i need her endgame to have a house with a red door

poor small folk

?

?

When the apricots represent Oliver's deepest and most hidden fragments of identity, and Oliver "trying to learn all he could about Anchise's grafts" shows his determination in understanding his contradictory bits of himself, that don't meet his confident, tan caubois mannerisms. Furthermore, the apricots were "larger, fleshier, jucier than most apricots in the region". Indicating his understanding of his identity allowed him to mature into such a beautiful fruit.

The skin motif comes up once again, where he appreciates and obsesses over the parts of Oliver that haven't been exposed, that haven't been shown, the multi-dimensionality of Oliver (?). Specifically, he fantasises about biting into the apricot, which he compares to Oliver's ass. This connects a desire for intimacy and a selfish (?) knowledge of the other regarding the dimensions of their identity. "Apricate" is to sunbathe.

remove

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what you get swap with intro - this is a shorter, clearer description

Put under name it

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so they can keep doing the meaningful work they love

amazing testimonials

change title, this is your how, your USP, your framework. title = why this works

layout same on each half of page

swap with logo/membership intro

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all great, shorten

reword so it's not double negative

great, make it bold

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change title to pain point

hide for sales page

関数呼び出しの中　のほうが意味が通りやすいと感じました

ここまではエラーにならなかったのですが、Windows環境ではここで

UnicodeDecodeError: 'cp932' codec can't decode byte 0x99 in position 205170: illegal multibyte sequence

というエラーが出ました。

path.read_text(encoding='utf-8')

と明示すればエラーは回避できました。訳注つけますか？

原文には「新しいファイル」というニュアンスは入ってないと思います

原文

Then we can open that file and scroll back and forth through the data more easily.

代案 そのファイルを開き、上下にスクロールすることでデータをより簡単に確認できます。

ここのas well asは「～だけでなく」というよりは単純な並列として訳したほうが意味が通じそうです。

information that we're interested inも「興味深い情報」というよりは「関心がある情報」のほうがしっくりきます。

代案 いくつかの辞書と、地震のマグニチュードや位置といった関心の対象となる情報が

レビューにはこちらからＤＬしたデータを使いました

https://ehmatthes.github.io/pcc_3e/

入念に～と否定形はつながりが悪いと感じました。

代案

十分調査されていません

https://www.theguardian.com/environment/article/2024/jun/25/newly-identified-tipping-point-for-ice-sheets-could-mean-greater-sea-level-rise

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put after Let's be real

don't need these headers

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give you

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love this

excellent

love that you're describing your audience. A membership for ...

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This is so true and something I don't always think about. I have recently been more aware of oppression in my school. I always try to assume best intent, but this makes me think there is more than just intent. I keep thinking we need to do better and just being aware of unintentional oppression seems like a good place to start.

Version 3 of this preprint has been peer-reviewed and recommended by Peer Community in Evolutionary Biology.<br /> See the peer reviews and the recommendation.

这是一个

Need to reinvent the web Indeed

4 - why ? IndyWeb - flipped web

上のtopics.htmlは日本語で書かれているので、スクショも日本語にしたほうがよさそうです。

ここだけハイパーリンクになっていないようです（本にするなら関係ないかもしれませんが念のため）。

上のbase.htmlは日本語で書かれているので、スクショも日本語にしたほうがよさそうです。

原文にはここに(1)があります。

Sphinxの構文が崩れているようです。

上に載っているindex.htmlは日本語なので、このスクショも日本語にしたほうがいいと思います。

Sphinxの構文が崩れているようです（この後ろの文にも同じものがあります）。

この章はDjangoの言語設定を英語にしている前提にしているように読めましたが（Djangoのデフォルトの言語設定は英語）、もしそうならここは英語で書いたほうがいいのではないでしょうか？

この章はDjangoの言語設定を英語にしている前提にしているように読めましたが（Djangoのデフォルトの言語設定は英語）、もしそうならここは英語で書いたほうがいいのではないでしょうか？

平仮名が多くて読みにくい気がしたので、「従う」にするとよさそうです。

| steɪd |

adjective

sedate, respectable, and unadventurous: staid law firms.

| ˈblʌɪðli |

adverb

in a way that shows a casual and cheerful indifference considered to be callous or improper: they blithely ignored any evidence that did not support their theory.

a strong negative reaction by a large number of people, especially to a social or political development

(of an argument or statement) not seeming reasonable or probable; failing to convince: this is a blatantly implausible claim.

(rack something up, rack up something) accumulate or achieve something, typically a score or amount: Japan is racking up record trade surpluses with the United States.

| heɪl |

verb

1 [with object] call out to (someone) to attract attention: I hailed her in English. • signal (an approaching taxi) to stop: she raised her hand to hail a cab.

2 [with object] praise (someone or something) enthusiastically: he has been hailed as the new James Dean.

3 (hail from) [no object] have one's home or origins in (a place): they hail from Turkey.

[Atrial septal defect (ASD)] https://doctube.com/watch/know-about-atrial-septal-defect-in-babies_H7812LYzcELgqQj.html) is a significant cardiac issue that can affect newborns, potentially impacting their lives significantly. Early detection and prompt treatment are crucial in managing this condition effectively. Gain a comprehensive understanding of ASD by watching this informative video, providing insights into its causes, symptoms, and the importance of timely intervention.

De acordo com o estudo "A Study Comparing Traditional and Hybrid Internet-Based Instruction in Introductory Statistics Classes" (https://doi.org/10.1080/10691898.2003.11910722) "Performance of students in the hybrid offering equaled that of the traditional students, but students in the hybrid were slightly less positive in their subjective evaluation of the course".

Talvez uma das razões para esta avaliação menos positiva seja a necessidade/dificuldade em haver esta conexão entre as modalidades referidas aqui e o próprio processo de adaptação dos docentes a esta modalidade de ensino.

Where is collaboration and co-operation here? This is not synergistic approach.

an underlying and often distinct theme in a piece of writing or conversation: in any biography the relationship of author to subject forms a haunting subtext.

It is curious.

Something curious, is that this type is within reality, everything we live in real life is composed of fragments, and this type is very dynamic because through these fragments that can be varied you can create your own history through it, and it is easy to apply.

https://www.hersenstichting.nl/hersenaandoeningen/Korsakov/?utm_source=google&utm_medium=cpc&campaignid=16244437132&adgroupid=128626087610&adid=645102432475&gad_source=1&gclid=CjwKCAjw1emzBhB8EiwAHwZZxYB1OABiaPwFGKA1JNzm3XE178Taw4NpGn5dW8xy7znQ8hCgMJ0lhBoCp8sQAvD_BwE

Something that I consider a good way to express our thoughts more potential, and more if you are a person who overthinks, is to write, and well nowadays this becomes even easier to do and it is very different to transmit our thoughts with others.

These new ways of navigating the literary world have become curious and although it is not something new to exist, it is something new to me, something I am working to understand and keep up with how things are evolving.

временно у меня?

значит, выгорел мб

25/06 - ЛБ

???

• Introduction to re:Invent 2021:

  • Werner Vogels, CTO of Amazon.com, celebrates the 10-year anniversary of re:Invent.
  • Emphasis on the evolution and innovation in cloud computing over the past decade.

• Historical Context and AWS Lambda:

  • Introduction of AWS Lambda as an event-driven compute service eliminating the need for servers: "introduce AWS Lambda, which is an event-driven compute service for a dynamic application, and you have to run no servers, no instances, server-free back ends."
  • Reflection on the cloud's impact on innovation and resource accessibility: "Suddenly, getting access to capacity was just a click of a button."

• Evolution of AWS Services:

  • Transition from physical to programmable resources, enhancing scalability and flexibility: "all the hardware pieces... now became virtually programmable."
  • Development of specialized EC2 instances for various needs (compute, memory, storage): "Now, you want to store the optimized ones, you want compute optimized, you want to have large memory instances where you could run your SAP online."

• Technological Innovations and Nitro Hypervisor:

  • Investment in data centers and introduction of the Nitro hypervisor for enhanced performance: "The Nitro hypervisor made it possible to introduce all these new hardware platforms for you."
  • Launch of Mac EC2 M1 instances, highlighting Apple's shift to custom silicon: "Today, I'm happy to announce that you will get your hands on the Mac EC2 M1 instances."

• Scaling and Utilization of EC2:

  • Significant increase in EC2 usage: "60 million launches of EC2 each day," doubling since 2019.
  • Importance of scaling both up and down to optimize resource use and energy consumption.

• AWS Global Footprint and Latency Reduction:

  • Expansion to 25 regions and 81 Availability Zones, with plans for more: "there are the nine more regions planned that we will bring online in the coming two years."
  • Development of Local Zones and AWS Wavelength for low-latency applications.

• Advances in Networking and AWS Cloud WAN:

  • Introduction of AWS Cloud WAN for building and managing global networks: "AWS Cloud WAN, which gives you the ability to build, manage, monitor global-private five wide area networks using AWS."
  • Shift from EC2-Classic to VPC as the default network model.

• Edge Computing and IoT:

  • FreeRTOS and AWS IoT Core for managing billions of devices: "for that, we give you FreeRTOS as a stable base as an operating system for these devices."
  • Introduction of AWS Panorama Appliance for video stream analysis in various environments.

• AWS in Space:

  • AWS Ground Station enabling space data processing and innovation: "Ground Station, data comes off to the Ground Station, you basically just went a 10-hour time to get your data of your satellites and move into AWS."
  • Case study of the Mohammed Bin Rashid Space Center using AWS for Mars exploration data.

• Development Tools and Services:

  • Launch of AWS Amplify Studio for low-code development: "AWS Amplify Studio, which is a completely visual environment to build feature rich apps in hours and weeks."
  • Introduction of new SDKs for Swift, Kotlin, and Rust, expanding developer tools.

• Customer Success and Best Practices:

  • Liberty Mutual's journey using AWS CDK for rapid, compliant development: "delivering industry leading capabilities like our unstructured data ingestion pipeline... all built using well architected CDK patterns."
  • Emphasis on reusable constructs and Well-Architected Framework to optimize development processes.

• Sustainability and Energy Efficiency:

  • AWS's commitment to reducing carbon footprints through efficient cloud solutions: "moving on premises workloads to AWS can lower your carbon footprint by 88%."
  • Introduction of the AWS Carbon Footprint Tool and new Sustainability Pillar in the Well-Architected Framework.

• Gaming and Distributed Systems:

  • Overview of New Worlds, an MMORPG game built on AWS architecture: "New Worlds is a game designed by Amazon Games... a massively online multiplayer game where players from all over the world can connect together and play together."
  • Details on the scalable, serverless architecture enabling immersive gameplay and rapid scaling: "in New Worlds, we were able to do this 30 times a second."

• Final Advice and Vision:

  • Importance of security, simplicity, and innovation in building modern applications: "protecting your customers will be forever, should be forever your number one priority."
  • Encouragement to build with AWS's broad array of services and tools, emphasizing sustainability and efficient resource use.

podendo ser até 4 anos

eLife assessment

This important work offers a thorough exploration of the molecular features of different cell types within the mouse vomeronasal organ, including the expression of chemosensory receptors, using single-cell transcriptomics. The data are thoughtfully analyzed and presented, although the evidence is incomplete and only partially supports some of the claims made by the authors.

Reviewer #1 (Public Review):

Summary:

The authors comprehensively present data from single-cell RNA sequencing and spatial transcriptomics experiments of the juvenile male and female mouse vomeronasal organ, with a particular emphasis on the neuronal populations found in this sensory tissue. The use of these two methods effectively maps the locations of relevant cell types in the vomeronasal organ at a level of depth beyond what is currently known. Targeted analysis of the neurons in the vomeronasal organ produced several important findings, notably the common co-expression of multiple vomeronasal type 1 receptors (V1Rs), vomeronasal type 2 receptors (V2Rs), and both V1R+V2Rs by individual neurons, as well as the presence of a small but noteworthy population of neurons expressing olfactory receptors (ORs) and associated signal transduction molecules. Additionally, the authors identify transcriptional patterns associated with neuronal development/maturation, producing lists of genes that can be used and/or further investigated by the field. Finally, the authors report the presence of coordinated combinatorial expression of transcription factors and axon guidance molecules associated with multiple neuronal types, providing the framework for future studies aimed at understanding how these patterns relate to the complex glomerular organization in the accessory olfactory bulb. Several of these conclusions have been reached by previous studies, partially limiting the overall impact of the current work. However, when combined, these results provide important insights into the cellular diversity in the vomeronasal organ that are likely to support multiple future studies of the vomeronasal system.

Strengths:

The comprehensive analysis of the data provides a wealth of information for future research into vomeronasal organ function. The targeted analysis of neuronal gene transcription demonstrates the co-expression of multiple receptors by individual neurons and confirms the presence of a population of OR-expressing neurons in the vomeronasal organ. Although many of these findings have been noted by others, the depth of analysis here validates and extends prior findings in an effective manner. The use of spatial transcriptomics to identify the locations of specific cell types is especially useful and produces a template for the field's continued research into the various cell types present in this complex sensory tissue. Overall, the manuscript's biggest strength is found in the richness of the data presented, which will not only support future work in the broader field of vomeronasal system function but also provide insights into others studying complex sensory tissues.

Weaknesses:

As noted above, several previous studies have identified co-expression of vomeronasal receptors by vomeronasal sensory neurons, and the expression of non-vomeronasal receptors, and this was not adequately addressed in the manuscript as presented. The inherent weaknesses of single-cell RNA sequencing studies based on the 10x Genomics platforms (need to dissociate tissues, limited depth of sequencing, etc.) are acknowledged. However, the authors document their extensive attempts to avoid making false positive conclusions through the use of software tools designed for this purpose. Because of its complexity, there are some portions of the manuscript where the data are difficult to interpret as presented, but this is a relatively minor weakness. The data resulting from the use of the Resolve Biosciences spatial transcriptomics platform are somewhat difficult to interpret, and the methods are somewhat opaque. That said, the resulting data provide useful links between transcriptional identities and cellular locations, which is not possible without the use of such tools.

Reviewer #2 (Public Review):

In their paper entitled "Molecular, Cellular, and Developmental Organization of the Mouse Vomeronasal Organ at Single Cell Resolution" Hills Jr. et al. perform single-cell transcriptomic profiling and analyze tissue distribution of a large number of transcripts in the mouse vomeronasal organ (VNO). The use of these complementary tools provides a robust approach to investigating many aspects of vomeronasal sensory neuron (VSN) biology based on transcriptomics. Harnessing the power of these techniques, the authors present the discovery of previously unidentified sensory neuron types in the mouse VNO. Furthermore, they report co-expression of chemosensory receptors from different clades on individual neurons, including the co-expression of VR and OR. Finally, they evaluated the correlation between transcription factor expression and putative surface axon guidance molecules during the development of different neuronal lineages. Based on such correlation analysis, authors further propose a putative cascade of events that could give rise to different neuronal lineages and morphological organization.

Taken together, Hills Jr. et al. present findings on (a) cell types in the VNO, (b) novel classes of sensory neurons, (c) developmental trajectories of the neuronal linage, (d) receptor expression in VSNs, (e) co-expression of chemosensory receptors, (f) a surface molecule code for individual receptor types, and (g) transcriptional regulation of receptor and axon guidance cues. Before outlining the major strengths and weaknesses of the manuscript, we need to disclose that, while we are comfortable reviewing aspects (a) to (e) of their work, we lack the expertise to provide constructive criticism on the two last points (f) and (g). Thus, we will not comment on these.

In general, interpretations/claims put forward by Hills Jr. et al. appear striking at first glance. Upon careful review of the manuscript, however, it becomes apparent that many of the groundbreaking discoveries lack compelling support. Several (not all) of the results presented in this work lack novelty, accurate interpretability, and corroboration. A recurrent theme throughout the manuscript is an incomplete, and somewhat misleading account of the current knowledge in the field. This is perhaps most apparent in the introductory paragraphs, where the authors present a biased report of previously published work, largely including only those results that do not overlap with their own findings, but ignoring results that would question the novelty of the data presented here. For example: "...In contrast, transcriptomic information of the VNO is rather limited (Ref 24,25)...". Indeed, transcriptomic information of the mouse VNO is limited. Here, however, the authors ignore recent reports of robust single-cell transcriptomic analysis from adult and juvenile mice. These papers are, in part, cited later in this manuscript (ref 88, 89, 90, 91), or are completely missing (doi.org/10.7554/eLife.77259). Regardless, previously published results on the same topics have to be included in the Introduction to put the background and novelty of the findings into perspective.

General comments on (a) cell types in the VNO

The authors performed single-cell transcriptomic analysis of a large number of cells from both adult and juvenile VNO, creating the largest dataset of its kind to date. This dataset contains a wealth of information and, once made public, could be a valuable resource to the community. However, the analysis implemented in this paper raises several questions:

Did the authors perform any cell selectivity, or any directed dissection, to obtain mainly neuronal cells? Previous studies reported a greater proportion of non-neuronal cells. For example, while Katreddi and co-workers (ref 89) found that the most populated clusters are identified as basal cells, macrophages, pericytes, and vascular smooth muscle, Hills Jr. et al. in this work did not report such types of cells. Did the authors check for the expression of marker genes listed in Ref 89 for such cell types?

The authors should report the marker genes used for cell annotation. This is important for data validation, comparison with other publicly available datasets, as well as future use of this dataset.<br /> The authors reported no differences between juvenile and adult samples, and between male and female samples. It is not clear how they evaluate statistically significant differences, which statistical test was used, or what parameters were evaluated.

"Based on our transcriptomic analysis, we conclude that neurogenic activity is restricted to the marginal zone." This conclusion is quite a strong statement, given that this study was not directed to carefully study neurogenesis distribution, and when neurogenesis in the basal zone has been proposed by other works, as stated by the authors.

General comments on (b) novel classes of sensory neurons

The authors report at least two new types of sensory neurons in the mouse VNO, a finding of huge importance that could have a substantial impact on the field of sensory physiology. However, the evidence for such new cell types is based solely on this transcriptomic dataset and, as such, is quite weak, since many crucial morphological and physiological aspects would be missing to clearly identify them as novel cell types. As stated before, many control and confirmatory experiments, and a careful evaluation of the results presented in this work must be performed to confirm such a novel and interesting discovery. The reported "novel classes of sensory neurons" in this work could represent previously undescribed types of sensory neurons, but also previously reported cells (see below) or simply possible single-cell sequencing artefacts.

The authors report the co-expression of V2R and Gnai2 transcripts based on sequencing data. That could dramatically change classical classifications of basal and apical VSNs. However, did the authors find support for this co-expression in spatial molecular imaging experiments?

Canonical OSNs: The authors report a cluster of cells expressing neuronal markers and ORs and call them canonical OSN. However, VSNs expressing ORs have already been reported in a detailed study showing their morphology and location inside the sensory epithelium (References 82, 83). Such cells are not canonical OSNs since they do not show ciliary processes, they express TRPC2 channels and do not express Golf. Are the "canonical OSNs" reported in this study and the OR-expressing VSNs (ref 82, 83) different? Which parameters, other than Gnal and Cnga2 expression, support the authors' bold claim that these are "canonical OSNs"? What is the morphology of these neurons? In addition, the mapping of these "canonical OSNs" shown in Figure 2D paints a picture of the negligible expression/role of these cells (see their prediction confidence).

Secretory VSN: The authors report another novel type of sensory neurons in the VNO and call them "secretory VSNs". Here, the authors performed an analysis of differentially expressed genes for neuronal cells (dataset 2) and found several differentially expressed genes in the sVSN cluster. However, it would be interesting to perform a gene expression analysis using the whole dataset including neuronal and non-neuronal cells. Could the authors find any marker gene that unequivocally identifies this new cell type?

When the authors evaluated the distribution of sVSN using the Molecular Cartography technique, they found expression of sVSN in both sensory and non-sensory epithelia. How do the authors explain such unexpected expression of sensory neurons in the non-sensory epithelium?

The low total genes count and low total reads count, combined with an "expression of marker genes for several cell types" could indicate low-quality beads (contamination) that were not excluded with the initial parameter setting. It looks like cells in this cluster express a bit of everything V1R, V2R, OR, secretory proteins...

General comments on (c) developmental trajectories of the neuronal linage

The authors evaluated a possible cascade of events leading to the development of different lineages of mature sensory neurons using GBCs as a starting point. They found the differential expression of several transcription factors at different stages of development. This analysis was performed correctly, and its interpretation is coherent. However, it is mysterious why the authors included only classical V1R and V2R-expressing neurons, while the novel sensory neurons, cOSN and sVSN, were not included. Furthermore, it is important to notice again the misreport of previously published works.

The authors wrote "...the transcriptomic landscape that specifies the lineages is not known...". This statement is not completely true, or at least misleading. There are still many undiscovered aspects of the transcriptomics landscape and lineage determination in VSNs. However, authors cannot ignore previously reported data showing the landscape of neuronal lineages in VSNs (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259). Expression of most of the transcription factors reported by this study (Ascl1, Sox2, Neurog1, Neurod1...) were already reported, and for some of them, their role was investigated, during early developmental stages of VSNs (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259). In summary, the authors should fully include the findings from previous works (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259), clearly state what has been already reported, what is contradictory and what is new when compared with the results from this work.

General comments on (d) receptor expression in VSNs

The authors evaluated the expression of chemosensory receptors in the VNO and correlated receptor expression with the expression of transcription factors. The analysis of such correlation showed that, while the expression of V1Rs is mainly correlated with the expression of the transcription factor Meis2, the expression of V2Rs is correlated with the combination of many transcription factors. These results are interesting, however, the co-expression of specific V2Rs with specific transcription factors does not imply a direct implication in receptor selection. Directed experiments to evaluate the VR expression dependent on a specific transcription factor must be performed.

This study reports that transcription factors, such as Pou2f1, Atf5, Egr1, or c-Fos could be associated with receptor choice in VSNs. However, no further evidence is shown to support this interaction. Based on these purely correlative data, it is rather bold to propose cascade model(s) of lineage consolidation.

General comments on (e) co-expression of chemosensory receptors

The authors use spatial molecular imaging to evaluate the co-expression of many chemosensory receptors in single VNO cells. Molecular Cartography is a powerful tool and the reported data in this work is truly interesting. The authors show some clear confirmation of previously reported V2R co-expression (Figure 5H), and new co-expression of chemosensory receptors including V1R, V2R, and Fpr (Figure 5G-K).

However, it is difficult to evaluate and interpret the results due to the lack of cell borders in spatial molecular imaging. The inclusion of cell border delimitation in the reported images (membrane-stained or computer-based) could be tremendously beneficial for the interpretation of the results.

It is surprising that the authors reported a new cell type expressing OR, however, they did not report the expression of ORs in Molecular Cartography technique. Did the authors evaluate the expression of OR using the cartography technique?

Reviewer #3 (Public Review):

This study presents a detailed examination of the molecular and cellular organization of the mouse VNO, unveiling new cell types, receptor co-expression patterns, lineage specification regulation, and potential associations between transcription factors, guidance molecules, and receptor types crucial for vomeronasal circuitry wiring specificity. The study identifies a novel type of VSN molecularly different from classic VSNs, which may serve as an accessory to other VSNs by secreting olfactory binding proteins and mucins in response to VNO activation. They also describe a previously undetected co-expression of multiple VRs in individual VSNs, providing an interesting view of the ongoing discussion on how receptor choice occurs in VSNs, either stochastic or deterministic. Finally, the study correlates the expression of axon guidance molecules associated with individual VRs, providing a putative molecular mechanism that specifies VSN axon projections and their connection with postsynaptic cells in the accessory olfactory bulb.

The conclusions of this paper are well supported by data, but some aspects of data analysis and acquisition need to be clarified and extended.

(1) The authors claim that they have identified two new classes of sensory neurons, one being a class of canonical olfactory sensory neurons (OSNs) within the VNO. This classification as canonical OSNs is based on expression data of neurons lacking the V1R or V2R markers but instead expressing ORs and signal transduction molecules, such as Gnal and Cnga2. Since OR-expressing neurons in the VNO have been previously described in many studies, it remains unclear to me why these OR-expressing cells are considered here a "new class of OSNs." Moreover, morphological features, including the presence of cilia, and functional data demonstrating the recognition of chemosignals by these neurons, are still lacking to classify these cells as OSNs akin to those present in the MOE. While these cells do express canonical markers of OSNs, they also appear to express other VSN-typical markers, such as Gnao1 and Gnai2 (Figure 2B), which are less commonly expressed by OSNs in the MOE. Therefore, it would be more precise to characterize this population as atypical VSNs that express ORs, rather than canonical OSNs.

(2) The second new class of sensory neurons identified corresponds to a group of VSNs expressing prototypical VSN markers (including V1Rs, V2Rs, and ORs), but exhibiting lower ribosomal gene expression. Clustering analysis reveals that this cell group is relatively isolated from V1R- and V2R-expressing clusters, particularly those comprising immature VSNs. The question then arises: where do these cells originate? Considering their fewer overall genes and lower total counts compared to mature VSNs, I wonder if these cells might represent regular VSNs in a later developmental stage, i.e., senescent VSNs. While the secretory cell hypothesis is compelling and supported by solid data, it could also align with a late developmental stage scenario. Further data supporting or excluding these hypotheses would aid in understanding the nature of this new cell cluster, with a comparison between juvenile and adult subjects appearing particularly relevant in this context.

(3) The authors' decision not to segregate the samples according to sex is understandable, especially considering previous bulk transcriptomic and functional studies supporting this approach. However, many of the highly expressed VR genes identified have been implicated in detecting sex-specific pheromones and triggering dimorphic behavior. It would be intriguing to investigate whether this lack of sex differences in VR expression persists at the single-cell level. Regardless of the outcome, understanding the presence or absence of major dimorphic changes would hold broad interest in the chemosensory field, offering insights into the regulation of dimorphic pheromone-induced behavior. Additionally, it could provide further support for proposed mechanisms of VR receptor choice in VSNs.

(4) The expression analysis of VRs and ORs seems to have been restricted to the cell clusters associated with the neuronal lineage. Are VRs/ORs expressed in other cell types, i.e. sustentacular, HBC, or other cells?

Author response:

We would like to thank all reviewers for their time, critical evaluation, recognition, and constructive comments of the manuscript. We will revise the manuscript accordingly. Below are our point-to-point response to the comments.

From Reviewer #1:

“…several previous studies have identified co-expression of vomeronasal receptors by vomeronasal sensory neurons, and the expression of non-vomeronasal receptors, and this was not adequately addressed in the manuscript as presented.”

We plan to add context and citations to the Introduction and Results sections relating to recent studies on the co-expression of vomeronasal receptors and the expression of non-vomeronasal receptors in VSNs.

“The data resulting from the use of the Resolve Biosciences spatial transcriptomics platform are somewhat difficult to interpret, and the methods are somewhat opaque.”

Unfortunately, detailed Molecular Cartography protocols remain proprietary at Resolve Biosciences and were not disclosed. We acknowledge this limitation. Our role in the acquisition and processing of data for this experiment is included in the current Methods section. We will clarify this in the revised manuscript. Additional figures produced by the Molecular Cartography analysis will also be added (See response to Reviewer #2, below) to the supplemental materials to help clarify interpretation of the results.

From Reviewer #2:

“…the authors present a biased report of previously published work, largely including only those results that do not overlap with their own findings, but ignoring results that would question the novelty of the data presented here.”

We had no intention of misleading the readers. In fact, we have discussed discrepancies between our results with other studies. However, we inadvertently left out a critical publication in preparing the manuscript. We plan to add context and citations (where missing) relating to recent studies that use single cell RNA sequencing in the vomeronasal organ, studies relating to the co-expression of vomeronasal receptors, and studies discussing V1R/V2R lineage determination.

“Did the authors perform any cell selectivity, or any directed dissection, to obtain mainly neuronal cells? Previous studies reported a greater proportion of non-neuronal cells. For example, while Katreddi and co-workers (ref 89) found that the most populated clusters are identified as basal cells, macrophages, pericytes, and vascular smooth muscle, Hills Jr. et al. in this work did not report such types of cells. Did the authors check for the expression of marker genes listed in Ref 89 for such cell types?”

For VNO dissections, we removed bones and blood vessels from VNO tissue and only kept the sensory epithelium. This procedure removed vascular smooth muscle cells, pericytes, and other non-neuronal cell types, which explains differences in cell proportions between out study and previous studies. We used a DAPI/Draq5 assay to sort live/nucleated cells for sequencing and no specific markers were used for cell selection. All cells in the experiment were successfully annotated using the cell-type markers shown in Fig. 1B, save for cells from the sVSN cluster, which were novel, and required further analysis to characterize.

“The authors should report the marker genes used for cell annotation.”

Marker genes used for cell annotation are shown in figure 1B. A full list of all marker genes used in the cell annotation process will be provided.

“The authors reported no differences between juvenile and adult samples, and between male and female samples. It is not clear how they evaluate statistically significant differences, which statistical test was used, or what parameters were evaluated.”

The claims made about male/female mice and P14/P56 mice directly pertain to the distribution of clusters and cells in UMAP space as seen in Figure 1 C & D. We have indeed performed differential gene expression analysis for male/female and P14/P56 comparisons using the FindMarkers function from the Seurat R package. Although we have found significant differential expression between male and female, and between P14 and P56 animals, the genes in this list do not appear to be influential for the neuronal lineage and cell type specification or related to cell adhesion molecules, which are the main focuses of this study. Nevertheless, we plan to add these results to the supplemental materials in a revised manuscript.

“‘Based on our transcriptomic analysis, we conclude that neurogenic activity is restricted to the marginal zone.’ This conclusion is quite a strong statement, given that this study was not directed to carefully study neurogenesis distribution, and when neurogenesis in the basal zone has been proposed by other works, as stated by the authors.”

Eighteen slides from whole VNO sections were used in Molecular Cartography analysis, while one representative slide was used to present findings. Across all slides, GBCs, INPs, and iVSNs show a pattern of proximity to the marginal zone (MZ), with GBCs presenting nearest to the MZ and iVSNs presented furthest. We believe that the full scope of our results justifies our claim that neurogenesis is restricted to the MZ. This claim is also supported by the 2021 study by Katreddi & Forni. We will provide additional figures to further support this claim.

“The authors report at least two new types of sensory neurons in the mouse VNO, a finding of huge importance that could have a substantial impact on the field of sensory physiology. However, the evidence for such new cell types is based solely on this transcriptomic dataset and, as such, is quite weak, since many crucial morphological and physiological aspects would be missing to clearly identify them as novel cell types. As stated before, many control and confirmatory experiments, and a careful evaluation of the results presented in this work must be performed to confirm such a novel and interesting discovery. The reported "novel classes of sensory neurons" in this work could represent previously undescribed types of sensory neurons, but also previously reported cells (see below) or simply possible single-cell sequencing artefacts.”

The reviewer is correct that detailed morphological and physiological studies are needed to further understand these cells. This is an opinion we share. Our paper is primarily intended as a resource paper to provide access to a large-scale single-cell RNA-sequenced dataset and discoveries based on the transcriptomic data that can support and inspire ongoing and future experiments in the field. Nonetheless, we are confident that neither of the novel cell clusters are the result of sequencing artefacts. We performed a robust quality-control protocol, including count correction for ambient RNA with the R package, SoupX, multiplet cell detection and removal with the Python module, Scrublet, and a strict 5% mitochondrial gene expression cut-off. Furthermore, the cell clusters in question show no signs of being the result of sequencing artefacts, as they are physically connected in a reasonable orientation to the rest of the neuronal lineage in modular clusters in 2D and 3D UMAP space. The OSN and sVSN (S1H) cell clusters each show distinct and self-consistent expressions of genes. Gene ontology (GO) analysis reveals significant GO term enrichment for both the sVSN (Fig. 2G) and mOSN clusters when compared to mature V1R and V2R VSNs, indicating functional differences. Additional figures for mOSN differential gene expression and gene ontology analysis results will be added to the supplemental figures.

“The authors report the co-expression of V2R and Gnai2 transcripts based on sequencing data. That could dramatically change classical classifications of basal and apical VSNs. However, did the authors find support for this co-expression in spatial molecular imaging experiments?” 

Genes with extremely high expression levels overwhelm signals from other genes, and therefore had to be removed from the experiment. This is a limitation of the Molecular Cartography platform. Unfortunately, Gnai2 was determined to be one of these genes and was not evaluated for this purpose.

“Canonical OSNs: The authors report a cluster of cells expressing neuronal markers and ORs and call them canonical OSN. However, VSNs expressing ORs have already been reported in a detailed study showing their morphology and location inside the sensory epithelium (References 82, 83). Such cells are not canonical OSNs since they do not show ciliary processes, they express TRPC2 channels and do not express Golf. Are the "canonical OSNs" reported in this study and the OR-expressing VSNs (ref 82, 83) different? Which parameters, other than Gnal and Cnga2 expression, support the authors' bold claim that these are "canonical OSNs"? What is the morphology of these neurons? In addition, the mapping of these "canonical OSNs" shown in Figure 2D paints a picture of the negligible expression/role of these cells (see their prediction confidence).” 

We observe OR expression in VSNs in our data; these cells cluster with VSNs. The putative mOSN cluster exhibits its own trajectory, distinct from VSN clusters. These cells express Gnal (Golf), which is not expressed in VSNs expressing ORs, nor in any other cell-type in the data. After performing differential gene expression on the putative mOSN cluster, comparing with V1R and V2R VSNs, independently, GO analysis returned the top significantly enriched GO molecular function, ‘olfactory receptor activity’, and the top significantly enriched cellular component, ‘cilium’. Because we were limited to list of 100 genes in Molecular Cartography probe panel, we have prioritized the detection of canonical VNO cell-types, vomeronasal receptor co-expression, and the putative sVSNs, and were not able to include a robust analysis of the putative OSNs.

“Secretory VSN: The authors report another novel type of sensory neurons in the VNO and call them "secretory VSNs". Here, the authors performed an analysis of differentially expressed genes for neuronal cells (dataset 2) and found several differentially expressed genes in the sVSN cluster. However, it would be interesting to perform a gene expression analysis using the whole dataset including neuronal and non-neuronal cells. Could the authors find any marker gene that unequivocally identifies this new cell type?”

We did not find unequivocal marker genes for sVSNs. We did perform differential analysis of the sVSN cluster with whole VNO data and with the neuronal subset, as well as against specific cell-types. We could not find a single gene that was perfectly exclusive to sVSNs. We used a combinatorial marker-gene approach to predicting sVSNs in the Molecular Cartography data. This required a larger subset of our 100 gene panel to be dedicated to genes for detecting sVSNs.

“When the authors evaluated the distribution of sVSN using the Molecular Cartography technique, they found expression of sVSN in both sensory and non-sensory epithelia. How do the authors explain such unexpected expression of sensory neurons in the non-sensory epithelium?” 

In our scRNA-Seq experiment, blood vessels were removed, limiting the power to distinguish between certain cell types. Because of the limited number of genes that we can probe using Molecular Cartography, the number of genes associated with sVSNs may be present in the non-sensory epithelium. This could lead to the identification of cells that may or may not be identical to the sVSNs in the non-neuronal epithelium. Indeed, further studies will need to be conducted to determine the specificity of these cells.

“The low total genes count and low total reads count, combined with an "expression of marker genes for several cell types" could indicate low-quality beads (contamination) that were not excluded with the initial parameter setting. It looks like cells in this cluster express a bit of everything V1R, V2R, OR, secretory proteins...”

We are confident that the putative sVSN cell cluster is not the result of low-quality cells. We performed a robust quality-control protocol, including count correction for ambient RNA with the R package, SoupX, multiplet cell detection and removal with the Python module, Scrublet, and a strict 5% mitochondrial gene expression cut-off. Furthermore, the cell clusters in question show no signs of being the result of sequencing artefacts, as they are connected in a reasonable orientation to the rest of the neuronal lineage in modular clusters in 2D and 3D UMAP space. The OSN and sVSN cell clusters each show distinct and self-consistent expressions of genes (Fig. S1H). Gene ontology (GO) analysis reveals significant GO term enrichment for both the sVSN (Fig. 2G) and mOSN clusters when compared to mature V1R and V2R VSNs, indicating functional differences. Moreover, while some genes were expressed at a lower level when compared to the canonical VSNs, others were expressed at higher levels, precluding the cause of discrepancy as resulting from an overall loss of gene counts.

“The authors wrote ‘...the transcriptomic landscape that specifies the lineages is not known...’. This statement is not completely true, or at least misleading. There are still many undiscovered aspects of the transcriptomics landscape and lineage determination in VSNs. However, authors cannot ignore previously reported data showing the landscape of neuronal lineages in VSNs (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259). Expression of most of the transcription factors reported by this study (Ascl1, Sox2, Neurog1, Neurod1...) were already reported, and for some of them, their role was investigated, during early developmental stages of VSNs (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259). In summary, the authors should fully include the findings from previous works (Ref ref 88, 89, 90, 91 and doi.org/10.7554/eLife.77259), clearly state what has been already reported, what is contradictory and what is new when compared with the results from this work.“

This is a difference in opinion about the terminology. Transcriptomic landscape in our paper refers to the genome-wide expression by individual cells, not just individual genes. The reviewer is correct that many of the genetic specifiers have been identified, which we cited and discussed. We consider these studies as providing a “genetic” underpinning, rather than the “transcriptomic landscape” in lineage progression. We will clarify this point in the revised manuscript. 

“…the co-expression of specific V2Rs with specific transcription factors does not imply a direct implication in receptor selection. Directed experiments to evaluate the VR expression dependent on a specific transcription factor must be performed.” 

The reviewer is correct, and we did not claim that the co-expression of specific transcription factors indicate a direct relationship with receptor selection. We agree that further directed experiments are required to investigate this question.

“This study reports that transcription factors, such as Pou2f1, Atf5, Egr1, or c-Fos could be associated with receptor choice in VSNs. However, no further evidence is shown to support this interaction. Based on these purely correlative data, it is rather bold to propose cascade model(s) of lineage consolidation.”

The reviewer is correct. As any transcriptomic study will only be correlative, additional studies will be needed to unequivocally determine the mechanistic link between the transcription factors with receptor choice. Our model provides a base for these studies.

“The authors use spatial molecular imaging to evaluate the co-expression of many chemosensory receptors in single VNO cells. […] However, it is difficult to evaluate and interpret the results due to the lack of cell borders in spatial molecular imaging. The inclusion of cell border delimitation in the reported images (membrane-stained or computer-based) could be tremendously beneficial for the interpretation of the results.”

The most common practice for cell segmentation of spatial transcriptomics data is to determine cell borders based on nuclear staining with expansion. We have tested multiple algorithms based on recent studies, but each has its own caveat. We will clarify this point in the revised manuscript.

“It is surprising that the authors reported a new cell type expressing OR, however, they did not report the expression of ORs in Molecular Cartography technique. Did the authors evaluate the expression of OR using the cartography technique?” 

We were limited to a 100-gene probe panel and only included one OR, the expression was not high enough for us to substantiate any claims.

From Reviewer #3:

“(1) The authors claim that they have identified two new classes of sensory neurons, one being a class of canonical olfactory sensory neurons (OSNs) within the VNO. This classification as canonical OSNs is based on expression data of neurons lacking the V1R or V2R markers but instead expressing ORs and signal transduction molecules, such as Gnal and Cnga2. Since OR-expressing neurons in the VNO have been previously described in many studies, it remains unclear to me why these OR-expressing cells are considered here a "new class of OSNs." Moreover, morphological features, including the presence of cilia, and functional data demonstrating the recognition of chemosignals by these neurons, are still lacking to classify these cells as OSNs akin to those present in the MOE. While these cells do express canonical markers of OSNs, they also appear to express other VSN-typical markers, such as Gnao1 and Gnai2 (Figure 2B), which are less commonly expressed by OSNs in the MOE. Therefore, it would be more precise to characterize this population as atypical VSNs that express ORs, rather than canonical OSNs.”

We observe OR expression in VSNs in our data; these cells cluster with VSNs. The putative mOSN cluster exhibits its own trajectory, distinct from VSN clusters. These cells express Gnal (Golf), which is not expressed in VSNs expressing ORs, nor in any other cell-type in the data. We have performed differential gene expression analysis on the putative mOSN cluster to compare with V1R and V2R VSNs. GO analysis returned the top significantly enriched GO terms include “olfactory receptor activity” and “cilium”., further supporting that these are OSNs Because we were limited to list of 100 genes in Molecular Cartography probe panels, we have prioritized the detection of canonical VNO cell-types, vomeronasal receptor co-expression, and the putative sVSNs, and were not able to include a robust analysis of the putative OSNs. With regard to Gnai2 and Go expression, we have examined our data from the OSNs dissociated from the olfactory epithelium and detected substantial expression of both. This new analysis provides additional support for our claim. We will update the information in a revised manuscript.

“(2) The second new class of sensory neurons identified corresponds to a group of VSNs expressing prototypical VSN markers (including V1Rs, V2Rs, and ORs), but exhibiting lower ribosomal gene expression. Clustering analysis reveals that this cell group is relatively isolated from V1R- and V2R-expressing clusters, particularly those comprising immature VSNs. The question then arises: where do these cells originate? Considering their fewer overall genes and lower total counts compared to mature VSNs, I wonder if these cells might represent regular VSNs in a later developmental stage, i.e., senescent VSNs. While the secretory cell hypothesis is compelling and supported by solid data, it could also align with a late developmental stage scenario. Further data supporting or excluding these hypotheses would aid in understanding the nature of this new cell cluster, with a comparison between juvenile and adult subjects appearing particularly relevant in this context.” 

We wholeheartedly agree with this assessment. Our initial thought was that these were senescent VSNs, but the trajectory analysis did not support this scenario, leading us to propose that these are putative secretive cells. Our analysis also shows that overall, 46% of the putative sVSNs were from the P14 sample and 54% from P56. These cells comprise roughly 6.4% of all P14 cells and 8.5% of P56 cells. In comparison, 28.4% of all cells are mature V1R VSNs at P14, but the percentage rise to 46.7% at P56. The significant presence of sVSNs at P14, and the disproportionate increase when compared with mature VSNs indicate that these are unlikely to be late developmental stage or senescent cells, although we cannot exclude these possibilities. We plan to clarify these points in the revised manuscript.   

We did not include sVSNs in the trajectory inference analysis because of inherent uncertainty about their developmental origins. However, PCA embeddings were the basis of the pseudotime analysis, and those embeddings that do include the sVSN cluster show that it is distributed evenly between the mature V1R and V2R clusters, with all mature clusters equidistant from GBC and INP clusters, indicating that they may indeed originate from the same stem cell populations. We plan to include trajectory analysis based on this assumption in the revised manuscript.

(3) The authors' decision not to segregate the samples according to sex is understandable, especially considering previous bulk transcriptomic and functional studies supporting this approach. However, many of the highly expressed VR genes identified have been implicated in detecting sex-specific pheromones and triggering dimorphic behavior. It would be intriguing to investigate whether this lack of sex differences in VR expression persists at the single-cell level. Regardless of the outcome, understanding the presence or absence of major dimorphic changes would hold broad interest in the chemosensory field, offering insights into the regulation of dimorphic pheromone-induced behavior. Additionally, it could provide further support for proposed mechanisms of VR receptor choice in VSNs. 

The reviewer raised a good point. We did not observe differences between male and female, or between P14 and P56 mice in the distribution of clusters and cells in UMAP space. Indeed, our differential expression analysis has revealed significantly differentially expressed genes in both comparisons. These genes have not been implicated in lineage or cell type determination and we decided not to include the analysis in the current version. In the revised manuscript, we plan to include the results.   

“(4) The expression analysis of VRs and ORs seems to have been restricted to the cell clusters associated with the neuronal lineage. Are VRs/ORs expressed in other cell types, i.e. sustentacular, HBC, or other cells?” 

Sparsely expressed low counts of VR and OR genes were observed in non-neuronal cell-types. When their expression as a percentage of cell-level gene counts is considered, however, the expression is negligible when compared to the neurons. The observed expression may be explained by stochastic base-level expression, or it may be the result of remnant ambient RNA that passed filtering. We will clarify this point in the revision.

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Love what you have done: Bayesian Inference is a good start, and I think a good fit for this work. I think you have a good justification for its implementation and it validity to explore your data, should give you at least a good overview of the work

the simplest answer is do all of it, but I would start without any sub grouping and see if these variables pop out; then work backwards (however that is just my preference and a case can be made that its wrong)

nope I would keep them

this depends on the final manuscript and how you will write it up; short answer is I would probably exclude them to reduc confounders

I guess you have to ask why not? would this not just be interesting to see, particularly vs non transfer patients?

i think it looks good, you may want to connect weather to diagnosis (as they will be linked slightly)

I think what you have done is great

Does it? I wonder if that is true, given your past work with the flight I would have thought fine it unlikely that both match. Though in the end you know best.

more of just I would like to know, Why?

you may want to include that you reduce this to only inter-hospital flights

Is it common to have 2 transfers?

Сомневаюсь в значении и, следовательно, знаках. Кажется, что тут больше на вводное похоже.

Выгрузить с Cisco? Если да, у нас, получается, ошибка в управлении.

Anki

Résumé de la vidéo [00:00:01][^1^][1] - [00:22:42][^2^][2]:

Cette vidéo présente des méthodes efficaces pour aider les enfants à apprendre et à se mettre au travail. Elle explique l'importance de se tester et d'espacer les révisions dans le temps pour une meilleure mémorisation et rétention à long terme.

Points forts: + [00:00:30][^3^][3] Introduction au sujet * Importance d'apprendre à apprendre * Difficultés rencontrées par les enfants et les parents * Objectif de partager des conseils utiles + [00:01:36][^4^][4] Les méthodes d'apprentissage dans les programmes scolaires * Présence dans les programmes depuis 2016 * Manque de mise en pratique dans les salles de classe * Nécessité de transmettre ces compétences aux enseignants, parents et enfants + [00:03:24][^5^][5] Techniques d'apprentissage efficaces selon la recherche * Se tester et étaler les révisions dans le temps * Alternance des contenus et élaboration sur les apprentissages * Inefficacité de méthodes courantes comme surligner ou relire plusieurs fois + [00:07:44][^6^][6] Planification des révisions pour les examens * Comparaison entre apprentissage massé et distribué * Meilleure rétention à long terme avec des révisions espacées * Adaptation du calendrier de révision en fonction des objectifs d'apprentissage + [00:12:25][^7^][7] Messages clés pour les parents et les enfants * Importance de la récupération active en mémoire * Espacement des séances de révision pour un apprentissage à long terme * Utilisation de méthodes génératives et élaboratives pour renforcer la compréhension + [00:18:34][^8^][8] Aider les enfants à se mettre au travail * Mise en place d'une routine après l'école * Utilisation des récompenses plutôt que des punitions * Automatisation des comportements souhaités par la répétition et la récompense

Résumé de la vidéo [00:19:00][^1^][1] - [00:22:42][^2^][2]:

La vidéo aborde des stratégies pour aider les enfants à apprendre plus efficacement et à établir des routines pour les devoirs. Elle souligne l'importance de se tester et d'espacer les révisions dans le temps pour une meilleure mémorisation à long terme. Des méthodes comportementales pour encourager les bonnes habitudes chez les enfants sont également discutées.

Points forts: + [00:19:00][^3^][3] Établir des routines pour les devoirs * Importance de commencer les devoirs juste après le goûter * Éviter de jouer avant les devoirs pour ne pas perturber la routine * Utiliser des récompenses pour encourager la bonne routine + [00:20:00][^4^][4] Utiliser des méthodes comportementales * Les punitions sont inefficaces et n'enseignent pas les bons comportements * Se concentrer sur les comportements souhaités et les récompenser * Renforcer positivement les bonnes habitudes jusqu'à ce qu'elles deviennent automatiques + [00:21:00][^5^][5] Transition vers l'autonomie * Passer progressivement de l'accompagnement à l'autonomie dans les devoirs * Rappeler à l'enfant la routine jusqu'à ce qu'il l'adopte de lui-même * L'objectif est d'automatiser le comportement souhaité + [00:22:00][^6^][6] Ressources supplémentaires * Présentation de livres et de blogs pour approfondir les techniques d'apprentissage * Suggestions de lectures pour mieux comprendre la gestion des comportements et l'apprentissage

Не нашла чёткой информации, что это за утилита. Сомневаюсь, строчными ли это писать.

А тут все или всё? Не совсем понимаю.

У меня в конце строки.

У меня в конце строки.

eLife assessment

This important study demonstrates a novel method for imaging glutamate receptors in situ via cryo-ET. The use of cutting-edge methods is well-described and is convincing, but there are minor concerns as to how generally this approach can be used in imaging cell surface receptors. This paper is broadly relevant to biophysicists and neuroscientists.

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

None noted.

Reviewer #2 (Public Review):

Summary:

The authors present a method that allows for the identification and localization of molecular machinery at chemical synapses in unstained, unfixed native brain tissue slices. They believe that this approach will provide a 3D structural basis for understanding different mechanisms of synaptic transmission, plasticity, and development. To achieve this, the group used genetically engineered mouse lines and generated thin brain slices that underwent high-pressure freezing (HPF) and focused ion beam (FIB) milling. Utilizing cryo-electron tomography (cryo-ET) and integrating it with cryo-fluorescence microscopy, they achieved micrometer resolution in identifying the glutamatergic synapses along with nanometer resolution to locate AMPA receptors GluA2-subunits using Fab-AuNP conjugates. The findings are summarized with detailed examples of successfully prepared substrates for cryo-ET, specific morphological identification and localization, and the detailed structural organization of excitatory synapses, including synaptic vesicle clusters close to the postsynaptic density and in the cleft.

Strengths:

The study advances previous work that used cultured neurons or synaptosomes. Combining cryo-electron tomography (cryo-ET) with fluorescence-guided targeting and labeling with Fab-AuNP conjugates enabled the study of synapses and molecular structures in their native environment without chemical fixation or staining. This preserves their near-native state, offering high specificity and resolution. The methods developed are generalizable, allowing adaptation for identifying and localizing other key molecules at glutamatergic synapses and potentially useful for studying a variety of synapses and cellular structures beyond the scope of this research.

Weaknesses

The preparation and imaging techniques are complex and require highly specialized equipment and expertise, potentially limiting their accessibility and widespread adoption.

Additionally, the methods might need further modifications/tweaks to study other types of synapses or molecular structures effectively.

The reliance on genetically engineered mouse lines may again impact the generalizability of the findings.

Similarly, the requirement of monoclonal, high-affinity antibodies/Fab fragments to specifically label receptors/proteins would limit the wider employment of these methods.

zcxvzxvzxv

eLife assessment

This study provides the first analysis of vascular stabilization on the critical and evolutionarily conserved structure around the Circle of Willis in the brain, strengthened by using parallel in vivo and in vitro experimental approaches. The evidence supporting the claims is solid and the work will be valuable for scientists studying developmental and disease-related vascular stabilization.

Reviewer #2 (Public Review):

Summary:

Cheng et al. explore the development of the arteries that form the circle of Willis and investigate how blood flow pulsatility influences vascular smooth muscle cell (VSMC) differentiation. Using live confocal imaging of the developing zebrafish, the authors show that endothelial cells in circle of Willis arteries transition from venous to arterial identity between 54 hours post-fertilization (hpf) and 3 days post-fertilization (dpf), and that this coincides with pdgfrb+ mural cell progenitor differentiation into acta2+ arterial VSMCs. They find that the anterior portions of the circle of Willis, including the internal carotid arteries (CaDI), establish acta2 expression earlier than posterior aspects, likely due to faster flow rate and increased pulsatility through the CaDI. Then, using computational fluid dynamics, an in vitro co-culture assay, and genetic and drug manipulations of blood flow, the authors provide evidence that pdgfrb+ differentiation is dependent upon pulsatile blood flow and klf2a activation. The results add to our understanding of vascular development and suggest that deficits in pulsatile flow could be potential drivers of arteriopathies.

Strengths:

(1) Longitudinal confocal imaging of live developing zebrafish makes the timeline of arterial development in the circle of Willis easy to understand. This is a strong approach to studying how vascular networks are altered with genetic and pharmacological manipulations.<br /> (2) Rigorous use of multiple techniques to test the hypothesis that pulsatile blood flow is required for smooth muscle cell differentiation. The microangiography experiment, in vitro co-culture assay, and genetic and drug manipulations of heart rate at various developmental timepoints yield outcomes that are consistent with the hypothesis.

Weaknesses:

(1) The authors should provide more information on how blood flow velocity and wall shear stress are calculated from circle of Willis vascular structure. It is presumed that these values are dependent upon the 3-D morphology of the vessel network, as labeled by intravenous dextran dye, but this is not clear. Small local differences in vessel diameter and shape will influence blood flow velocity, but these morphological changes are not clearly articulated. Further, it is unclear how flow input levels to the CaDI and basilar arteries are decided across time-points. In general, descriptions of the blood flow modeling are very sparse.<br /> (2) Is it possible to measure the blood flow speed empirically with line-scanning or high-speed tracking of labeled blood cells? This would provide some validation of the modeling results.<br /> (3) Does the cardiac injection of dextran itself affect the diameter or flow of the arteries, given the invasiveness of the procedure? This could be examined in fish with a transgenic endothelial label and with vs. without dextran.<br /> (4) The data from the microangiography experiment in Figure 3 does not fully support the stated results. The authors report that the CaDI had the highest blood flow speed starting from 54 hfp, but it does not appear to be higher than the other arteries at this time point. Additionally, there is not sufficient evidence that wall shear stress coincides with smooth muscle cell differentiation in the CaDI. Wall shear stress appears to be similar between 54 hpf and 3 dpf in the CaDI, only increasing between 3 dpf and 4 dpf, while differentiation is shown to begin at 3 dpf.<br /> (5) The genetic and drug manipulations of heart rate are important experiments, but more detail is required to understand the effects of the manipulations. At least, a discussion on the limitations of these manipulations is needed. For example, how does one separate the pulsatile versus nutritive effects of blood flow/heart rate reduction? It is possible that off-target or indirect effects of Nifedipine decrease smooth muscle cell proliferation, or that altered cardiac contractility fundamentally alters many aspects of vascular development other than blood flow. Nifedipine is also likely to act upon VSMC calcium handling in the circle of Willis, which may in turn affect cell maturation.<br /> (6) It is unclear if acta2 expression is conferring vascular tone, as would be expected if the cells are behaving as mature VSMCs. Does arterial diameter decrease with an increase in acta2 expression? Are acta2 positive mural cells associated with more dynamic changes in arteriole diameter under basal or stimulated conditions?

Reviewer #3 (Public Review):

Summary:

Cheng et al. studied if and how blood flow regulates differentiation of vascular smooth muscle cells (VSMC) in the Circle of Willis (CW) in zebrafish embryos. They show that CW vessels gradually acquire arterial identity. VSMCs also undergo gradual differentiation, which correlates with blood flow velocity. Using cell culture they show that pulsatile blood flow promotes pericyte differentiation into smooth muscle cells. They further identify transcription factor klf2a as differentially regulated by blood flow, and show that klf2a inhibition results in VSMC differentiation. The authors conclude that pulsatile flow promotes VSMC differentiation through klf2a activation.

Strengths:

Overall this is an important study, because VSMC differentiation in CW has not been previously studied, although analogous observations regarding the role of blood flow and klf2 involvement have been previously made in other systems and other vascular beds, for example, mouse klf2 mutants, which have deficient VSMC coverage of the dorsal aorta (Wu et al., 2008, JBC 283: 3942-50). The results convincingly show that VSMC differentiation in CW depends on the blood flow, and that klf2a flow dependent function regulates VSMC differentiation.

Weaknesses:

(1) The provided data do not support correlation between wall shear stress (WSS) and acta2+ cell number. The number of acta2+ cells in CaDI increases dramatically between 54 hpf and 3 dpf (Fig. 2F). However, the graph provided in the response to reviewers shows that WSS in CaDI is actually lower at 3 dpf compared to 54 hpf. Authors argue that Pearson correlation analysis shows that both variables increase together, but this is calculated over the stage between 54 hpf and 4 dpf. acta2+ cells appear by 3 dpf, and at this stage WSS in CaDI is not increased (or even lower), which argues agains WSS being the cause of acta2+ cell differentiation. Furthermore, data in Fig. 3I-K show that WSS actually decreases in BCA and PCS between 54 hpf and 4 dpf, while the number of acta2+ increases in BCA and PCS by 4 dpf. This also argues against the argument that WSS affects differentiation of acta2+ cells.<br /> (2) In multiple instances, results are based on a single independent experiment (Fig. 3, Fig. 4H, I, Fig. S2 and Fig. S3) with only a few embryos analyzed in many cases. This falls short of expected standards in the field, and it is unclear if these results are reproducible.

Author response:

The following is the authors’ response to the original reviews.

Weaknesses to be addressed: 

(1) More detail is required to understand the effects of genetic and drug manipulations on heart rate as these are important experiments. At the very least, a discussion on the limitations of these manipulations is needed. 

- For example, how does one separate the pulsatile versus nutritive effects of blood flow/heartrate reduction? 

- The conclusion that arterial SMC differentiation is driven by pulsatile blood flow needs to be toned down. Indeed, this conclusion is mainly supported by in vitro cell co-cultures exposed to laminar versus pulsatile flow. In vivo, reducing Tnnt2a expression affects cardiac contractility and blood flow does not selectively affect pulsatility. To make this conclusion, the authors would need an experimental means to selectively dampen the pulsatility of blood flow.

We understand this concern and we toned down the statements related to the pulsatile flow of our conclusion by using 'flow' instead of 'pulsatile flow' in all text except for the in vitro co-cultures part. We also added a paragraph to discuss the limited capability of qualitatively reduce blood flow in vivo, and acknowledge that the effects of nutrients and flow reduction could not be uncoupled in live zebrafish embryos. We proposed that in the future, in vitro 3D vascular culture models may be combined with microfluidics to precisely calibrate nutrient composition in culture media, flow velocity and pulse; these methods would help address these questions more thoroughly. See page 11-12 line 312-322.

(2) Since mural cells are sensitive to transmural pressure, could the authors elaborate on the potential role of raised intravascular pressure in SMC differentiation? This would better parallel rodents and humans. 

We thank you for this suggestion. We added a paragraph to discuss the potential role of raised intravascular pressure in VSMC differentiation in the discussion section (see page 11 line 296-311).

(3) The authors use nifedipine to reduce blood flow. Nifedipine is a specific and potent inhibitor of voltage-dependent calcium channels (VDCC) which are expressed in SMCs. Prior studies (PMID: 35588738) showed that VDCC blockers increased rather than inhibited SMC differentiation. Nifedipine is also likely to act upon VSMC calcium handling in the circle of Willis, which may in turn affect cell maturation. Could the authors comment on this seeming discrepancy?

It is possible that off-target or indirect effects of Nifedipine decrease smooth muscle cell proliferation, or that altered cardiac contractility fundamentally alters aspects of vascular development other than blood flow. 

- Additionally, it would be helpful to report the quantitative heart rate reduction achieved with Nifedipine. This would clear up concerns that the heart rate reduction is too large for normal vascular development to occur, and thus decrease proliferation rate independent of changes in blood flow pulsatility. 

We concur with these comments, which is why our experimentation with Nifedipine is reinforced by employing an alternative, non-pharmacological strategy to inhibit blood flow: the use of morpholino against tnnt2a gene. The results with either Nifedipine or tnnt2a support the lack of VSMCs maturation. In addition, we provided the quantitative heart rate reduction achieved with Nifedipine shown in new Figure S2A-S2C, suggesting that the drug is not completely halting the heart rate but decreasing it. Nevertheless, we report that Zebrafish embryos can survive and develop a normal blood vascular system without any heartbeat. Hence, we exclude that the effect on VSMCs maturation is linked non-specifical effects caused by the loss of heartbeat. Nevertheless, we now acknowledged in our discussion the limitation of nifedipine, as it may affect VSMC through VDCCs (page 12, line 323-334).

We also added a paragraph in the discussion section to compare nifedipine, an L-type VDCC blocker, and ML218, a T-type VDCC selective inhibitor from the previous study (Ando et al., 2022). We noted that in this previous study, the increase in VSMC differentiation only occur on anterior metencephalic central arteries (AMCtAs) that are more than 40 mm away from the BCA; these AMCtAs are much smaller than CoW arteries and have different geometry hence possible different kinetics of VSMC maturation (Ando et al., 2022) as our manuscript discovery would suggest.

(4) The authors should provide more information on how blood flow velocity and wall shear stress are calculated from the Circle of Willis vascular structure. It is presumed that these values are dependent upon the 3-D morphology of the vessel network, as labeled by intravenous dextran dye, but this is not clear. (a second reviewer similarly comments: I was unclear how flow velocity values were obtained in Fig. 3E. Are they based on computational simulation, or are they experimentally calculated following the dextran injection?) Small local differences in vessel diameter and shape will influence blood flow velocity, but these morphological changes are not clearly articulated. Further, it is unclear how flow input levels to the CaDI and basilar arteries are decided across time points. For instance, is it possible to measure the blood flow speed empirically with line-scanning or high-speed tracking of labeled blood cells or particles? This would provide validation of the modeling results. 

The computational fluid dynamic simulation was performed according to previous study from our lab (Barak et al., 2021). Blood flow velocity and wall shear stress are dependent upon the 3D morphology of the vessel network labeled by intravascular dextran. Details on how the computational fluid dynamic simulation was performed are added in method section page 17 line 433-449.

Moreover, to address this reviewer concern we have now provided new experimental measurement of blood flow using the red blood cell (RBC) velocity via axial line scanning microscopy in Tg(kdrl:gfp;gata1:DsRed)zn1/sd2 zebrafish embryos at 54 hpf, 3 dpf, and 4 dpf. By using the experimental RBC velocity, we re-simulated the computational fluid dynamic. The new findings align with our conclusion and are further elaborated upon in response to this reviewer comment listed as point 6. Details on how RBC velocity calculated is added in method section page 16 line 414-431.

(5) Does the cardiac injection of dextran itself affect the diameter of the arteries, given the invasiveness of the procedure? This could be examined in fish with a transgenic endothelial label with and without dextran. 

Here, we performed an experiment on wildtype zebrafish at 5 days post-fertilization (dpf) with and without Dextran injection, examining the effects of Dextran injection on vessel diameters. As shown in the representative image below, the XZ panel clearly illustrates a Dextran-filled PCS vessel with no alteration in vessel size. Dextran microangiography, a technique employed to obtain vessel geometry with fluorescent microsphere, has been well established in zebrafish (Kamei et al., 2010). Our findings, demonstrating that Dextran does not affect vessel size, are consistent with previous studies utilizing Dextran microangiography.

Author response image 1.

(6) The data from the microangiography experiment in Figure 3 does not fully support the stated results. The authors report that the CaDI had the highest blood flow speed starting from 54 hpf, but it does not appear to be higher than the other arteries at this time point. Additionally, there is not sufficient evidence that wall shear stress coincides with smooth muscle cell differentiation in the CaDI. Wall shear stress appears to be similar between 54 hpf and 3 dpf in the CaDI, only increasing between 3 dpf and 4 dpf, while differentiation is shown to begin at 3 dpf. The authors need to address this and/or soften conclusions. 

First, In response to this specific reviewer concern, we measured red blood cell (RBC) velocity by used axial line scanning microscopy to analyze Tg(kdrl:gfp;gata1:DsRed)zn1/sd2 zebrafish embryos (the detailed method was added in Method section in the manuscript). We replaced the computational simulated blood flow velocity by RBC velocity in new Figure 3E-3G, and re-run the computational simulated wall shear stress (WSS) using the RBC velocity in new Figure 3I-3K. We compared RBC velocity and WSS among different vessels at each time point. We confirmed that CaDI has the highest RBC velocity starting from 54 hpf to 4 dpf (new Figure 3A-3C, and 3E-3G) and found an overall increase in average WSS from 54 hpf to 4 dpf (new Figure 3A-3C, and 3H). Further, WSS in CaDI was significantly higher than BCA and PCS at 54 hpf, 3 dpf, and 4 dpf (new Figure 3A-3C, 3I-3K). Altogether, the CFD simulation suggests that CoW arteries experience different hemodynamic WSS that is associated with spatiotemporal pattern of VSMC differentiation on CoW arteries.”.  (Page 6, line 153-162)

Second, to identify the correlation of WSS and VSMC differentiation in CaDI, we performed Pearson correlation analysis. In the image provided here, we plotted a linear regression with normalized # of acta2+ cells in CaDI and WSS with developmental stages (54 hpf, 3 and 4 dpf), and performed Pearson correlation coefficient analysis by using GraphPad Prism 10.0.3. The correlation coefficient r = 0.595, suggesting that the two variables (acta2+ cells and WSS) tend to increase together with developmental stages (54 hpf, 3 and 4 dpf).

Author response image 2.

Third, we softened our conclusion as the RBC velocity across CoW arteries was differentially distributed while VSMC differentiation occurred in these vessels.

(7) It is unclear if acta2 expression is conferring vascular tone, as would be expected if the cells are behaving as mature VSMCs. Does arterial diameter decrease with an increase in acta2 expression? Are acta2-positive mural cells associated with more dynamic changes in arteriole diameter under basal or stimulated conditions? 

Thanks for this interesting question. VSMC maturation and its vasoactivity could be further investigated in the future. Our study focused on early stage of VSMC differentiation, in which pdgfrb+ progenitors started to express VSMC marker acta2. We discussed the onset of transgelin expression and loss of abcc9 expression as markers of VSMC maturation. In addition, a previous study found that VSMC covered vessels in zebrafish brain dilate as early as 4 dpf and constrict at 6 dpf (Bahrami & Childs, 2020). Future study may focus on the association between expression of different VSMC markers and VSMC functional maturation. (page 10, line 272-279)

(8) The authors argue that CoW vessels transition from venous to arterial identity (Fig. 1). However, kdrl is not an ideal arterial marker for this experiment as it is expressed in both arteries and veins. While it is true that many arterial beds have stronger kdrl expression than the veins, its expression in both arteries and veins changes with developmental stage, and its expression level may vary depending on the type of vessel. Therefore, showing that kdrl increases from 32 hpf - 4 dpf in CoW vessels is not convincing because its expression may increase in both venous or arterial vasculature as the vessels mature. In addition, flt4 expression is not exclusively venous; for example, it has noticeable expression in the dorsal aorta at 24-32 hpf stages. It would be helpful to confirm this transition by analyzing additional arterial and venous markers. 

We acknowledge this and we added a paragraph to discuss the limitation. We combined loss of flt4 and increase in kdrl to establish the temporal sequence of circle of Willis morphogenesis, arterial specification, and VSMC differentiation. We acknowledge that additional arterial and venous markers need to be analyzed for a more thorough characterization of arterial specification in vertebrate brain vascular development. See page 12 line 335-341.

(9) The authors show that acta2+ VSMCs are absent in tnnt2a MO embryos, concluding that blood flow is required for their differentiation from pericytes. However, there is no data showing that pericytes are still present in tnnt2a MO embryos. Although this has been previously shown by Ando et al 2016, it would be beneficial to confirm in the current study as this is a critical piece of evidence needed for this conclusion. 

To determine if blood flow is dispensable for pdgfrb+ progenitor recruitment, we performed tnnt2a MO (0.35 ng/embryo) injection in Tg(pdgrb:egfp, kdrl:ras-mcherry) ncv22/s896. Loss of blood flow did not affect pdgfrb+ progenitor emergence around the CoW (new Figure S2G-S2H) at 3 days post fertilization (dpf). This is consistent with previous observation in Ando et al 2016 Figure S2C (Ando et al., 2016).

(10) The authors show that klf2a MO injected embryos have a reduced number of VSMCs at 3 dpf but a normal number at 4 dpf (Fig. 6), concluding that klf2a is only important to initiate CaDI muscularization. If this is true, it would raise important questions about how VSMCs differentiate at a later stage in the absence of klf2a. For instance, is blood flow not required to differentiate at a later stage, or is there another factor that compensates in the absence of klf2a? The alternative explanation/ caveat is that klf2a MO loses efficacy with development, leading to the recovery of VSMCs at this stage. Therefore, it would be important to confirm this result using a genetic klf2a mutant. 

Thank you for pointing this out.  We note that based on the klf2a reporter line, klf2a activity in CoW arterial endothelial cells is highly correlated with the number of acta2+ VSMCs in CaDI, BCA and PCS at 3 dpf (r = 0.974, new Figure S5J). Interestingly however, klf2a activity remained stable from 3 dpf to 4 dpf, well beyond initiation of VSMC differentiation. Thus, we speculate sustained klf2a expression may support further maturation of VSMCs, as acta2+ VSMCs showed distinct morphology at 4 dpf compared with 3 dpf. (Page 10, line 268-272). As for the observation that klf2a morphants have normal number of VSMCs at 4 dpf, we think that in addition to the temporary effect of morpholino, a proximal explanation is compensation by paralogous klf2b in zebrafish. We acknowledge that further characterization of CoW VSMC development in klf2a and klf2b double genetic mutants (Rasouli et al., 2018; Steed et al., 2016) may help determine whether klf2b compensates klf2a in CoW VSMC differentiation beyond 4 dpf. See page 10-11 line 292-295.

(11) A large part of the discussion focuses on Notch and Wnt signaling, as downstream Klf2 effectors. While these are reasonable hypotheses to propose, there is no data on the involvement of these pathways in the current study. It seems excessive to speculate on detailed mechanisms of how Klf2 activates Notch and Wnt signaling in the absence of data showing that these pathways are affected in CoW vessels. Therefore, the discussion could be shortened here unless additional data can be obtained to demonstrate the involvement of these pathways in VSMCs in CoW.

We concur and have condensed the discussion on Notch and Wnt signaling as downstream klf2 effectors.

Minor comments: 

(1) Line 138 "CaDI is the only vessels in the CoW receiving pulsatile arterial blood low ... ". Adding a reference to support this statement would be useful. 

We agree and revised this sentence into ‘CaDI receive proximal arterial feed through lateral dorsal aorta from cardiac outflow tract (Isogai et al., 2001)’. It was also based on our general observation of zebrafish vascular anatomy and blood flow under a confocal microscope.

(2) The image insets in Figs. 1A, 2A, 4E-L, 5A, 6A are quite small. Please make them larger to help the reader interpret the findings. 

We agree. We maximized the image size to help the reader interpret the finding, and to visualize confocal images and schematics side-by-side.

(3) The schematics in Figs. 1-2, and 4-6 are helpful, but the different cell types are difficult to see because they are small and their colors/shapes are not very distinct. 

We agree. We increased the size and color contrast to provide better visualization of the schematics in new schematic Figures. 1-2 and 4-6.

(4) It is stated that there are no diameter differences between different arteries, but statistics are not reported. 

The statistics in Figure 3D were performed by ordinary two-way ANOVA followed by Tukey’s multiple comparisons test, with a single pooled variance. Here we added pairwise comparisons among vessels in the CoW. Hence when non indicated the difference are non-significant.

(5) Figure 3F would be better visualized on a log scale, as it is difficult to see the differences between each post-fertilization timepoint. 

We agree. In the new Figure 3H, the average wall shear stress (WSS) in CoW arteries is presented on log scale in y axis to see the differences between each post-fertilization timepoint.

(6) Please provide more background and validation on the pericyte cell line, and their use for the questions in this study. 

Thank you for the question, TgBAC(pdgfrb:egfp)ncv22 was generated and described by Ando et al 2016 to clarify mural cell coverage of vascular endothelium in zebrafish (Ando et al., 2016). We added a describe in the method section to provide background and validation on this pericyte line (see page 13 line 368-372).

(7) Flow velocity and WSS changes are shown in each vessel in Figs. 3E,G. However, the comparison should be made between different types of vessels to see if there is a statistical difference and PCS, for example, which would explain differences in VSMC coverage. 

We agreed. We compared the difference among arteries in the CoW at each developmental timepoint and performed ordinary one-way ANOVA with Tukey’s multiple comparisons test. Figure. 3E is replaced by new Figure. 3E-G and Figure. 3G is replaced by new Figure. 3I-K.

(8) Similarly, between CaDI, the number of klf2a cells in Fig. 5B should be compared between different vessels, not between different stages of the same vessel. 

We agree. In new Figure 5B-E, the number of klf2a+ cells per 100 μm vessel length are compared among different vessels at each developmental stage and analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons test.

(9) When quantifying klf2+ cells in Fig. 5, it would be helpful to quantify klf2 expression level between cells in different vessels. This could be done by quantifying GFP expression in existing images. The difference in expression level may explain the variation between CaDI and PCS more accurately than just the difference in cell number. 

The GFP expression reflect the stability of GFP protein expression and labels discrete nuclei with active klf2a expression. Hence the quantification of GFP level might not give an accurate readout of klf2a expression per se but rather of its activity. For this reason we don’t think that this experiment will add accurate measurement of klf2a expression.

(10) Do data points in Figure 4D correspond to different cells in the same chamber experiment? If so, they cannot be treated as independent replicates. Each data point should correspond to an independent replicate experiment. 

We agree. Now in the figure legend, we report the number of cells analyzed.

(11) Graph placement is confusing in Figs. 4I, M. An adjacent Fig. 4G shows Nifedipine treated embryos, while the graph next to (Fig. 4I) shows acta+ cell number from tnnt2a 4 dpf experiment. Similarly, the bottom Fig. 4K tnn2a 4 dpf MO experiment has an adjacent graph Fig. 4M, which shows nifedipine treatment quantification, which makes it very confusing. 

We agreed. We rearranged Figure 4E (representative images of control embryos at 3 dpf and 4 dpf), Figure 4F (tnnt2a MO embryos at 3 dpf and 4 dpf), Figure 4G (nifedipine treated embryos at 3 dpf and 4 dpf).

Reference:

Ando, K., Fukuhara, S., Izumi, N., Nakajima, H., Fukui, H., Kelsh, R. N., & Mochizuki, N. (2016). Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish. Development, 143(8), 1328-1339. https://doi.org/10.1242/dev.132654

Ando, K., Tong, L., Peng, D., Vazquez-Liebanas, E., Chiyoda, H., He, L., Liu, J., Kawakami, K., Mochizuki, N., Fukuhara, S., Grutzendler, J., & Betsholtz, C. (2022). KCNJ8/ABCC9-containing K-ATP channel modulates brain vascular smooth muscle development and neurovascular coupling. Dev Cell, 57(11), 1383-1399 e1387. https://doi.org/10.1016/j.devcel.2022.04.019

Bahrami, N., & Childs, S. J. (2020). Development of vascular regulation in the zebrafish embryo. Development, 147(10). https://doi.org/10.1242/dev.183061

Barak, T., Ristori, E., Ercan-Sencicek, A. G., Miyagishima, D. F., Nelson-Williams, C., Dong, W., Jin, S. C., Prendergast, A., Armero, W., Henegariu, O., Erson-Omay, E. Z., Harmanci, A. S., Guy, M., Gultekin, B., Kilic, D., Rai, D. K., Goc, N., Aguilera, S. M., Gulez, B., . . . Gunel, M. (2021). PPIL4 is essential for brain angiogenesis and implicated in intracranial aneurysms in humans. Nat Med, 27(12), 2165-2175. https://doi.org/10.1038/s41591-021-01572-7

Isogai, S., Horiguchi, M., & Weinstein, B. M. (2001). The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol, 230(2), 278-301. https://doi.org/10.1006/dbio.2000.9995

Kamei, M., Isogai, S., Pan, W., & Weinstein, B. M. (2010). Imaging blood vessels in the zebrafish. In Methods in cell biology (Vol. 100, pp. 27-54). Elsevier.

Rasouli, S. J., El-Brolosy, M., Tsedeke, A. T., Bensimon-Brito, A., Ghanbari, P., Maischein, H. M., Kuenne, C., & Stainier, D. Y. (2018). The flow responsive transcription factor Klf2 is required for myocardial wall integrity by modulating Fgf signaling. Elife, 7. https://doi.org/10.7554/eLife.38889

Steed, E., Faggianelli, N., Roth, S., Ramspacher, C., Concordet, J. P., & Vermot, J. (2016). klf2a couples mechanotransduction and zebrafish valve morphogenesis through fibronectin synthesis. Nat Commun, 7, 11646. https://doi.org/10.1038/ncomms11646

find a way over or through (an obstacle or difficult route): she cautiously negotiated the hairpin bend

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noun

a person who investigates crimes; a detective:

Verb carry out a careful investigation into a crime or mystery: there's nothing you can do but sleuth around until you find the answer.

[with adverbial] informal succeed in achieving something by a very narrow margin: the bill squeaked through with just six votes to spare.

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a person who claims to have a supernatural ability to perceive events in the future or beyond normal sensory contact: she has had a message from a clairvoyant that her son is alive and well.

| ˈseɪvjə | (savior US English)

noun

a person who saves someone or something from danger or difficulty: politicians of the era usually portray themselves as the nation's saviours.

British English a tournament in which the loser in each round is eliminated.

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[predicative] ready, prepared, or likely to do something: the first family was set for a quiet night of rest

eLife assessment

This important study demonstrates that combining AlphaFold2 with the author's sampling method AF2-RAVE improves protein-ligand docking for three protein kinases and their inhibitors. The evidence is compelling but would benefit from a more complete description of the methodology and a clear assessment of the method's range of applicability. The work will be of interest to researchers who work on computer-aided drug design.

Reviewer #1 (Public Review):

The development of effective computational methods for protein-ligand binding remains an outstanding challenge to the field of drug design. This impressive computational study combines a variety of structure prediction (AlphaFold2) and sampling (RAVE) tools to generate holo-like protein structures of three kinases (DDR1, Abl1, and Src kinases) for binding to type I and type II inhibitors. Of central importance to the work is the conformational state of the Asp-Phy-Gly "DFG motif" where the Asp points inward (DFG-in) in the active state and outward (DFG-out) in the inactive state. The kinases bind to type I or type II inhibitors when in the DFG-in or DFG-out states, respectively.

It is noted that while AlphaFold2 can be effective in generating ligand-free apo protein structures, it is ineffective at generating holo-structures appropriate for ligand binding. Starting from the native apo structure, structural fluctuations are necessary to access holo-like structures appropriate for ligand binding. A variety of methods, including reduced multiple sequence alignment (rMSA), AF2-cluster, and AlphaFlow may be used to create decoy structures. However, those methods can be limited in the diversity of structures generated and lack a physics-based analysis of Boltzmann weight critical to their relative evaluation.

To address this need, the authors combine AlphaFold2 with the Reweighted Autoencoded Variational Bayes for Enhanced Sampling (RAVE) method, to explore metastable states and create a Boltzmann ranking. With that variety of structures in hand, grid-based docking methods Glide and Induced-Fit Docking (IFD) were used to generate protein-ligand (kinase-inhibitor) complexes.

The authors demonstrate that using AlphaFold2 alone, there is a failure to generate DFG-out structures needed for binding to type II inhibitors. By applying the AlphaFold2 with rMSA followed by RAVE (using short MD trajectories, SPIB-based collective variable analysis, and enhanced sampling using umbrella sampling), metastable DFG-out structures with Boltzmann weighting are generated enabling protein-ligand binding. Moreover, the authors found that the successful sampling of DFG-out states for one kinase (DDR1) could be used to model similar states for other proteins (Abl1 and Src kinase). The AF2RAVE approach is shown to result in a set of holo-like protein structures with a 50% rate of docking type II inhibitors.

Overall, this is excellent work and a valuable contribution to the field that demonstrates the strengths and weaknesses of state-of-the-art computational methods for protein-ligand binding. The authors also suggest promising directions for future study, noting that potential enhancements in the workflow may result from the use of binding site prediction models and free energy perturbation calculations.

Reviewer #2 (Public Review):

Summary:

This manuscript explores the utility of AlphaFold2 (AF2) and the author's own AF2-RAVE method for drug discovery. As has been observed elsewhere, the predictive power of docking against AF2 structures is quite limited, particularly for proteins like kinases that have non-trivial conformational dynamics. However, using enhanced sampling methods like RAVE to explore beyond AF2 starting structures leads to a significant improvement.

Strengths:

This is a nice demonstration of the utility of the authors' previously published RAVE method.

Weaknesses:

My only concern is the authors' discussion of induced fit. I'm quite confident the structures discussed are present in the absence of ligand binding, consistent with conformational selection. It seems the author's own data also argues for an important role in conformational selection. It would be nice to acknowledge this instead of going along with the common practice in drug discovery of attributing any conformational changes to induced fit without thoughtful consideration of conformational selection.

Reviewer #3 (Public Review):

In this manuscript, the authors aim to enhance AlphaFold2 for protein conformation-selective drug discovery through the integration of AlphaFold2 and physics-based methods, focusing on improving the accuracy of predicting protein structures ensemble and small molecule binding of metastable protein conformations to facilitate targeted drug design.

The major strength of the paper lies in the methodology, which includes the innovative integration of AlphaFold2 with all-atom enhanced sampling molecular dynamics and induced fit docking to produce protein ensembles with structural diversity. Moreover, the generated structures can be used as reliable crystal-like decoys to enrich metastable conformations of holo-like structures. The authors demonstrate the effectiveness of the proposed approach in producing metastable structures of three different protein kinases and perform docking with their type I and II inhibitors. The paper provides strong evidence supporting the potential impact of this technology in drug discovery. However, limitations may exist in the generalizability of the approach across other structures, especially complex structures such as protein-protein or DNA-protein complexes.

The authors largely achieved their aims by demonstrating that the AF2RAVE-Glide workflow can generate holo-like structure candidates with a 50% successful docking rate for known type II inhibitors. This work is likely to have a significant impact on the field by offering a more precise and efficient method for predicting protein structure ensemble, which is essential for designing targeted drugs. The utility of the integrated AF2RAVE-Glide approach may streamline the drug discovery process, potentially leading to the development of more effective and specific medications for various diseases.

李笑来老师的英语辅助app

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Résumé de la vidéo [00:00:00][^1^][1] - [00:42:59][^2^][2]:

Cette vidéo présente un débat sur l'échec scolaire et le rôle de l'école dans la société française. Les intervenants discutent des causes multifactorielles du décrochage scolaire, de l'impact des inégalités sociales internalisées par l'école, et de la nécessité d'une approche plus individualisée pour répondre aux besoins des élèves. Ils soulignent également l'importance de la formation des enseignants et de l'engagement parental dans la réussite éducative.

Points forts: + [00:00:00][^3^][3] Le contexte du débat * Introduction des participants et réactions au documentaire sur l'échec scolaire * Discussion sur la complexité du décrochage scolaire et son lien avec les inégalités sociales * Évaluation de l'approche actuelle de l'école face à l'échec scolaire + [00:14:00][^4^][4] Les facteurs du décrochage * Analyse des causes individuelles, sociales et institutionnelles du décrochage * Importance de l'orientation et de l'évaluation des élèves dans la prévention du décrochage * Rôle des dispositifs de remédiation et de la formation des enseignants + [00:26:36][^5^][5] La perception des enseignants et des parents * Témoignages sur la réalité des enseignants et des parents face à l'échec scolaire * Débat sur la responsabilité partagée entre l'école, les parents et la société * Discussion sur la médicalisation du décrochage et la recherche de solutions adaptées + [00:35:12][^6^][6] L'émancipation de l'élève * Importance de l'émancipation de l'élève et de la prise en compte de son environnement familial * Débat sur le rôle des parents dans l'échec scolaire et la nécessité d'une collaboration école-famille * Réflexion sur la normativité de l'école et la diversité des besoins des élèves

А тут пробел после запятой не нужен?)

В сведениях об авторе перед открывающей скобкой пробела не хватает.

我入职的第一家公司，桂林市啄木鸟医疗器械有限公司，入职时也有军训。无论任何岗位，所有的应届生都需要参加，学到了什么对工作有用的东西吗？啥也没有。

induct: admit (someone) formally to a post or organization

eLife assessment

The mechanisms that ensure accurate chromosome segregation are key for genome integrity and defects therein can cause human disease. Although the involvement of MAP kinases in modulating mitosis is known, this manuscript makes a valuable contribution by going to some lengths to reveal links between Spindle Assembly Checkpoint dynamics and stress-responsive MAP-kinase pathways. The strength of the evidence is solid but there are minor weaknesses, which need to be addressed.

Reviewer #1 (Public Review):

Summary:

This manuscript addresses two main issues:<br /> (i) do MAPKs play an important role in SAC regulation in single-cell organism such as S pombe?<br /> (ii) what is the nature of their involvement and what are their molecular targets?

The authors have extensively used the cold-sensitive β-tubulin mutant to activate or inactivate SAC employing an arrest-release protocol. Localization of Cdc13 (cyclin B) to the SPBs is used as a readout for the SAC activation or inactivation. The roles of two major MAPK pathways i.e. stress-activated pathway (SAP) and cell integrity pathway (CIP), have been explored in this context (with CIP more extensively than SAP). Sty1Δ or pmk1Δ mutants were used to inactivate the SAP or CIP pathways and wis1DD or pek1DD expression was utilized to constitutively activate these pathways, respectively. Lowering of Slp1Cdc20 abundance (by phosphorylation of Slp1-Thr 480) is revealed as the main function of MAPK to augment the robustness of the spindle assembly checkpoint.

Strengths:

The experiments are generally well-conducted, and the results support the interpretations in various sections. The experimental data clearly supports some of the key conclusions:

(1) While inactivation of SAP and CIP compromises SAC-imposed arrest, their constitutive activation delays the release from the SAC-imposed arrest.<br /> (2) CIP signaling, but not SAP signaling, attenuates Slp1Cdc20 levels.<br /> (3) Pmk1 and Cdc20 physically interact and Pmk1-docking sequences in Slp1 (PDSS) are identified and confirmed by mutational/substitution experiments.<br /> (4) Thr480 (and also S76) is identified as the residue phosphorylated by Pmk1. S28 and T31 are identified as Cdk1 phosphorylation sites. These are confirmed by mutational and other related analyses.<br /> (5) Functional aspects of the phosphorylation sites have been elucidated to some extent: (a) Phosphorylation of Slp1-T480 by Pmk1 reduces its abundance thereby augmenting the SAC-induced arrest (b) S28, T31 (also S59) are phosphorylated by Cdk1(c) K472 and K479 residues are involved in ubiquitylation of Slp.

Weaknesses:

(1) Cdc13 localization to SPBs has been used as a readout for SAC activation/inactivation throughout the manuscript. However, the only image showing such localization (Figure 1C) is of poor quality where the Cdc13 localization to SPBs is barely visible. This should be replaced by a better image.

(2) The overlapping error bars in Cdc13-localization data in some figures (for instance Figure 3E and 4H) make the effect of various mutations on SAC activation/inactivation rather marginal. In some of these cases, Western-blotting data support the authors' conclusions better.

(3) This specific point is not really a weakness but rather a loose end:<br /> One of the conclusions of this study is that MAPK (PMK1) contributes to the robustness of SAC-induced arrest by lowering the abundance of Slp1Cdc20. The authors have used pmk1Δ or constitutively activating the MAPK pathways (Pek1DD) and documented their effect on SAC activation/inactivation dynamics. It is not clear if SAC activation also leads to activation of MAPK pathways for them to contribute to the SAC robustness. To tie this loose end, the author could have checked if the MAPK pathway is also activated under the conditions when SAC is activated. Unless this is shown, one must assume that the authors are attributing the effect they observe to the basal activity of MAPKs.

(4) This is also a loose end:<br /> The authors show that activation of stress pathways (by addition of KCl for instance) causes phosphorylation-dependent Slp1Cdc20 downregulation (Figure 6) under the SAC-activating condition. Does activation of the stress pathway cause phosphorylation-dependent Slp1Cdc20 downregulation under the non-SAC-activation condition or does it occur only under the SAC-activating condition?

(5) Although the authors have gone to some length to identify S28 and T31 (also S59) as phosphorylation sites for Cdk1, their functional significance in the context of MAPK involvement is not yet clear. Perhaps it is outside the scope of this study to dig deeper into this aspect more than the authors have.

(6) In its current state, the Discussion section is quite disjointed. The first section "Involvement of MAPKs in cell cycle regulation" should be in the Introduction section (very briefly, if at all). It certainly does not belong to the Discussion section. In any case, the Discussion section should be more organized with a better flow of arguments/interpretations.

Reviewer #2 (Public Review):

Summary:

This study by Sun et al. presents a role for the S. pombe MAP kinase Pmk1 in the activation of the Spindle Assembly Checkpoint (SAC) via controlling the protein levels of APC/C activator Cdc20 (Slp1 in S. pombe). The data presented in the manuscript is thorough and convincing. The authors have shown that Pmk1 binds and phosphorylates Slp1, promoting its ubiquitination and subsequent degradation. Since Cdc20 is an activator of APC/C, which promotes anaphase entry, constitutive Pmk1 activation leads to an increased percentage of metaphase-arrested cells. The authors have used genetic and environmental stress conditions to modulate MAP kinase signalling and demonstrate their effect on APC/C activation. This work provides evidence for the role of MAP kinases in cell cycle regulation in S. pombe and opens avenues for exploration of similar regulation in other eukaryotes.

Strengths:

The authors have done a very comprehensive experimental analysis to support their hypothesis. The data is well represented, and including a model in every figure summarizes the data well.

Weaknesses:

As mentioned in the comments, the manuscript does not establish that MAP kinase activity leads to genome stability when cells are subjected to genotoxic stressors. That would establish the importance of this pathway for checkpoint activation.

eLife assessment

The study is noteworthy for its effort to achieve a deeper understanding of PTH-1 Receptor signaling. This molecular pathway which underpins the control of calcium and phosphate metabolism throughout life in land-dwelling animals, can be targeted to the therapeutic benefit of people with osteoporosis. We consider the significance of the findings in this paper to be valuable to the community of investigators working on PTH receptor and PTH ligand signaling. The strength of the evidence is solid and it could become even stronger by addressing a few shortcomings.

Reviewer #1 (Public Review):

Summary:

In this work, the authors investigate the functional difference between the most commonly expressed form of PTH, and a novel point mutation in PTH identified in a patient with chronic hypocalcemia and hyperphosphatemia. The value of this mutant form of PTH as a potential anabolic agent for bone is investigated alongside PTH(1-84), which is a previously used anabolic therapy. The authors have achieved the aims of the study. Their conclusion, however, that this suggests a "new path of therapeutic PTH analog development" seems unfounded; the benefit of this PTH variant is not clear, but the work is still interesting.

The work does not identify why the patient with this mutation has hypocalcemia and hyperphosphatemia; this was not the goal of the study, but the data are useful for helping to understand that.

Strengths:

The work is novel, as it describes the function of a novel, naturally occurring, variant of PTH in terms of its ability to dimerise, to lead to cAMP activation, to increase serum calcium, and its pharmacological action compared to normal PTH.

Weaknesses:

(1) The use of very young, 8-10 week old, mice as a model of postmenopausal osteoporosis is a major limitation of this study. At 8 weeks, the effect of ovariectomy leads to lack of new trabecular bone formation, rather than trabecular bone loss due to a defect in bone remodelling. Although the findings here provide a comparison between two forms of PTH, it is unlikely to be of direct relevance to the patient population. For example, the authors find an inhibitory effect of PTH on osteoclast surface, which is very unusual. Adding to this concern is that the authors have not described the regions used for histomorphometry, and from their figures (particularly the TRAP stain), it seems that the primary spongiosa (which is a region of growth) has been used for histomorphometry, rather than the secondary spongiosa (which more accurately reflects bone remodelling). Much further detail is needed to justify the use of this very young model, and a section on the limitations of this model is needed. Please provide that section in the revised manuscript.

(2) It is also somewhat concerning that the age range is from 8-10 weeks, increasing the variability within the model. Did the age of mice differ between the groups analysed?

(3) Methods are not sufficiently detailed. For example, the regions used for histomorphometry are not described, there is no information on micro-CT thresholds, no detail on the force used for mechanical testing. Please address this request.

(4) There are three things unclear about the calvarial injection mouse model. Firstly, were the mice injected over the calvariae or with a standard subcutaneous injection (e.g. at the back of the neck)? If they were injected over the calvaria, why were both surfaces measured? Secondly, why was the dose of the R25C-PTH double that of PTH(1-34)? Thirdly, there is no justification for the use of "more intense coloration" as a marker of new bone; this requires calcein labelling to prove it new bone. It would be more reliable to measure and report the thickness of the calvaria. Please address these technical questions.

(5) The presentation of mechanical testing data is not sufficient. Example curves should be shown, and data corrected for bone size needs to be shown. The difference in mechanical behaviour is interesting, but does it stem from a difference in the amount of bone, or two a difference in the quality of the bone? Please explain this matter better in the manuscript.

(6) The micro-CT analysis of the cortical bone in the OVX model is insufficient. Please indicate whether cross-sectional area has increased. Is there an increase in the size of the bones, or is the increase in cortical thickness due to a narrowing of the marrow space? This may help resolve the apparent contradiction between the cortical thickness data (where there is no difference between the two PTH formulations) and the mechanical testing data (where there is a difference). Please explain this matter better in the manuscript.

(7) The evidence that dimeric PTH has a different effect to monomeric PTH is very slim; I am not sure this is a real effect. Such differences take a long time to sort out (e.g. the field is still trying to determine whether teriparatide and abaloparatide are different). I think the authors need to look more carefully at their data - almost all effects are the same. Ultimately, the statement that dimeric PTH may be a more effective anabolic therapy than monomeric PTH are not supported by the data, and this should be removed. There is little to no difference found between normal PTH and the variant in their effects on calcium and phosphate homeostasis or on bone mass. However, the analysis has been somewhat cursory, with insufficient mechanical testing or cortical data presented. Many of the effects seem to be the same (e.g. cortical thickness, P1NP, ALP, vertebral BV/TV and MAR), but the way it is written it sounds like there is a difference. Please remove some of the unfounded claims that you have made in this manuscript.

(8) Statistical analysis used multiple t-tests. ANOVA would be more appropriate.

Reviewer #2 (Public Review):

Summary:

The study conducted by Noh et al. investigated the effects of parathyroid hormone (PTH) and a dimeric PTH peptide on bone formation and serum biochemistry in ovariectomized mice as a model for postmenopausal osteoporosis. The authors claimed that the dimeric PTH peptide has pharmacological benefits over PTH in promoting bone formation, despite both molecules having similar effects on bone formation and serum Ca2+. However, after careful evaluation, I am not convinced that this manuscript adds a significant contribution to the literature on bone and mineral research.

Strengths:

Experiments are well performed, but strengths are limited to the methodology used to evaluate bone formation and serum biochemical analysis.

Weaknesses:

(1) Limited significance of this study:<br /> • this study follows a previous study (not cited) reporting the effect of the dimeric R25CPTH(1-34) on bone regeneration in an osteoporotic dog (Beagle) model (Jeong-Oh Shin et al., eLife 13:RP93830, 2024). It's unclear why the authors tested the dimeric R25C-PTH peptide on a rodent animal model, which has limitations because the healing mechanism of human bone is more similar in dogs than in mice.<br /> • the authors should clarify why they tested the effects of dimeric R25CPTH(1-34) and not dimeric R25CPTH(1-84)?<br /> • The study is descriptive with no mechanism.

(2) Statistics are inadequately described or performed for the experimental design:<br /> • the statistical analysis in Figure 5 needs to be written in a way that makes it clearer how statistics were done; t-test or one-way ANOVA?<br /> • Statistics in Figures 6 and 7 should be performed by one-way ANOVA to compare the mean values of one variable among three or more groups, and not t-test.

(3) Misleading and confused discussion:<br /> • The first paragraph lacks clarity in the PTH nomenclature and the authors should provide a clear statement that the PTH mutant found in patients is likely a monomeric R25CPTH(1-84), considering that there has been no proof of a dimeric form.<br /> • Moreover, the authors should discuss the study by White et al. (PNAS 2019), which shows that there are defective PTH1R signaling responses to monomeric R25CPTH(1-34). This results in faster ligand dissociation, rapid receptor recycling, a short cAMP time course, and a loss of calcium ion allosteric effect.<br /> • The authors should also clarify what they mean by "the dimeric form of R25CPTH can serve as a new peptide ...(lines 328-329)" The dimeric R25CPTH(1-34) induces similar bone anabolic effects and calcemic responses to PTH(1-34), so it is unclear what the new benefit of the dimeric PTH is.

Please address these concerns.

Ce problème est à signaler, car ca n'est qu'en cas d'égalité des votes que le président a double voix

en effet c'est très variable, quand le nombre de dossier favorable à la famille est important, c'est à cause des vices de procédure

c'est oublié les travaux de recherche qui montre que l'école renforce de façon biaisée les déterminismes et prend ces décision avec des aprioris (cf colloque au collège de France)

Les statistiques montrent que c'est entre 25% et 75%

Тут не нужна *, как в функции выше?

Мне по-прежнему кажется, что здесь и в абзаце выше написание аргументов должно совпадать. Либо тут dest, либо там dst.

Просмотр дизассемблера, так?

У меня оторвано.

Dead link. I was able to find an archive version here for replacement: https://web.archive.org/web/20180917203934/https://publicdomainmanifesto.org/manifesto.html

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