells. Using Mb1-Cre x Bcl11a floxed mice, the authors demonstrated that the development of this subset of pDCs, referred to as "B-pDCs," relied on the transcription factor BCL11a. Functionally, B-pDCs exhibited a diminished capacity to produce IFN-I in response to TLR9 agonists but secreted more IL-12 compared to conventional pDCs. Moreover, B-pDCs, either spontaneously or upon activation, exhibited increased expression of activation markers (CD80/CD86/MHC-II) and a heightened ability to activate T-cell responses in vitro compared to conventional pDCs. Finally, Araujo et al. characterized these B-pDCs at the transcriptomic level using bulk and single-cell RNA sequencing, revealing them as a unique subset of pDCs expressing certain B cell markers such as Mb1, as well as specific markers (Axl) associated with cells recently described as transitional DCs.
Thus, in contrast to previous findings, this study posits that a small proportion of pDCs derive from B cell-committed lymphoid progenitors, and this subset of B-pDCs exhibits distinct functional characteristics, being less specialized in IFN-I production but rather in T cell activation.
Strengths:
Previously, the same research group delineated the significance of BCL11a as a critical transcription factor in pDC development (Ippolito et al., PNAS, 2014). This study elucidates the precise stage during hematopoiesis at which BCL11a expression becomes essential for the emergence of a distinct subset of pDCs, substantiated by robust genetic evidence in vivo. Furthermore, it underscores the shared developmental origin between pDCs and B cells, reinforcing prior research in the field that suggests a lymphoid origin of pDCs. Finally, this work attributes specific functional properties to pDCs originating from these lymphoid progenitors shared with B cells, emphasizing the early imprinting of functional heterogeneity during their development.
Weaknesses:
The authors delineate a subset of pDCs dependent on the BCL11a transcription factor, originating from lymphoid progenitors, and compare it to conventional pDCs, which they suggest differentiate from common DC progenitors of myeloid origin. However, this interpretation lacks support from the authors' data. Their single-cell RNA sequencing data identifies cells corresponding to progenitors (Prog2), from which the majority of pDCs, termed conventional pDCs, likely originate. This progenitor cell population expresses Il7r, Siglech, and Ly6D, but not Csfr1. The authors describe this progenitor as resembling a "pro-pDC myeloid precursor," yet these cells align more closely with lymphoid (Il7r+) progenitors described by Rodrigues et al. (Nature Immunology, 2018) and Dress et al. (Nature Immunology, 2019). Furthermore, analysis of their Mb1 reporter mice reveals that only a fraction of common lymphoid progenitors (CLP) express YFP, giving rise to a fraction of YFP+ pDCs. However, this does not exclude the possibility that YFP- CLP could also give rise to pDCs. The authors could address this caveat by attempting to differentiate pDCs from both YFP+ and YFP- CLPs in vitro in the presence of FLT3L. Additionally, transfer experiments using these lymphoid progenitors could be conducted in vivo to assess their differentiation potential in competitive settings.
Using their Mb1-reporter mice, the authors demonstrate that YFP pDCs originating from lymphoid progenitors are functionally distinct from conventional pDCs, mostly in vitro, but their in vivo relevance remains unknown. It is crucial to investigate how Bcl11a conditional deficiency in Mb1-expressing cells affects the anti-viral immune response, for example, using the M-CoV infection model as described by Sulczewski et al. in Nature Immunology, 2023. Particularly, the authors suggest that their B-pDCs act as antigen-presenting cells involved in T-cell activation compared to conventional pDCs. However, these findings contrast with those of Rodrigues et al., who have shown that pDCs of myeloid origin are more effective than pDCs of lymphoid origin in activating T-cell responses. The authors should discuss these discrepancies in greater detail. It is also notable that B-PDCs acquire the expression of ID2 (Figure S3A), commonly a marker of conventional/myeloid DCs. The authors could analyze in more detail the acquisition of specific myeloid features (CD11c, CX3CR1) by this B-PDCs subset and discuss how the expression of ID2 may impair classical pDC features, as ID2 is a repressor of E2-2, a master regulator of pDC fate.
Finally, through the analysis of their single-cell RNA sequencing data, the authors show that the subset of B-pDCs they identified expresses Axl, confirmed at the protein level. Given this specific expression profile, the authors suggest that B-pDCs are related to a previously described subset of transitional DCs, which were reported to share a common developmental path with pDCs, (Sulczewski et al. in Nature Immunology, 2023). While intriguing, this observation requires further phenotypic and functional characterization to substantiate this claim.
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Reviewer #1 (Public Review):
Summary:
C. elegans NHL-2 is a member of the conserved TRIM-NHL RNA binding protein family, with known functions in promoting small regulatory RNA function, including the conserved let-7 family microRNAs. Since NHL-2 promotes microRNA function, the authors seek to address if this function is due to direct binding of a mRNA target shared with the miRNA pathway. They successfully solve the crystal structure of NHL-2's NHL domain and discover residues Tyr935/Arg978 are required for RNA binding in vitro. In C. elegans, they establish that Tyr935/Arg978 are required for nhl-2 to promote let-7 microRNA function. Processing body (P body) size is increased in nhl-2 (Y935A R978A) and null mutants. The microRNA Argonautes, ALG-1 and ALG-2, also show increased binding to known let-7 mRNA targets in nhl-2 null mutants. Together these data suggest a lack of mRNA turnover in the absence of functional NHL-2. NHL-2 may function with CGH-1 and IFET-1 to promote let-7 miRISC function.
Strengths:
The authors successfully solve the structure of NHL-2's NHL domain. Although unable to crystalize it bound to RNA they are able to predict residues important for RNA binding based on charge, position and comparison with other known NHL domain structures crystalized with RNA. In vitro RNA binding assays confirm that Tyr935/Arg978 are required for RNA binding in vitro.
Weaknesses:
(1) In vivo, authors use a combination of established let-7 microRNA genetics and a 3' UTR reporter assay to establish that Tyr935/Arg978 are required for nhl-2 to promote let-7 microRNA function. However, they do not demonstrate that full length NHL-2 actually binds RNA directly in vivo in the Tyr935/Arg978 mutated background. While the presented genetic evidence suggests nhl(RBlf) acts much like the nhl-2 null, it is never demonstrated that full length NHL-2(RBlf) is actually RNA binding defective/dead in vivo. Yet several times in the text this is implied or stated. For example,<br /> o page 8, section title. "RNA binding is essential for NHL-2 function in heterochronic pathway"<br /> o page 9 - line 13-14. "Together, these data indicate that the RING and NHL domains are required for the normal function of NHL-2, but that the loss of RNA-binding activity has a more pronounced phenotype, suggesting that RNA-binding is critical for NHL-2 function."<br /> o page 11, line 3-4. "Together these experiments support the conclusion that... RNA binding is essential for its function"<br /> The language should be softened (e.g., page 8: "Residues required for RNA binding in vitro are required for NHL-2 function in heterochronic path") or additional experiments should be performed to support that NHL-2(RBlf) is in fact RNA binding defective/dead, like wild-type NHL-2 vs NHL-2(RBlf) RIP-qPCR for let-7 targets.
(2) Authors report that Processing body (P body) size is dependent on nhl-2 and the Tyr935/Arg978 residues. microRNA Argonautes, ALG-1 and ALG-2, also show increased binding to known let-7 mRNA targets in nhl-2 null mutants (unfortunately requirement of Tyr935/Arg978 is not tested). However total levels of these mRNAs are unchanged. Authors propose these data together support a role for nhl-2 in promoting microRNA target turnover. Unfortunately, it is unclear how increased P body size with no observed increase of microRNA target levels are to be resolved.
(3) The authors propose a model where NHL-2, CGH-1(DDX6) and IFET-1(eIF4E-transporter/4E-T) promote microRNA mediated translational repression and possibly turnover based on nhl-2-dependent IFET-1 interaction with ALG-1, cgh-1's synthetic interaction with both nhl-2 and ifet-1 to enhance let-7-mediated alae development, and conservation of known interactions between Dead Box helicases and eiF4A, which is supplemented by ALPHAFold modelling of IFET-1. The Boag lab previously characterized ifet-1 as a translational repressor required for germline P granule formation (Sengupta 2013 J Cell Sci). The role of NHL-2 RNA binding is unclear in this model as is any more molecular evidence of direct NHL-2, CGH-1 and IFET-1 interaction.
(4) In Figure 5, adult nhl-2(ok818) worms express the mCherry when the putative NHL-2 binding sites in the lin-28 3'UTR reporter are mutated. Couldn't this be interpreted as suggesting that the observed phenotype is nhl-2 independent? The authors mention this as an "interesting" observation in text, but I find it concerning. The authors should address this issue more directly. The reporter expression data needs to be quantified.
(5) I am frankly confused at the direction the manuscript takes in the Discussion section. The role of NHL-2 RNA binding, which has been the core of the paper, is seemingly disregarded and exchanged for what is mainly speculation about protein-protein level regulation with CGH-1 and IFET-1. This is all based on only a few pieces of data that do not include any analysis using the nhl-2(RBlf): nhl-2-dependent IFET-1 interaction with ALG-1, cgh-1's synthetic interaction with both nhl-2 and ifet-1 to enhance let-7-mediated alae development, and conservation of known interactions between Dead Box helicases and eiF4A, which is supplemented by ALPHAFold modelling of IFET-1. I'd strongly suggest reworking the text to better integrate IFET-1 or skip it and refocus the Discussion around the majority of the data characterizing NHL-2 RNA binding.
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Reviewer #1 (Public Review):
Summary:
The "optorepressilator", an optically controllable genetic oscillator based on the famous E. coli 3-repressor (LacI, TetR, CI) oscillator "repressilator", was developed. An individual repressilator shows a stable oscillation of the protein levels with a relatively long period that extends a few doubling times of E. coli, but when many cells oscillate, their phases tend to desynchronize. The authors introduced an additional optically controllable promoter through a conformal change of CcaS protein and let it control how much additional CI is produced. By tightly controlling the leak from the added promoter, the authors successfully kept the original repressilator oscillation when the added promoter was not activated. In contrast, the oscillation was stopped by expressing the additional CI. Using this system, the authors showed that it is possible to synchronise the phase of the oscillation, especially when the activation happens as a short pulse at the right phase of the repressilator oscillation. The authors further show that, by changing the frequency of the short pulses, the repressilator was entrained to various ratios to the pulse period, and the author could reconstruct the so-called "Arnold tongues", the signature of entrainment of the nonlinear oscillator to externally added periodic perturbation. The behaviour is consistent with the simplified mathematical model that simulates the protein concentration using ordinary differential equations.
Strengths:
Optical control of the oscillation of the protein clock is a powerful and clean tool for studying the synthetic oscillator's response to perturbation in a well-controlled and tunable manner. The article utilizes the plate reader setup for population average measurements and the mother machine setup for single-cell measurements, and they compensate nicely to acquire necessary information.
Weaknesses:
The current paper added the optogenetically controlled perturbation to control the phase of oscillation and entrainment, but there are a few other works that add external perturbation to a collection of cells that individually oscillate to study phase shift and/or entrainment. The current paper lacks discussion about the pros and cons of the current system compared to previously analyzed systems.
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Reviewer #1 (Public Review):
The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).
The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth considering and exploring further.
After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revision. However, one piece of new data is perplexing to me. The new Figure 7 presents the results of a model analysis of the strength of the EI caused by a second fish to localize when the focal fish is chirping. From my understanding of this type of model, EOD frequency is not a parameter in the model since it evaluates the strength of the field at a given point in time. Therefore the only thing that matters is the phase relationship and strength of the EOD. Assuming that the second fish's EOD is kept constant and the phases relationship is also the same, the only difference during a chirp that could affect the result of the calculation is the potential decrease in EOD amplitude during the chirp. It is indeed logical that if the focal fish decreased its EOD amplitude the target fish's EOD becomes relatively stronger. Where things are harder to understand is why the different types of chirps (e.g. type 1 vs type 2) lead to the same increase in signal even though they are typically associated with different levels of amplitude modulations. Also, it is hard to imagine that a type 2 chirps that is barely associated with any decrease in EOD amplitude (0-10% maybe), would cause doubling of the EI strength. There might be something I don't understand but the authors should provide a lot more details on how this result is obtained and convince us that it makes sense.
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Reviewer #1 (Public Review):
Summary:
The authors present tviblindi, a computational workflow for trajectory inference from molecular data at single-cell resolution. The method is based on (i) pseudo-time inference via expecting hitting time, (ii) sampling of random walks in a directed acyclic k-NN where edges are oriented away from a cell of origin w.r.t. the involved nodes' expected hitting times, and (iii) clustering of the random walks via persistent homology. An extended use case on mass cytometry data shows that tviblindi can be used elucidate the biology of T cell development.
Strengths:
- Overall, the paper is very well written and most (but not all, see below) steps of the tviblindi algorithm are explained well.
- The T cell biology use case is convincing (at least to me: I'm not an immunologist, only a bioinformatician with a strong interest in immunology).
Weaknesses:
- The main weakness of the paper is that a systematic comparison of tviblindi against other tools for trajectory inference (there are many) is entirely missing. Even though I really like the algorithmic approach underlying tviblindi, I would therefore not recommend to our wet-lab collaborators that they should use tviblindi to analyze their data. The only validation in the manuscript is the T cell development use case. Although this use case is convincing, it does not suffice for showing that the algorithms's results are systematically trustworthy and more meaningful (at least in some dimension) than trajectories inferred with one of the many existing methods.
- The authors' explanation of the random walk clustering via persistent homology in the Results (subsection "Real-time topological interactive clustering") is not detailed enough, essentially only concept dropping. What does "sparse regions" mean here and what does it mean that "persistent homology" is used? The authors should try to better describe this step such that the reader has a chance to get an intuition how the random walk clustering actually works. This is especially important because the selection of sparse regions is done interactively. Therefore, it's crucial that the users understand how this selection affects the results. For this, the authors must manage to provide a better intuition of the maths behind clustering of random walks via persistent homology.
- To motivate their work, the authors write in the introduction that "TI methods often use multiple steps of dimensionality reduction and/or clustering, inadvertently introducing bias. The choice of hyperparameters also fixes the a priori resolution in a way that is difficult to predict." They claim that tviblindi is better than the original methods because "analysis is performed in the original high-dimensional space, avoiding artifacts of dimensionality reduction." However, in the manuscript, tviblindi is tested only on mass cytometry data which has a much lower dimensionality than scRNA-seq data for which most existing trajectory inference methods are designed. Since tviblindi works on a k-NN graph representation of the input data, it is unclear if it could be run on scRNA-seq data without prior dimensionality reduction. For this, cell-cell distances would have to be computed in the original high-dimensional space, which is problematic due to the very high dimensionality of scRNA-seq data. Of course, the authors could explicitly reduce the scope of tviblindi to data of lower dimensionality, but this would have to be stated explicitly.
- Also tviblindi has at least one hyper-parameter, the number k used to construct the k-NN graphs (there are probably more hidden in the algorithm's subroutines). I did not find a systematic evaluation of the effect of this hyper-parameter.
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Reviewer #1 (Public Review):
Summary:
Meissner-Bernard et al present a biologically constrained model of telencephalic area of adult zebrafish, a homologous area to the piriform cortex, and argue for the role of precisely balanced memory networks in olfactory processing.
This is interesting as it can add to recent evidence on the presence of functional subnetworks in multiple sensory cortices. It is also important in deviating from traditional accounts of memory systems as attractor networks. Evidence for attractor networks has been found in some systems, like in the head direction circuits in the flies. However, the presence of attractor dynamics in other modalities, like sensory systems, and their role in computation has been more contentious. This work contributes to this active line of research in experimental and computational neuroscience by suggesting that, rather than being represented in attractor networks and persistent activity, olfactory memories might be coded by balanced excitation-inhibitory subnetworks.
Strengths:
The main strength of the work is in: (1) direct link to biological parameters and measurements, (2) good controls and quantification of the results, and (3) comparison across multiple models.
(1) The authors have done a good job of gathering the current experimental information to inform a biological-constrained spiking model of the telencephalic area of adult zebrafish. The results are compared to previous experimental measurements to choose the right regimes of operation.<br /> (2) Multiple quantification metrics and controls are used to support the main conclusions and to ensure that the key parameters are controlled for - e.g. when comparing across multiple models.<br /> (3) Four specific models (random, scaled I / attractor, and two variant of specific E-I networks - tuned I and tuned E+I) are compared with different metrics, helping to pinpoint which features emerge in which model.
Weaknesses:
Major problems with the work are: (1) mechanistic explanation of the results in specific E-I networks, (2) parameter exploration, and (3) the functional significance of the specific E-I model.
(1) The main problem with the paper is a lack of mechanistic analysis of the models. The models are treated like biological entities and only tested with different assays and metrics to describe their different features (e.g. different geometry of representation in Fig. 4). Given that all the key parameters of the models are known and can be changed (unlike biological networks), it is expected to provide a more analytical account of why specific networks show the reported results. For instance, what is the key mechanism for medium amplification in specific E/I network models (Fig. 3)? How does the specific geometry of representation/manifolds (in Fig. 4) emerge in terms of excitatory-inhibitory interactions, and what are the main mechanisms/parameters? Mechanistic account and analysis of these results are missing in the current version of the paper.
(2) The second major issue with the study is a lack of systematic exploration and analysis of the parameter space. Some parameters are biologically constrained, but not all the parameters. For instance, it is not clear what the justification for the choice of synaptic time scales are (with E synaptic time constants being larger than inhibition: tau_syn_i = 10 ms, tau_syn_E = 30 ms). How would the results change if they are varying these - and other unconstrained - parameters? It is important to show how the main results, especially the manifold localisation, would change by doing a systematic exploration of the key parameters and performing some sensitivity analysis. This would also help to see how robust the results are, which parameters are more important and which parameters are less relevant, and to shed light on the key mechanisms.
(3) It is not clear what the main functional advantage of the specific E-I network model is compared to random networks. In terms of activity, they show that specific E-I networks amplify the input more than random networks (Fig. 3). But when it comes to classification, the effect seems to be very small (Fig. 5c). Description of different geometry of representation and manifold localization in specific networks compared to random networks is good, but it is more of an illustration of different activity patterns than proving a functional benefit for the network. The reader is still left with the question of what major functional benefits (in terms of computational/biological processing) should be expected from these networks, if they are to be a good model for olfactory processing and learning.<br /> One possibility for instance might be that the tasks used here are too easy to reveal the main benefits of the specific models - and more complex tasks would be needed to assess the functional enhancement (e.g. more noisy conditions or more combination of odours). It would be good to show this more clearly - or at least discuss it in relation to computation and function.
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Reviewer #1 (Public Review):
Assessment:
This important work advances our understanding of navigation and path integration in mammals by using a clever behavioral paradigm. The paper provides compelling evidence that mice are able to create and use a cognitive map to find "short cuts" in an environment, using only the location of rewards relative to the point of entry to the environment and path integration, and need not rely on visual landmarks.
Summary:
The authors have designed a novel experimental apparatus called the 'Hidden Food Maze (HFM)' and a beautiful suite of behavioral experiments using this apparatus to investigate the interplay between allothetic and idiothetic cues in navigation. The results presented provide a clear demonstration of the central claim of the paper, namely that mice only need a fixed start location and path integration to develop a cognitive map. The experiments and analyses conducted to test the main claim of the paper -- that the animals have formed a cognitive map -- are conclusive. While I think the results are quite interesting and sound, one issue that needs to be addressed is the framing of how landmarks are used (or not), as discussed below, although I believe this will be a straightforward issue for the authors to address.
Strengths:
The 90-degree rotationally symmetric design and use of 4 distal landmarks and 4 quadrants with their corresponding rotationally equivalent locations (REL) lends itself to teasing apart the influence of path integration and landmark-based navigation in a clever way. The authors use a really complete set of experiments and associated controls to show that mice can use a start location and path integration to develop a cognitive map and generate shortcut routes to new locations.
Weaknesses:
I have two comments. The second comment is perhaps major and would require rephrasing multiple sentences/paragraphs throughout the paper.
(1) The data clearly indicate that in the hidden food maze (HFM) task mice did not use external visual "cue cards" to navigate, as this is clearly shown in the errors mice make when they start trials from a different start location when trained in the static entrance condition. The absence of visual landmark-guided behavior is indeed surprising, given the previous literature showing the use of distal landmarks to navigate and neural correlates of visual landmarks in hippocampal formation. While the authors briefly mention that the mice might not be using distal landmarks because of their pretraining procedure - I think it is worth highlighting this point (about the importance of landmark stability and citing relevant papers) and elaborating on it in greater detail. It is very likely that mice do not use the distal visual landmarks in this task because the pretraining of animals leads to them not identifying them as stable landmarks. For example, if they thought that each time they were introduced to the arena, it was "through the same door", then the landmarks would appear to be in arbitrary locations compared to the last time. In the same way, we as humans wouldn't use clouds or the location of people or other animate objects as trusted navigational beacons. In addition, the animals are introduced to the environment without any extra-maze landmarks that could help them resolve this ambiguity. Previous work (and what we see in our dome experiments) has shown that in environments with 'unreliable' landmarks, place cells are not controlled by landmarks - https://www.sciencedirect.com/science/article/pii/S0028390898000537, https://pubmed.ncbi.nlm.nih.gov/7891125/. This makes it likely that the absence of these distal visual landmarks when the animal first entered the maze ensured that the animal does not 'trust' these visual features as landmarks.
(2) I don't agree with the statement that 'Exogenous cues are not required for learning the food location'. There are many cues that the animal is likely using to help reduce errors in path integration. For example, the start location of the rat could act as a landmark/exogenous cue in the sense of partially correcting path integration errors. The maze has four identical entrances (90-degree rotationally symmetric). Despite this, it is entirely plausible that the animal can correct path integration errors by identifying the correct start entrance for a given trial, and indeed the distance/bearing to the others would also help triangulate one's location. Further, the overall arena geometry could help reduce PI error. For example, with a food source learned to be "near the middle" of the arena, the animal would surely not estimate the position to be near the far wall (and an interesting follow-on experiment would be to have two different-sized, but otherwise nearly identical arenas). As the rat travels away from the start location, small path integration errors are bound to accumulate, these errors could be at least partially corrected based on entrance and distal wall locations. If this process of periodically checking the location of the entrance to correct path integration errors is done every few seconds, path integration would be aided 'exogenously' to build a cognitive map. While the original claim of the paper still stands, i.e. mice can learn the location of a hidden food size when their starting point in the environment remains constant across trials. I would advise rewording portions of the paper, including the discussion throughout the paper that states claims such as "Exogenous cues are not required for learning the food location" to account for the possibility that the start and the overall arena geometry could be used as helpful exogenous cues to correct for path integration errors.
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Reviewer #1 (Public Review):
The paper submitted by Yogesh and Keller explores the role of cholinergic input from the basal forebrain (BF) in the mouse primary visual cortex (V1). The study aims to understand the signals conveyed by BF cholinergic axons in the visual cortex, their impact on neurons in different cortical layers, and their computational significance in cortical visual processing. The authors employed two-photon calcium imaging to directly monitor cholinergic input from BF axons expressing GCaMP6 in mice running through a virtual corridor, revealing a strong correlation between BF axonal activity and locomotion. This persistent activation during locomotion suggests that BF input provides a binary locomotion state signal. To elucidate the impact of cholinergic input on cortical activity, the authors conducted optogenetic and chemogenetic manipulations, with a specific focus on L2/3 and L5 neurons. They found that cholinergic input modulates the responses of L5 neurons to visual stimuli and visuomotor mismatch, while not significantly affecting L2/3 neurons. Moreover, the study demonstrates that BF cholinergic input leads to decorrelation in the activity patterns of L2/3 and L5 neurons.
This topic has garnered significant attention in the field, drawing the interest of many researchers actively investigating the role of BF cholinergic input in cortical activity and sensory processing. The experiments and analyses were thoughtfully designed and conducted with rigorous standards, providing evidence of layer-specific differences in the impact of cholinergic input on neuronal responses to bottom-up (visual stimuli) and top-down inputs (visuomotor mismatch).
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Reviewer #1 (Public Review):
The impact of this paper is that it shows conclusively the bone defects caused by ninein depletion, albeit transient defects, which has been indirectly deduced in past studies. The paper is largely descriptive including the cytoskeletal analysis of osteoclasts thus it remains unclear how ninein reduction causes bone defects and why this defect is transient. The Discussion includes several unfounded potential mechanisms that really need to be thoroughly analyzed to gain a mechanistic understanding of the bone defects in ninein-null mice.
Other points:<br /> Data showing normal osteoblasts in ninein-null mice was qualitative and requires further in-depth analysis and quantification of osteoblast and osteocyte presence and activity in ninein del/del mice to strengthen the study.
In ninein knock-out mice, reduced TRAP+ve multinuclear cells were observed (Figure 6A and 6B). However, the magnitude of difference (about 5% decrease in multinucleated cells) is not consistent with the skeletal deformities reported in Figures 2-4, potentially suggesting the contribution of additional mechanisms.
The fusion assay in Figure 6C needs further clarification. How was the syncytia perimeter defined to measure cell surface? The x-axis suggests that there are syncytia that contain up to 160 nuclei at day 3. How were the nuclei differentially stained and quantified?
Some text needs clarification. For instance, "On days 3 and 4, we found only about half as many large syncytia in cultures from ninein-deleted mice, compared to controls, but on day 5 large syncytia lacking ninein exceeded 90% of control levels. Altogether, this suggests that fusion deficiencies are a transient phenomenon in in vitro-induced adult osteoclasts. On later days of culture, fusion efficiency started to diminish." What is the definition of "large syncytia"? Is the fusion index increase by day 5 diminished in later days? A graph of the syncytia size/ nuclei number or fusion index in the above-mentioned days will be helpful.
Assessment of resorption was qualitative in Figure 6E and since the fusion deficiencies are transient, quantification of a corresponding resorption activity is needed. This should be described in the Materials and Methods section.
Further experiments are needed to show connections between reduced centrosome clustering and reduced osteoclast formation as there is no evidence to date that suggest centrosome clustering is required for cell fusion. Multi-color live imaging and dynamic analysis can be used to determine if the ninein deficient cells show defective movement/migration/ fusion dynamics.
Quantification of the % of multinucleated osteoclasts that contain clustered and dispersed centrosomes is needed.
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Reviewer #1 (Public Review):
Summary:
Previous work in humans and non-human animals suggests that during offline periods following learning, the brain replays newly acquired information in a sequential manner. The present study uses a MEG-based decoding approach to investigate the nature of replay/reactivation during a cued recall task directly following a learning session, where human participants are trained on a new sequence of 10 visual images embedded in a graph structure. During retrieval, participants are then cued with two items from the learned sequence, and neural evidence is obtained for the simultaneous or sequential reactivation of future sequence items. The authors find evidence for both sequential and clustered (i.e., simultaneous) reactivation. Replicating previous work, low-performing participants tend to show sequential, temporally segregated reactivation of future items, whereas high-performing participants show more clustered reactivation. Adding to previous work, the authors show that an image's reactivation strength varies depending on its proximity to the retrieval cue within the graph structure.
Strengths:
As the authors point out, work on memory reactivation has largely been limited to the retrieval of single associations. Given the sequential nature of our real-life experiences, there is clearly value in extending this work to structured, sequential information. State-of-the-art decoding approaches for MEG are used to characterize the strength and timing of item reactivation. The manuscript is very well written with helpful and informative figures in the main sections. The task includes an extensive localizer with 50 repetitions per image, allowing for stable training of the decoders and the inclusion of several sanity checks demonstrating that on-screen items can be decoded with high accuracy.
Weaknesses:
Of major concern, the experiment is not optimally designed for analysis of the retrieval task phase, where only 4 min of recording time and a single presentation of each cue item are available for the analyses of sequential and non-sequential reactivation. In their revision, the authors include data from the learning blocks in their analysis. These blocks follow the same trial structure as the retrieval task, and apart from adding more data points could also reveal a possible shift from sequential to clustered reactivation as learning of the graph structure progresses. The new analyses are not entirely conclusive, maybe given the variability in the number of learning blocks that participants require to reach the criterion. In principle, they suggest that reactivation strength increases from learning (pre-rest) to final retrieval (post-rest).
On a more conceptual note, the main narrative of the manuscript implies that sequential and clustered reactivation are mutually exclusive, such that a single participant would show either one or the other type. With the analytic methods used here, however, it seems possible to observe both types of reactivation. For example, the observation that mean reactivation strength (across the entire trial, or in a given time window of interest) varies with graph distance does not exclude the possibility that this reactivation is also sequential. In fact, the approach of defining one peak time window of reactivation may bias towards simultaneous, graded reactivation. It would be helpful if the authors could clarify this conceptual point. A strong claim that the two types of reactivation are mutually exclusive would need to be substantiated by further evidence, for instance, a suitable metric contrasting "sequenceness" vs "clusteredness".
On the same point, the non-sequential reactivation analyses use a time window of peak decodability that is determined based on the average reactivation of all future items, irrespective of graph distance. In a sequential forward cascade of reactivations, it could be assumed that the reactivation of near items would peak earlier than the reactivation of far items. In the revised manuscript, the authors now show the "raw" timecourses of item decodability at different graph distances, clearly demonstrating their peak reactivation times, which show convincingly that reactivation for near and far items occurs at very similar time points. The question that remains, therefore, is whether the method of pre-selecting a time window of interest described above could exert a bias towards finding clustered reactivation.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Chang et al. provide glutamate co-expression profiles in the central noradrenergic system and test the requirement of Vglut2-based glutamatergic release in respiratory and metabolic activity under physiologically relevant gas challenges. Their experiments show that conditional deletion of Vglut2 in NA neurons does not impact steady-state breathing or metabolic activity in room air, hypercapnia, or hypoxia. Their observations challenge the importance of glutamatergic signaling from Vglut2 expressing NA neurons in normal respiratory homeostasis in conscious adult mice.
Strengths:
The comprehensive Vglut1, Vglut2, and Vglut3 co-expression profiles in the central noradrenergic system and the combined measurements of breathing and oxygen consumption are two major strengths of this study. Observations from these experiments provide previously undescribed insights into (1) expression patterns for subtypes of the vesicular glutamate transporter protein in the noradrenergic system and (2) the dispensable nature of Vglut2-dependent glutamate signaling from noradrenergic neurons to breathing responses to physiologically relevant gas challenges in adult conscious mice.
Weaknesses:
Although the cellular expression profiles for the vesicular glutamate transporters are provided, the study does not document that glutamatergic-based signaling originating from noradrenergic neurons is evident at the cellular level under normal, hypoxic, and/or hypercapnic conditions. The authors effectively recognize this issue and appropriately discuss their findings in this context.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors constructed a novel HSV-based therapeutic vaccine to cure SIV in a primate model. The novel HSV vector is deleted for ICP34.5. Evidence is given that this protein blocks HIV reactivation by interference with the NFkappaB pathway. The deleted construct supposedly would reactivate SIV from latency. The SIV genes carried by the vector ought to elicit a strong immune response. Together the HSV vector would elicit a shock and kill effect. This is tested in a primate model.
Strengths and weaknesses:
(1) Deleting ICP34.5 from the HSV construct has a very strong effect on HIV reactivation. Why is no eGFP readout given in Figure 1C as for WT HSV? The mechanism underlying increased activation by deleting ICP34.5 is only partially explored. Overexpression of ICP34.5 has a much smaller effect (reduction in reactivation) than deletion of ICP34.5 (strong activation); so the story seems incomplete.
(2) No toxicity data are given for deleting ICP34.5. How specific is the effect for HIV reactivation? An RNA seq analysis is required to show the effect on cellular genes.
(3) The primate groups are too small and the results to variable to make averages. In Figure 5, the group with ART and saline has two slow rebounders. It is not correct to average those with a single quick rebounder. Here the interpretation is NOT supported by the data.
Discussion
HSV vectors are mainly used in cancer treatment partially due to induced inflammation. Whether these are suitable to cure PLWH without major symptoms is a bit questionable to me and should at least be argued for.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The authors study age-related changes in the excitability and firing properties of sympathetic neurons, which they ascribe to age-related changes in the expression of KCNQ (Kv7, "M-type") K+ currents in rodent sympathetic neurons, whose regulation by GPCRs has been most thoroughly studied for over 40 years.
Strengths:
The strengths include the rigor of the current-clamp and voltage-clamp experiments and the lovely, crisp presentation of the data, The separation of neurons into tonic, phasic and adapting classes is also interesting, and informative. The ability to successfully isolate and dissociate peripheral ganglia from such older animals is also quite rare and commendable! There is much useful detail here.
Weaknesses:
Whereas the description of the data are very nice and useful, the manuscript does not provide much in the way of mechanistic insights. As such, the effect is more of an epi-phenomenon of unclear insight, and the authors cannot ascribe changes in signaling mechanisms, such as that of M1 mAChRs to the phenomena that is supported by data.
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www.biorxiv.org www.biorxiv.org
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Joint Public Review:
Summary:
The paper explores chemosensory behaviour in surface and cave morphs and F2 hybrids in the Mexican cave fish Astyanax mexicanus. The authors develop a new behavioural assay for the long-term imaging of individual fish in a parallel high-throughput setup. The authors first demonstrate that the different morphs show different basal exploratory swimming patterns and that these patterns are stable for individual fish. Next, the authors test the attraction of fish to various concentrations of alanine and other amino acids. They find that the cave morph is a lot more sensitive to chemicals and shows directional chemotaxis along a diffusion gradient of amino acids. Surface fish, although can detect the chemicals, do not show marked chemotaxis behaviour and have an overall lower sensitivity. These differences have been reported previously but the authors report longer-term observations on many individual fish of both morphs and their F2 hybrids. The data also indicate that the observed behaviour is a quantitative genetic trait. The approach presented will allow the mapping of genes contribution to these traits. The work will be of general interest to behavioural neuroscientists and those interested in olfactory behaviours and the individual variability in behavioural patterns.
Strengths:
The authors provide a large dataset of swimming behaviour for surface fish and cave fish and also their F2 hybrids, demonstrating large differences in chemosensory behaviour and indicating that this is a quantitative genetic trait.
One strength of the paper is the development of a new and improved setup for the behavioural imaging of individual fish for extended periods and under chemosensory stimulation. The authors show that cave fish need up to 24 h of habituation to display a behavioural pattern that is consistent and unlikely to be due to the stressed state of the animals. The setup also uses relatively large tanks that allows the build-up of chemical gradients.
With their new system, the authors could generate cleaner results without mechanical disturbances. The authors characterize multiple measurements to score the odour response behaviours and also developed a new personality analysis. Their conclusion that cave fish evolved as a specialist to sense alanine and histidine among 6 tested amino acids was well supported by their data.
Weaknesses:
Further work will be needed to pinpoint the nature of the genetic changes and neurobiological mechanisms that underlie the differences between the two forms and the large individual variation of behaviours.<br /> The authors did not measure the concentrations of alanine and other amino acids in the local cave water and surface water.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
The manuscript aims to provide insights into the mediators and mechanisms underlying tardigrade radiation tolerance. The authors start by assessing the effect of ionizing radiation (IR) on the tardigrade lab species, H. exemplaris, as well as the ability of this organism to recover from this stress - specifically they look at DNA double and single strand breaks. They go on to characterize the response of H. exemplaris and two other tardigrade species to IR at the transcriptomic level. Excitingly, the authors identify a novel gene/protein called TDR1 (tardigrade DNA damage response protein 1). They carefully assess the induction of expression/enrichment of this gene/protein using a combination of transcriptomics and biochemistry - even going so far as to use a translational inhibitor to confirm the de novo production of this protein. TDR1 binds DNA in vitro and co-localizes with DNA in tardigrades.
Reverse genetics in tardigrades is difficult, thus the authors use a heterologous system (human cells) to express TDR1 in. They find that when transiently expressed TDR1 helps improve human cell resistance to IR.
This work is a masterclass in integrative biology incorporating a holistic set of approaches spanning next-gen sequencing, organismal biology, biochemistry, and cell biology. I think the importance of the findings is suitable and honestly, I find very little to critique in their experimental approaches.
Overall, I find this to be one of the more compelling papers on tardigrade stress-tolerance I have read.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This work by Leclercq and colleagues performed metabolomics on biospecimens collected from 96 patients diagnosed with several types of alcohol use disorders (AUD). The authors discovered strong alterations in circulating glycerophospholipids, bile acids, and some gut microbe-derived metabolites in AUD patients compared to controls. An exciting part of this work is that metabolomics was also performed in frontal cortex of post-mortem brains and cerebrospinal fluid of heavy alcohol users, and some of the same metabolites were seen to be altered in the central nervous system. This is an important study that will form the basis for hypothesis generation around diet-microbe-host interactions in alcohol use disorder. The work is done in a highly rigorous manner, and the rigorously collected human samples are a clear strength of this work. Overall, many new insights may be gained by this work, and it is poised to have a high impact on the field.
Strengths:
(1) The rigorously collected patient-derived samples.
(2) There is high rigor in the metabolomics investigation.
(3) Statistical analyses are well-described and strong.
(4) An evident strength is the careful control of taking blood samples at the same time of the day to avoid alterations in meal- and circadian-related fluctuations in metabolites.
Weaknesses:
(1) Some validation in animal models of ethanol exposure compared to pair-fed controls would help strengthen causal relationships between metabolites and alterations in the CNS.
(2) The classification of "heavy alcohol users" based on autopsy reports may not be that accurate.
(3) The fact that most people with alcohol use disorder choose to drink over eating food, there needs to be some more discussion around how dietary intake (secondary to heavy drinking) most likely has a significant impact on the metabolome.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Arman Angaji and his team delved into the intricate world of tumor growth and evolution, utilizing a blend of computer simulations and real patient data from liver cancer.
Strengths:
Their analysis of how mutations and clones are distributed within tumors revealed an interesting finding: tumors don't just spread from their edges as previously believed. Instead, they expand both from within and the edges simultaneously, suggesting a unique growth mode. This mode naturally indicates that external forces may play a role in cancer cells dispersion within the tumor. Moreover, their research hints at an intriguing phenomenon - the high death rate of progenitor cells and extremely slow pace in growth in the initial phase of tumor expansion. Understanding this dynamic could significantly impact our comprehension of cancer development.
Weaknesses:
It's important to note, however, that this study relies on specific computer models, metrics derived from inferred clones, and a limited number of patient data. While the insights gained are promising, further investigation is essential to validate these findings. Nonetheless, this work opens up exciting avenues for comprehending the evolution of cancers.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study explores the neural control of muscle by decomposing the firing activity of constituent motor units from the grid of surface electromyography (EMG) in the Tibialis (TA) Anterior and Vastus Lateralis (VL) during isometric contractions. The study involves extensive samples of motor units across the broadest range of voluntary contraction intensities up to 80% of MVC. The authors examine the rate coding of the population of motor units, which describes the instantaneous firing rate of each motor unit as a function of muscle force. This relationship is characterized by a natural logarithm function that delineates two distinct phases: an initial phase with a steep acceleration in firing rate, particularly pronounced in low-threshold motor units, and a subsequent modest linear increase in firing rate, more significant in high-threshold motor units.
Strengths:
The study makes a significant contribution to the field of neuromuscular physiology by providing a detailed analysis of motor unit behavior during muscle contractions in a few ways.
(1) The significance lies in its comprehensive framework of motor unit activity during isometric contractions in a broad range of intensities, providing insights into the non-linear relationship between the firing rate and the muscle force. The extensive sample of motor units across the pool confirms the observation in animal studies in which the spinal motoneuron exhibits a discharge consisting of distinct phases in response to synaptic currents, under the influence of persistent inward currents. As such, it is now reasonable to state the human motor units across the pool are also under the control of gain modulation via some neuromodulatory effects in addition to synaptic inputs arising from ionotropic effects.
(2) The firing scheme across the entire motoneuron pool revealed in this study reconciles the discrepancy in firing organization under debate; i.e., whether it is 'onion skin' like or not (Heckman and Enoka 2012). The onion skin like model states that the low threshold motor units discharge higher than high threshold motor units and have been held for a long time because the firing behaviors were examined in a partial range of contraction force range due to technical limitations. This reconciliation is crucial because it is fundamental to modelling the organization of motor unit recruitment and rate coding to achieve a desired force generation to advance our understanding of motor control.
(3) The extensive data collection with a novel blind source separation algorithm on the expanded number of channels of surface EMG signal provides a robust dataset that enhances the reliability and validity of findings, setting a new standard for empirical studies in the field.
Collectively, this study fills several knowledge gaps in the field and advances our understanding of the mechanism underlying the isometric force generation.
Weaknesses:
Although the findings and claims based on them are mostly well aligned, some accounts of the methods and claims need to be clarified.
(1) The authors examine the input-output function of a motor unit by constructing models, using force as an input and discharge rate as an output. It sounds circular, or the other way around to use the muscle force as an input variable, because the muscle force is the result of motor unit discharges, not the cause that elicits the discharges. More specifically, as a result of non-linear interactions of synchronous and/or asynchronous discharges of a population of a given motoneuron pool that give rise to transient increase/maintenance in twitch force, the gross muscle force is attained. I acknowledge that it is extremely challenging experimentally to measure synaptic currents impinging upon the spinal motoneurons in human subjects and the author has an assumption that the force could be used as a proxy of synaptic currents. However, it is necessary to explicitly provide the caveats and rationale behind that. Force could be used as the input variable for modelling.
2) The authors examine the firing organizations in TA and VL in this study without explicit purposes and rationale for choosing these muscles. The lack of accounts makes it hard for the readers to interpret the data presented, particularly in terms of comparing the results from the different muscles.
(3) In the methods, the author described the manual curation process after applying the blind source separation algorithm. For the readers to understand the whole process of decomposition and to secure rigor and robustness of the analyses, it would be necessary to provide details on what exact curation is performed with what criteria.
(4) In Figure 3, the early recruited units tend to become untraceable in the higher range of contraction. This is more pronounced in the muscle VL. This limitation would ambiguate the whole firing curve along the force axis and therefore limitation and the applicability in the different muscles needs to be discussed.
(5) It is unclear how commonly the notion "the long-held belief that rate coding is similar across motor units from the same pool" is held among the community without a reference. Different firing organizations have been modelled and discussed in the seminal paper by Fuglevand et al. (1993), and as far as I understand, the debate has not converged to a specific consensus. As such, any reference would be required to support the claim the notion is widely recognized.
(6) The authors claim that the firing behavior as a function of force is well characterized by a natural logarithmic function, which consists of initial steep acceleration followed by a modest increase in firing rate. Arguably the gain modulation in firing rate could be attributed to a neuromodulatory effect on the spinal motoneuron, which has been suggested by a number of animal studies. However, the complexity of the interactions between ionotropic and neuromodulatory inputs to motoneurons may require further elucidation to fully understand the mechanisms of neural control; it is possible to consider the differential acceleration among different threshold motor units as a differential combinatory effect of ionotropic and neuromodulatory inputs, but it is not trivially determined how differentially or systematically the inputs are organized. Likewise, the authors make an account for the difference in firing rate between TA and VL in terms of different amounts or balances of excitatory and inhibitory inputs to the motoneuron pool, but again this could be explained by other factors, such as a different extent of neuromodulatory effects. To determine the complexity of the interactions, further studies will be warranted.
(7) It is unclear with the account " ... the bandwidth of muscle force is < 10Hz during isometric contraction" in the manuscript alone, and therefore, it is difficult to understand the following claim. It appears very interesting and crucial for motor unit discharge and force generation and maintenance because it would pose a question of why the discharge rate of most motor units is higher than 10Hz, despite the bandwidth being so limited, but needs to be elaborated.
(References)
Heckman, C. J. & Enoka, R. M. Motor unit. Comprehensive Physiology 2, 2629-2682 (2012).
Fuglevand, A. J., Winter, D. A. & Patla, A. E. Models of recruitment and rate coding organization in motor-unit pools. J Neurophysiol 70, 2470-2488 (1993).
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
In this valuable study, the authors found that the macrolide drug rapamycin, which is an important pharmacological tool in the clinic and the research lab, is less specific than previously thought. They provide solid functional evidence that rapamycin activates TRPM8 and develop an NMR method to measure the specific binding of a ligand to a membrane protein.
Strengths:
The authors use a variety of complementary experimental techniques in several different systems, and their results support the conclusions drawn.
Weaknesses:
Controls are not shown in all cases, and a lack of unity across the figures makes the flow of the paper disjointed. The proposed location of the rapamycin binding pocket within the membrane means that molecular docking approaches designed for soluble proteins alone do not provide solid evidence for a rapamycin binding pocket location in TRPM8, but the authors are appropriately careful in stating that the model is consistent with their functional experiments.
Impact:
This work provides still more evidence for the polymodality of TRP channels, reminding both TRP channel researchers and those who use rapamycin in other contexts that the adjective "specific" is only meaningful in the context of what else has been explicitly tested.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
Motivated by the existence of different behavioral strategies (e.g. model-based vs. model-free), and potentially different neural circuits that underlie them, Venditto et al. introduce a new approach for inferring which strategies animals are using from data. In particular, they extend the mixture of agents (MoA) framework to accommodate the possibility that the weighting among different strategies might change over time. These temporal dynamics are introduced via a hidden Markov model (HMM), i.e. with discrete state transitions. These state transition probabilities and initial state probabilities are fit simultaneously along with the MoA parameters, which include decay/learning rate and mixture weightings, using the EM algorithm. The authors test their model on data from Miller et al., 2017, 2022, arguing that this formulation leads to (1) better fits and (2) improved interpretability over their original model, which did not include the HMM portion. Lastly, they claim that certain aspects of OFC firing are modulated by the internal state as identified by the MoA-HMM.
Strengths:
The paper is very well written and easy to follow, especially for one with a significant modeling component. Furthermore, the authors do an excellent job explaining and then disentangling many threads that are often knotted together in discussions of animal behavior and RL: model-free vs. model-based choice, outcome vs. choice-focused, exploration vs. exploitation, bias, preservation. Each of these concepts is quantified by particular parameters of their models. Model recovery (Fig. 3) is mostly convincing and licenses their fits to animal behavior later (although see below). While the specific claims made about behavior and neural activity are not especially surprising (e.g. the animals begin a session, in which rare vs. common transitions are not yet known, in a more exploratory mode), the MoA-HMM framework seems broadly applicable to other tasks in the field and useful for the purpose of quantification here.
Weaknesses:
The authors sometimes seem to equivocate on to what extent they view their model as a neural (as opposed to merely behavioral) description. For example, they introduce their paper by citing work that views heterogeneity in strategy as the result of "relatively independent, separable circuits that are conceptualized as supporting distinct strategies, each potentially competing for control." The HMM, of course, also relates to internal states of the animal. Therefore, the reader might come away with the impression that the MoA-HMM is literally trying to model dynamic, competing controllers in the brain (e.g. basal ganglia vs. frontal cortex), as opposed to giving a descriptive account of their emergent behavior. If the former is really the intended interpretation, the authors should say more about how they think the weighting/arbitration mechanism between alternative strategies is implemented, and how it can be modulated over time. If not, they should make this clearer.
Second, while the authors demonstrate that model recovery recapitulates the weight dynamics and action values (Fig. 3), the actual parameters that are recovered are less precise (Fig. 3 Supplement 1). The authors should comment on how this might affect their later inferences from behavioral data. Furthermore, it would be better to quantify using the R^2 score between simulated and recovered, rather than the Pearson correlation (r), which doesn't enforce unity slope and zero intercept (i.e. the line that is plotted), and so will tend to exaggerate the strength of parameter recovery.
Finally, the authors are very aware of the difficulties associated with long-timescale (minutes) correlations with neural activity, including both satiety and electrode drift, so they do attempt to control for this using a third-order polynomial as a time regressor as well as interaction terms (Fig. 7 Supplement 1). However, on net there does not appear to be any significant difference between the permutation-corrected CPDs computed for states 2 and 3 across all neurons (Fig. 7D). This stands in contrast to the claim that "the modulation of the reward effect can also be seen between states 2 and 3 - state 2, on average, sees a higher modulation to reward that lasts significantly longer than modulation in state 3," which might be true for the neuron in Fig. 7C, but is never quantified. Thus, while I am convinced state modulation exists for model-based (MBr) outcome value (Fig. 7A-B), I'm not convinced that these more gradual shifts can be isolated by the MoA-HMM model, which is important to keep in mind for anyone looking to apply this model to their own data.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
The authors studied how hippocampal connectivity gradients across the lifespan, and how these relate to memory function and neurotransmitter distributions. They observed older age with less distinct transitions and observed an association between gradient de-differentiation and cognitive decline.
This is overall an innovative and interesting study to assess gradient alterations across the lifespan and its associations to cognition.
The paper is well-written, and the methods appear sound and thoughtful. There are several strengths, including the inclusion of two independent cohorts, the use of gradient mapping and alignment techniques, and an overall sound statistical and analysis framework. There are several areas for potential improvements in the paper, and these are listed below:
(1) The reported D1 associations appear a bit post-hoc in the current work and I was unclear why the authors specifically focussed on dopamine here, as other transmitter systems are similar present at the level of the hippocampus and implicated in aging.
Moreover, the authors may be aware that multiple PET tracers are somewhat challenged in the mesiotemporal region. Is this the case for the D1 receptor as well? The hippocampus is a small and complex structure, and PET more of a low res technique so one would want to highlight and discuss the limitations of the correlations with PET maps here and/or evaluate whether the analysis adds necessary findings to the study.
From my (perhaps somewhat biased) perspective, it might be valuable to instead or in addition look at measures of hippocampal microstructure and how these relate to the functional aging effects. This could be done, if available, using data from the same subjects (eg based on quantitative MRI contrasts and/or structural MRI) and/or using contextualization findings as implemented in eg hippomaps.readthedocs.io
(2) Can the authors clarify why they did not replicate based on cohorts that are more widely used in the community and open access, such as CamCAN and/or HCP-Aging? It might connect their results with other studies if an attempt was made to also show that findings persist in either of these repositories.
(3) The authors applied TSM and related these parameters to topographic changes in the gradients. I was wondering whether and how such an approach controls for autocorrelation present in both the PET map and gradients. Could the authors clarify?
(4) The TSM approach quantifies the gradients in terms of x/y/z direction in a cartesian coordinate system. Wouldn't a shape intrinsic coordinate system in the hippocampus also be interesting, and perhaps even be more efficient to look at here (see eg DeKraker 2022 eLife or Paquola et al 2020 eLife)?
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www.researchsquare.com www.researchsquare.com
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Reviewer #1 (Public Review):
Summary:
The paper describes a program developed to identify PPI-hot spots using the free protein structure and compares it to FTMap and SPOTONE, two webservers that they consider as competitive approaches to the problem. On the positive side, I appreciate the effort in providing a new webserver that can be tested by the community but have two major concerns as follows.
(1) The comparison to the FTMap program is wrong. The authors misinterpret the article they refer to, i.e., Zerbe et al. "Relationship between hot spot residues and ligand binding hot spots in protein-protein interfaces" J. Chem. Inf. Model. 52, 2236-2244, (2012). FTMap identifies hot spots that bind small molecular ligands. The Zerbe et al. article shows that such hot spots tend to interact with hot spot residues on the partner protein in a protein-protein complex (emphasis on "partner"). Thus, the hot spots identified by FTMap are not the hot spots defined by the authors. In fact, because the Zerbe paper considers the partner protein in a complex, the results cannot be compared to the results of Chen et al. This difference is missed by the authors, and hence the comparison of the FTMap is invalid. I did not investigate the comparison to SPOTONE, and hence have no opinion.
(2) Chen et al. use a number of usual features in a variety of simple machine-learning methods to identify hot spot residues. This approach has been used in the literature for more than a decade. Although the authors say that they were able to find only FTMap and SPOTONE as servers, there are dozens of papers that describe such a methodology. Some examples are given here: (Higa and Tozzi, 2009; Keskin, et al., 2005; Lise, et al., 2011; Tuncbag, et al., 2009; Xia, et al., 2010). There are certainly more papers. Thus, while I consider the web server as a potentially useful contribution, the paper does not provide a fundamentally novel approach.
Higa, R.H. and Tozzi, C.L. Prediction of binding hot spot residues by using structural and evolutionary parameters. Genet Mol Biol 2009;32(3):626-633.
Keskin, O., Ma, B.Y. and Nussinov, R. Hot regions in protein-protein interactions: The organization and contribution of structurally conserved hot spot residues. J Mol Biol 2005;345(5):1281-1294.
Lise, S., et al. Predictions of Hot Spot Residues at Protein-Protein Interfaces Using Support Vector Machines. PLoS One 2011;6(2).
Tuncbag, N., Gursoy, A. and Keskin, O. Identification of computational hot spots in protein interfaces: combining solvent accessibility and inter-residue potentials improves the accuracy. Bioinformatics 2009;25(12):1513-1520.
Xia, J.F., et al. APIS: accurate prediction of hot spots in protein interfaces by combining protrusion index with solvent accessibility. BMC Bioinformatics 2010;11:174.
Strengths:<br /> A new web server was developed for detecting protein-protein interaction hot spots.
Weaknesses:<br /> The comparison to FTMap results is wrong. The method is not novel.
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Reviewer #1 (Public Review):
Summary:
Balasubramanian et al. characterized the cell types comprising mouse Schlemm's canal (SC) using bulk and single-cell RNA sequencing (scRNA-seq). The results identify expression patterns that delineate the SC inner and outer wall cells and two inner wall 'states'. Further analysis demonstrates expression patterns of glaucoma-associated genes and receptor-ligand pairs between SEC's and neighboring trabecular meshwork.
Strengths:
While mouse SC has been profiled in previous scRNA-seq studies (van Zyl et al 2020, Thomson et al 2021), these data provide higher resolution of SC cell types, particularly endothelial cell (SEC) populations. SC is an important regulator of anterior chamber outflow and has important consequences for glaucoma.
Weaknesses:
(1) Since SC has previously been characterized in mouse, human, and other species by scRNA-seq in other studies, this study would benefit from more direct comparisons to published datasets. For example, Table 4 could be expanded to list the SC cell numbers profiled in each study. Expression patterns highlighted in this study could be independently verified by plotting in publicly available mouse SC datasets. Further, a comparison to human expression patterns would assess whether type-specific expression patterns are conserved. Alternatively, an integrated analysis could be performed. Indeed, the authors mention that an integrated analysis was attempted but the data is not shown. It is unclear if this was because of a lack of agreement between datasets or other reasons.
(2) Figure 1 presents bulk RNA seq results comparing SEC, BEC, and LEC expression patterns. These populations were isolated using cell surface markers and enrichment by FACS. Since each EC population is derived from the same sample, the accuracy of this data hinges on the purity of enrichment. However, a reference is not given for this method and it is not clear how purity was validated. The authors later note that marker Emcn, which was used to identify BECs, is also expressed in SECs and LECs at lower levels. It should be demonstrated that these populations are clearly separated by flow cytometry.
(3) Bulk RNA-seq analysis infers similarity from the number of DEGs between samples, however, this is not a robust indicator. A correlation analysis should be run to verify conclusions.
(4) Figures 2-4 present three different datasets targeting the same tissue: 1) C57bl/6j scRNA-seq, 2) C57bl/6j snRNA-seq, 3) 129/sj scRNA-seq. Integrated analysis comparing datasets #1 to #2 and #3 is also presented. Integration methods are not described beyond 'normalization for cell numbers'. It is unclear if additional alignment methods were used. Integration across each of these datasets needs careful consideration, especially since different filtering methods were used (e.g. <20% mito in scRNA-seq and <5% in snRNA-seq). Improper integration could affect the ability to cluster or exaggerate differences between cell/types and states. It would be useful to demonstrate the contribution of different samples and datasets to each cell type/state to verify that these are not driven by batch effects, mouse strain, or collection platform.
(5) IW1 and IW2 are not well separated, and it is unclear if these represent truly different cell states. Figure 5b shows the staining of CCL21A and describes expression in the 'posterior portion' but in the image there are no DAPI+ nuclei in the anterior portion, suggesting the sampling in this section is different from Figure 5a. This would be improved by co-staining NPNT and CCL21A to demonstrate specificity.
(6) The substructures observed within clusters in sc/snRNA-seq data suggest that overall profiling may still not be comprehensive. This should be noted in the discussion.
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www.biorxiv.org www.biorxiv.org
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Reviewer #1 (Public Review):
Summary:
This study reports single-cell RNA sequencing results of lung adenocarcinoma, comparing 4 treatment-naive and 5 post-neoadjuvant chemotherapy tumor samples.<br /> The authors claim that there are metabolic reprogramming in tumor cells as well as stromal and immune cells after chemotherapy.<br /> The most significant findings are in the macrophages that there are more pro-tumorigenic cells after chemotherapy, i.e. CD45+CD11b+ARG+ cells. In the treatment-naive samples, more anti-tumorigenic CD45+CD11b+CD86+ macrophages are found. They sorted each population and performed functional analyses.
Strengths:
Comparison of the treatment-naive and post-chemotherapy samples of lung adenocarcinoma.
Weaknesses:
(1) Lengthy descriptive clustering analysis, with indistinct direct comparisons between the treatment-naive and the post-chemotherapy samples.<br /> (2) No statistical analysis was performed for the comparison.<br /> (3) Difficult to match data to the text.<br /> (4) ARG1 is a cytosolic enzyme that can be detected by intracellular staining after fixation. It is unclear how the staining and sorting was performed to measure function of sorted cells.
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