Author response:

Response to Reviewer #1:

“Claiming a possible therapeutic role for this gene is a bit far-fetched at the present state of the art”.

We agree that while the therapeutic relevance of Svep1 is not clear at this point, this potential is always something we consider in interpreting our data.

Response to Reviewer #2:

a. “The weakness of this paper is that it does not present a convincing explanation for how Svep1 regulates any of the phenotypes described. In this regard, a demonstration of a genetic interaction between Svep1 and FGF9 mutants or a careful characterization of a tissue-specific knockdown of Svep1, could be insightful. In addition, a comparison of the phenotype of Svep1 mutants and the phenotypes of other mutants affecting ECM components would be worthwhile”. 

We agree that additional experiments are needed to determine how exactly Svep1 contributes to the phenotypes described. While our preliminary data point to an interaction of Svep1 and Fgf9, we agree that additional data are needed to prove that such interaction is a primary driver of the phenotypes observed.

b. “A minor weakness is that the title of the paper is not fully supported by the data presented. While the defects in the morphogenesis of the distal lung in Svep1 mutants presage a defect in alveolar differentiation, this cannot be formally demonstrated since the animals die soon after birth”

The reviewer is correct that we cannot formally demonstrate this in the current model. The profound defects observed in Svep1 mutants lead to early death, making it challenging to study the full process of alveolarization. However, it is important to note that lung morphogenesis is a continuous process in which earlier developmental phases lay the groundwork for subsequent stages. During the branching phase, the fate of alveolar cell types is established, while the saccular stage serves as a critical foundation for alveolar development, where alveolar cells begin to differentiate. We believe that the significant abnormalities in cellular differentiation observed prior to the bulk of alveolarization indicate likely defects in the later stages of alveolar differentiation. Therefore, while the model limits our ability to directly assess alveolarization, we anticipate that defects in cellular differentiation will continue to manifest beyond the saccular stage in Svep1 mice.

for - adjacency - the largest companies in the world have more capital than many countries - the society of the spectacle - lobby industry

adjacency - between - the largest multi-national companies in the world have more capital than many countries - the society of the spectacle - adjacency relationship - It is a well publicized fact that the world's largest multi-national companies have more capital than many countries - This fact is a prime example of the conclusions of the society of the spectacle, - Governments are coopted to serve the needs of the multi-nationals through corporate lobbyists - In fact, multi-national corporations are called "multi-national" precisely because they are so large that they exceed the boundaries of nation states, they are LARGER than nation states - Advertising, movies and products all flow trans-nationally across political boundaries - Military weapons developed by the military industrial complex and sold to nation states make modern warefare between them exponentially more harmful - In the end, the elites within such corporations benefit from the most from the consumption - The diversion is towards maximizing their profits at the expense of all else: - people - the environment - life on earth

for - adjacency - the society of the spectacle - internet society of modernity - Deep Humanity - BEing journey - to discover the society of the spectacle

Being journey - to discover the society of the spectacle - Modernity, so steeped in social media and the internet is INDEED a new mode of living and perceiving the world - To discover the extend to which we have socially normalized a social pathology, we can introduce BEing journeys that help us explore how a life that is freed from the social norm feels like

for - definition - the spectacle - context - the society of the spectacle - cacooning - the spectacle - social media - the spectacle

definition - the spectacle - context - the society of the spectacle - A society where images presented by mass media / mass entertainment not only dominate - but replaces real experiences with a superficial reality that is - focused on appearances designed primarily to distract people from reality - This ultimately disconnects them from - themselves and - those around them

comment - How much does our interaction with virtual reality of - written symbols - audio - video - two dimensional images - derived from our screens both large and small affect our direct experience of life? - When people are distracted by such manufactured entertainment, they have less time to devote to important issues and connecting with real people - We can sit for hours in social isolation, ignoring our bodies need for exercise and our emotional need for real social connection - We can ignore the real crisis going on in the world and instead numb ourselves out with contrived entertainment

for - insight - society of the spectacle - secrecy and diversion is inherent to it

insight - society of the spectacle - secrecy and diversion is inherent to it - it's a society where things happen normally like in any other society but - where we don't know who is pulling the strings - Its main objective is - to divert people's attention by - hiding the real and - promoting the Irrelevant

for - who is - Guy Debord - to - Wikipedia - Situationist International

who is - Guy Debord - Guy Debord was - French - born in 1931 in Paris - Marxist theorist, - philosopher - filmmaker - founding member of the Situationist International

to - Wikipedia - Situationist International - https://hyp.is/muSjgIR4Ee-IdBeTNYoyrg/en.wikipedia.org/wiki/Situationist_International

We're already there; GPT injected with enough domain-specific contexts beats out incompetent software engineers -- many of whom have real jobs at real companies. Asking AI to roll an implementation is far easier than asking an intern today.

Author is dodging the topic: AI will replace many agronomists. A language model competent enough to surface any data you need with a question can replace people; the productivity of one person is multiplied, eliminating the need for other people in that role. Language models disproportionately reward experts, and multiplying the productivity of an expert eliminates jobs.

What is manas Sarovar ?

What is Angsi

What is Barzakh ??

Who is Juno? How this name came from?

(Experimental question)

I don't believe this. You can't develop these models without web-scale data.

eLife Assessment

The study presents a very well-illustrated specimens of the artiopodan Cindarella eucalla from the Chengjiang Biota, using computer tomography (CT) scanning to illustrate multiple specimens with preserved appendages, a rarity in artiopodans. The description of these fossils is important for expanding our understanding of this taxon and its relatives. The imaging and morphological description are followed by a discussion of how this morphology relates to other Cambrian arthropods and its potential ecological function. The evidence provided in this section about resulting function and ecology is presently incomplete and the conclusions are put forward too strongly. This assessment could be improved if the work is revised with more careful wording and additional data.

Reviewer #1 (Public review):

Summary:

The manuscript by Zhang et al. analyzed 17 specimens of Cindarella eucalla with 3D technology and discussed the anatomical findings, the relationship to other artiopods, and the ecology of the animal. The results are excellent and the findings are very interesting. However, the discussion needs to be extended, as the point the authors are trying to make is not always clear. I also recommend some restructuring of the discussion. Overall this is an important manuscript, and I'm looking forward to reading the edited version.

Strengths:

The analyses, the 3D data is excellent and provides new information.

Weaknesses:

The discussion - the authors provide information for the findings, but do not discuss them in detail. More information is needed.

Reviewer #2 (Public review):

Summary:

Zhang et al. present very well-illustrated specimens of the artiopodan Cinderella eucalla from the Chengjiang Biota. Multiple specimens are shown with preserved appendages, which is rare for artiopodans and will greatly help our understanding of this taxon. The authors use CT scanning to reveal the ventral organization of this taxon. The description of the taxon needs some modification, specifically expansion of the gut and limb morphology. The conclusion that Cinderella was a fast-moving animal is very weak, comparisons with extant fast animals and possibly FEA analyses are necessary to support such a claim. Although the potential insights provided by such well-preserved fossils could be valuable, the claims made are tenuous and based on the available evidence presented herein.

Strengths:

The images produced through CT scanning specimens reveal the very fine detail of the appendages and are well illustrated. Specimens preserve guts and limbs, which are informative both for the phylogenetic position and ecology of this taxon. The limbs are very well preserved, with protopodite, exopodite, and endopodites visible. Addressing the weaknesses below will make the most of this compelling data that demonstrates the morphology of the limbs well.

Weaknesses:

Although this paper includes very well-illustrated fossils, including new information on the eyes, guts, and limbs of Cinderella, the data are not fully explained, and the conclusions are weakly supported.

The authors suggest the preservation of complex ramifying diverticular, but it should be better illustrated and the discussion of the gut diverticulae should be longer, especially as gut morphology can provide insights into the feeding strategy.

The conclusion that Cinderella eucalla was fast, sediment feeding in a muddy environment, is not well supported. These claims seem to be tenuously made without any evidence to support them. The authors should add a new section in the discussion focused on feeding ecology where they explicitly compare the morphology to suspension-feeding artiopodans to justify whether it fed that way or not. To further explore feeding, the protopodite morphology needs to be more carefully described and compared to other known taxa. The function of endites on the endopodite to stir up sediment for particle feeding in a muddy environment would also need to be more thoroughly discussed and compared with modern analogs. The impact of their findings is not highlighted in the discussion, which is currently more of a review of what has been previously said and should focus more on what insights are provided by the great fossils illustrated by the authors.

The authors argue that their data supports fast escaping capabilities, but it is not clear how they reached that conclusion based on the data available. Is there a way this can be further evaluated? The data is impressive, so including comparisons with extant taxa that display fast escaping strategies would help the authors make their case more compelling. The authors also claim that the limbs of Cinderella are strong, again this conclusion is unclear. Comparison with the limbs of other taxa to show their robustness would be useful. To actually test how these limbs deal with the force and strain applied to them by a sudden burst of movement, the authors could conduct Finite Element Analyses.

Reviewer #3 (Public review):

This paper provides an interesting description of the ventral parts of the Cambrian xandarellid Cindarella eucalla, derived from exceptionally preserved specimens of the Chengjiang Biota. These morphological data are useful for our broad understanding and future research on Xandarellida, and are generally well-represented in the description and accompanying figures. The strengths of this work rest in this morphological description of exceptional fossil material, and this is generally well supported. In addition, the authors put this description in the context of the morphology of other xandarellids and Cambrian arthropod groups, with most of these parallels being useful and reasonably supported, though in several places homology is assumed and this currently lacks evidence. The manuscript goes on to use these morphological data and comparisons to other groups (particularly trilobites) to make suggestions for the ecology of Cindarella eucalla and other xandarellids. The majority of my comments on this work relate to this latter aim - the ecological conclusions drawn are generally derived through morphological comparisons, where a specific morphology has been suggested as an adaption to a particular ecological function in another extinct arthropod group. However, the original suggestions for ecological function are untested, and so remain hypotheses. Despite this, they are frequently presented as truisms to enable ecological conclusions to be drawn for Cindarella eucalla. I have listed my comments and queries on the study below for the authors to address or respond to, and I hope they are useful to the authors.

Comments:

There are a number of ecological and functional morphology conclusions stated that seem put too strongly to be considered sufficiently supported by the evidence given. These relate to both the description of C. eucalla, and comparisons to other extinct arthropod taxa (notably trilobites). Many of these latter statements are assumptions of functional morphology, and should not be repeated as truisms, rather than they represent suggested functions and ecologies based on the known morphological descriptions. This aspect occurs throughout the article, and, for me, is the primary concern.

Author response:

Reviewer #1 (Public review):

Summary:

The manuscript by Zhang et al. analyzed 17 specimens of Cindarella eucalla with 3D technology and discussed the anatomical findings, the relationship to other artiopods, and the ecology of the animal. The results are excellent and the findings are very interesting. However, the discussion needs to be extended, as the point the authors are trying to make is not always clear. I also recommend some restructuring of the discussion. Overall this is an important manuscript, and I'm looking forward to reading the edited version.

Strengths:

The analyses, the 3D data is excellent and provides new information.

Weaknesses:

The discussion - the authors provide information for the findings, but do not discuss them in detail. More information is needed.

We are committed to enhancing the quality of our manuscript further and, in response to your comments, will implement the following improvements:

(1) Comparative Analysis of Eyes: We will expand our discussion to include a detailed comparative analysis of the eyes of Cindarella eucalla with those of other artiopods (e.g. Xandarellids, trilobites, living insects), focusing on morphology, size, and other relevant characteristics.

(2) Segmental Mismatch Discussion: We will provide an in-depth exploration of the specifics and significance of the segmental mismatch to offer a clearer understanding of its implications. We will also compare the characteristics of this mismatch in our focal species with those observed in extant arthropods, such as spiders and myriapods. This comparison will be further enriched by integrating our phylogenetic analysis, thereby providing a broader evolutionary context.

(3) Methodological Clarity: We will provide more detailed information on the parameters used for the analyses in the Methods section, especially the phylogenetic sections and the X-ray tomography section.

(4) Phylogenetic Analysis: We will engage in a more in-depth discussion of certain characters (e.g. anterior sclerite, hypostome, endopodite, segmental mismatch, etc.) within our phylogenetic analyses to clarify their relevance and contribution to our findings.

Reviewer #2 (Public review):

Summary:

Zhang et al. present very well-illustrated specimens of the artiopodan Cinderella eucalla from the Chengjiang Biota. Multiple specimens are shown with preserved appendages, which is rare for artiopodans and will greatly help our understanding of this taxon. The authors use CT scanning to reveal the ventral organization of this taxon. The description of the taxon needs some modification, specifically expansion of the gut and limb morphology. The conclusion that Cinderella was a fast-moving animal is very weak, comparisons with extant fast animals and possibly FEA analyses are necessary to support such a claim. Although the potential insights provided by such well-preserved fossils could be valuable, the claims made are tenuous and based on the available evidence presented herein.

Strengths:

The images produced through CT scanning specimens reveal the very fine detail of the appendages and are well illustrated. Specimens preserve guts and limbs, which are informative both for the phylogenetic position and ecology of this taxon. The limbs are very well preserved, with protopodite, exopodite, and endopodites visible. Addressing the weaknesses below will make the most of this compelling data that demonstrates the morphology of the limbs well.

Weaknesses:

Although this paper includes very well-illustrated fossils, including new information on the eyes, guts, and limbs of Cinderella, the data are not fully explained, and the conclusions are weakly supported.

The authors suggest the preservation of complex ramifying diverticular, but it should be better illustrated and the discussion of the gut diverticulae should be longer, especially as gut morphology can provide insights into the feeding strategy.

The conclusion that Cinderella eucalla was fast, sediment feeding in a muddy environment, is not well supported. These claims seem to be tenuously made without any evidence to support them. The authors should add a new section in the discussion focused on feeding ecology where they explicitly compare the morphology to suspension-feeding artiopodans to justify whether it fed that way or not. To further explore feeding, the protopodite morphology needs to be more carefully described and compared to other known taxa. The function of endites on the endopodite to stir up sediment for particle feeding in a muddy environment would also need to be more thoroughly discussed and compared with modern analogs. The impact of their findings is not highlighted in the discussion, which is currently more of a review of what has been previously said and should focus more on what insights are provided by the great fossils illustrated by the authors.

The authors argue that their data supports fast escaping capabilities, but it is not clear how they reached that conclusion based on the data available. Is there a way this can be further evaluated? The data is impressive, so including comparisons with extant taxa that display fast escaping strategies would help the authors make their case more compelling. The authors also claim that the limbs of Cinderella are strong, again this conclusion is unclear. Comparison with the limbs of other taxa to show their robustness would be useful. To actually test how these limbs deal with the force and strain applied to them by a sudden burst of movement, the authors could conduct Finite Element Analyses.

Here are the key points we plan to address:

(1) Gut and Limb Morphology: We will expand our description of the gut and limb morphology of C. eucalla, providing a more detailed comparison and analysis. This will include a revised discussion on the function and ecological implications of these features.

(2) Fast-Moving Animal Claim: We acknowledge your concern about the conclusion that C. eucalla was a fast-moving animal. We will conduct a more detailed comparison among C. eucalla and other Cambrian artiopods and living arthropods, focusing on morphological and functional aspects. We will also reconsider our claim and will be more cautious in our conclusions. If the comparison proves insufficient, we will remove this assertion from the manuscript. But we may no longer conduct Finite Element Analysis, as a comprehensive and cautious analysis would require a massive project to complete.

(3) Sediment Feeding in a Muddy Environment: We will revise the section discussing the feeding ecology of C. eucalla. We will enhance this section by comparing the morphology of C. eucalla to that of suspension-feeding artiopods, which will help to substantiate our claims. Additionally, we will expand the discussion to include a more detailed examination of endites, gnathobases, and other relevant anatomical structures.

(4) Impact of Findings: We will endeavor to highlight the impact of our findings in the discussion, focusing on the insights provided by the well-preserved fossils illustrated in our study.

Reviewer #3 (Public review):

This paper provides an interesting description of the ventral parts of the Cambrian xandarellid Cindarella eucalla, derived from exceptionally preserved specimens of the Chengjiang Biota. These morphological data are useful for our broad understanding and future research on Xandarellida, and are generally well-represented in the description and accompanying figures. The strengths of this work rest in this morphological description of exceptional fossil material, and this is generally well supported. In addition, the authors put this description in the context of the morphology of other xandarellids and Cambrian arthropod groups, with most of these parallels being useful and reasonably supported, though in several places homology is assumed and this currently lacks evidence. The manuscript goes on to use these morphological data and comparisons to other groups (particularly trilobites) to make suggestions for the ecology of Cindarella eucalla and other xandarellids. The majority of my comments on this work relate to this latter aim - the ecological conclusions drawn are generally derived through morphological comparisons, where a specific morphology has been suggested as an adaption to a particular ecological function in another extinct arthropod group. However, the original suggestions for ecological function are untested, and so remain hypotheses. Despite this, they are frequently presented as truisms to enable ecological conclusions to be drawn for Cindarella eucalla. I have listed my comments and queries on the study below for the authors to address or respond to, and I hope they are useful to the authors.

Comments:

There are a number of ecological and functional morphology conclusions stated that seem put too strongly to be considered sufficiently supported by the evidence given. These relate to both the description of C. eucalla, and comparisons to other extinct arthropod taxa (notably trilobites). Many of these latter statements are assumptions of functional morphology, and should not be repeated as truisms, rather than they represent suggested functions and ecologies based on the known morphological descriptions. This aspect occurs throughout the article, and, for me, is the primary concern.

We plan to address the following points in upon revision:

(1) Homology Assumptions: You pointed out that we have assumed homology in certain instances without sufficient evidence. We will revise the manuscript to include a more detailed analysis of the anterior sclerite and exite, considering phylogenetic relationships and morphological comparisons to provide a more robust discussion.

(2) Ecological and Functional Morphology: We acknowledge that our conclusions regarding the ecological function were presented with too much certainty. We will adopt a more cautious approach in our discussion, ensuring that our ideas are clearly labeled as such and are supported by a comparison of relevant studies on Cambrian artiopods and extant arthropods, including fluid dynamics, functional morphology, etc. We will re-evaluate the ecological function section, and if it does not adds value and clarity to the manuscript—our speculations do not contribute to the understanding of the specimen or may lead to misunderstandings—we will remove the relevant parts. We believe future changes reflect a more cautious and rigorous approach to the ecological and functional interpretations of C. eucalla.

Résumé de la vidéo [00:00:00][^1^][1] - [00:21:39][^2^][2]:

Cette vidéo traite de l'augmentation inquiétante des cancers du côlon chez les jeunes. Elle présente des témoignages de patients et des explications médicales sur les causes possibles et les efforts de recherche en cours.

Moments forts: + [00:00:45][^3^][3] Témoignage de Christina * Diagnostiquée à 31 ans avec un cancer du côlon stade 4 * Subit une opération majeure et une chimiothérapie * Rémission après 5 ans + [00:03:47][^4^][4] Augmentation des cas chez les jeunes * Augmentation des cancers du côlon chez les moins de 50 ans * 13% d'augmentation en 2022 en Europe * Nécessité d'une nouvelle approche médicale + [00:05:01][^5^][5] Symptômes et diagnostic * Symptômes courants : sang dans les selles, douleurs abdominales, perte de poids * Importance de consulter en cas de symptômes * Retard de diagnostic fréquent chez les jeunes + [00:09:01][^6^][6] Études et recherches * Études en cours pour comprendre les causes * Hypothèses sur l'inflammation et le microbiome * Différences immunitaires entre jeunes et âgés + [00:16:42][^7^][7] Facteurs de risque environnementaux * Pollution, pesticides, microplastiques * Études pour identifier les liens avec le cancer * Importance de la recherche pour des solutions préventives

Вот пример, и на него можно ответить ниже.

https://web.archive.org/web/20241007071434/https://www.dbreunig.com/2024/10/03/we-need-help-with-discovery-more-than-generation.html

Author says generation isn't a problem to solve for AI, there's enough 'content' as it is. Posits discovery as a bigger problem to solve. The issue there is, that's way more personal and less suited for VC funded efforts to create a generic tool that they can scale from the center. Discovery is not a thing, it's an individual act. It requires local stuff, tuned to my interests, networks etc. Curation is a personal thing, providing intent to discovery. Same why [[Algemene event discovery is moeilijk 20150926120836]], as [[Event discovery is sociale onderhandeling 20150926120120]] Still it's doable, but more agent like than central tool.

for - definition - the spectacle - Situationist International - adjacency - the spectacle - manufacturing consent

definition - the spectacle - Sittuationist International - A unified critique offered by the Situationist International of advanced capitalism - The critique was concerned with the insidious use of mass media and entertainment to subvert individual expression through lived experience by - substituting it with mass media images as proxies to directly lived experiences - which leads to mass consumption of commodities produced by advanced captalism - creates far-reaching passive second-hand alienation that harms both the individual and society

adjacency - between - the spectacle - manufacturing consent - adjacency relationship - The spectacle is closely related to Noam Chomsky's work on manufacturing consent

for - book - The Society of the Spectacle - definition - recuperation - from - youtube - The Society of the Spectacle - politics - Marxist group - Situationist International

definition - recuperation - A technique of the spectacle whereby - Official culture is considered a "rigged game" - Conservative powers forbid subversive ideas to have direct access to public discourse - Subversive ideas must first - get trivialized - get sterilized - before they are safely incorporated back within mainstream society - where they lose their agential power and - they can be exploited to add new flavors and bolster the status quo dominant ideas of the rigged game

from - youtube - The Society of the Spectacle - https://hyp.is/K2b2OIR5Ee-khSfaPJUKWg/www.youtube.com/watch?v=93jXDJhi6_c

eLife Assessment

The paper presents a new pipeline for functional validation of genes known to underlie fragile bone disorders, using CRISPR-mediated knockouts and a number of phenotypic assessments in zebrafish. The solid data demonstrate the feasibility and validity of the approach, which presents a valuable tool for rapid functional validation of candidate gene(s) associated with heritable bone diseases identified from genetic studies.

Reviewer #1 (Public review):

Summary:

In this work, a screening platform is presented for rapid and cost-effective screening of candidate genes involved in Fragile Bone Disorders. The authors validate the approach of using crispants, generating FO mosaic mutants, to evaluate the function of specific target genes in this particular condition. The design of the guide RNAs is convincingly described, while the effectiveness of the method is evaluated to 60% to 92% of the respective target genes being presumably inactivated. Thus, injected F0 larvae can be directly used to investigate the consequences of this inactivation.

Skeletal formation is then evaluated at 7dpf and 14dpf, first using a transgenic reporter line revealing fluorescent osteoblasts, and second using alizarin-red staining of mineralized structures. In general, it appears that the osteoblast-positive areas are more often affected in the crispants compared to the mineralized areas, an observation that appears to correlate with the observed reduced expression of bglap, a marker for mature osteoblasts, and the increased expression of col1a1a in more immature osteoblasts.

Finally, the injected fish (except two lines that revealed high mortality) are also analyzed at 90dpf, using alizarin red staining and micro-CT analysis, revealing an increased incidence of skeletal deformities in the vertebral arches, fractures, as well as vertebral fusions and compressions for all crispants except those for daam2. Finally, the Tissue Mineral Density (TMD) as determined by micro-CT is proposed as an important marker for investigating genes involved in osteoporosis.

Taken together, this manuscript is well presented, the data are clear and well analyzed, and the methods are well described. It makes a compelling case for using the crispant technology to screen the function of candidate genes in a specific condition, as shown here for bone disorders.

Strengths:

Strengths are the clever combination of existing technologies from different fields to build a screening platform. All the required methods are comprehensively described.

Weaknesses:

One may have wished to bring one or two of the crispants to the stage of bona fide mutants, to confirm the results of the screening, however, this is done for some of the tested genes as laid out in the discussion.

Reviewer #2 (Public review):

Summary:

More and more genes and genetic loci are being linked to bone fragility disorders like osteoporosis and osteogenesis imperfecta through GWAS and clinical sequencing. In this study, the authors seek to develop a pipeline for validating these new candidate genes using crispant screening in zebrafish. Candidates were selected based on GWAS bone density evidence (4 genes) or linkage to OI cases plus some aspect of bone biology (6 genes). NGS was performed on embryos injected with different gRNAs/Cas9 to confirm high mutagenic efficacy and off-target cutting was verified to be low. Bone growth, mineralization, density, and gene expression levels were carefully measured and compared across crispants using a battery of assays at three different stages.

Strengths:

(1) The pipeline would be straightforward to replicate in other labs, and the study could thus make a real contribution towards resolving the major bottleneck of candidate gene validation.

(2) The study is clearly written and extensively quantified.

(3) The discussion attempts to place the phenotypes of different crispant lines into the context of what is already known about each gene's function.

(4) There is added value in seeing the results for the different crispant lines side by side for each assay.

Weaknesses:

(1) The study uses only well-established methods and is strategy-driven rather than question/hypothesis-driven.

(2) Some of the measurements are inadequately normalized and not as specific to bone as suggested:

(a) The measurements of surface area covered by osteoblasts or mineralized bone (Figure 1) should be normalized to body size. The authors note that such measures provide "insight into the formation of new skeletal tissue during early development" and reflect "the quantity of osteoblasts within a given structure and [is] a measure of the formation of bone matrix." I agree in principle, but these measures are also secondarily impacted by the overall growth and health of the larva. The surface area data are normalized to the control but not to the size/length of each fish - the esr1 line in particular appears quite developmentally advanced in some of the images shown, which could easily explain the larger bone areas. The fact that the images in Figure S5 were not all taken at the same magnification further complicates this interpretation.

(b) Some of the genes evaluated by RT-PCR in Figure 2 are expressed in other tissues in addition to bone (as are the candidate genes themselves); because whole-body samples were used for these assays, there is a nonzero possibility that observed changes may be rooted in other, non-skeletal cell types.

(3) Though the assays evaluate bone development and quality at several levels, it is still difficult to synthesize all the results for a given gene into a coherent model of its requirement.

(4) Several additional caveats to crispant analyses are worth noting:

(a) False negatives, i.e. individual fish may not carry many (or any!) mutant alleles. The crispant individuals used for most assays here were not directly genotyped, and no control appears to have been used to confirm successful injection. The authors therefore cannot rule out that some individuals were not, in fact, mutagenized at the loci of interest, potentially due to human error. While this doesn't invalidate the results, it is worth acknowledging the limitation.

(b) Many/most loci identified through GWAS are non-coding and not easily associated with a nearby gene. The authors should discuss whether their coding gene-focused pipeline could be applied in such cases and how that might work.

Reviewer #3 (Public review):

Summary:

The manuscript "Crispant analysis in zebrafish as a tool for rapid functional screening of disease-causing genes for bone fragility" describes the use of CRISPR gene editing coupled with phenotyping mosaic zebrafish larvae to characterize functions of genes implicated in heritable fragile bone disorders (FBDs). The authors targeted six high-confident candidate genes implicated in severe recessive forms of FBDs and four Osteoporosis GWAS-implicated genes and observed varied developmental phenotypes across all crispants, in addition to adult skeletal phenotypes.

A major strength of the paper is the streamlined method that produced significant phenotypes for all candidate genes tested.

A major weakness is a lack of new insights into underlying mechanisms that may contribute to disease phenotypes, nor any clear commonalities across gene sets. This was most evident in the qRT-PCR analysis of select skeletal developmental genes, which all showed varied changes in fold and direction, but with little insight into the implications of the results.

Ultimately, the authors were able to show their approach is capable of connecting candidate genes with perturbation of skeletal phenotypes. It was surprising that all four GWAS candidate genes (which presumably were lower confidence) also produced a result. These authors have previously demonstrated that crispants recapitulate skeletal phenotypes of stable mutant lines for a single gene, somewhat reducing the novelty of the study.

from: https://hypothes.is/a/M6gibIR6Ee-bBR9gG2hplA

!search - peergos ck editor spell checker

eLife Assessment

This important work advances our understanding of trained immunity, especially in the context of Bacillus Calmette-Guérin (BCG) administration and host-pathogen interactions. The evidence supporting the conclusions are convincing, based on a combination of state-of-the-art omics techniques such as bulk and single-cell RNA sequencing with the use of JAK/STAT signaling inhibitors. The work will be of broad interest to immunologists and infection biologists.

Reviewer #1 (Public review):

Summary:

In the submitted manuscript, Solomon et al carefully detail shifts in tissue-specific myeloid populations associated with trained immunity using intraperitoneal BCG injection as a model for induction. They define the kinetics of shifts in myeloid populations within the spleen and the transcriptional response associated with IP BCG exposure. In lineage tracing experiments, they demonstrate that tissue-resident macrophages, red-pulp macrophages (RPM) that are rapidly depleted after BCG exposure, are replenished from recruited monocytes and expansion of tissue-resident cells; they use transcriptional profiling to characterize those cells. In contrast to previous descriptions of BCG-driven immune training, they do not find BCG in the bone marrow in their model, suggesting that there is not direct training of myeloid precursor populations in the bone marrow. They then link the observed trained immunity phenotype (restriction of heterologous infection with ST) with early activation of STAT1 through IFN-γ.

Strengths:

The work includes careful detaining of shifts and origins of myeloid populations within tissue associated with trained immunity and is a meaningful advance for the field. Given that the temporality of exposure relative to trained immunity phenotypes is a major focus of the work, there are some additional experiments that would make the work stronger.

Weaknesses:

(1) The contribution of persistent BCG in spleen to the observed trained immunity phenotypes is not clear: The trained immunity phenotypes are interpreted as being driven by the early (within days) response to BCG exposure. While the fedratinib data generally support this interpretation, the authors show that BCG remains present in spleen albeit at low levels all the way out to 60 days post exposure. Given that the focus in the paper is on tissue-specific immune training, it would be helpful to know whether the ongoing presence of BCG at low levels in the profiled tissue contributes to the trained immunity phenotypes observed.

(2) Unclear temporality of STAT1/IFN-γ requirement for the trained immunity phenotype: The data demonstrate that STAT1/IFN-γ are required at the earliest time points post-BCG exposure for trained immunity to be initiated. Related to the point about BCG above, it would be helpful to understand whether this is a specifically time-limited requirement when trained immunity is first induced, or whether ongoing signaling through this axis is required for maintenance of the observed trained immunity phenotypes.

Reviewer #2 (Public review):

Summary:

In this study, Solomon and colleagues demonstrate that trained immunity induced by BCG can reprogram myeloid cells within localised tissue, which can sustain prolonged protective effects. The authors further demonstrate an activation of STAT1-dependent pathways.

Strengths:

The main strength of this paper is the in-depth investigation of cell populations affected by BCG training, and how their transcriptome changes at different time points post-training. Through use of flow cytometry and sequencing methods, the authors identify a new cell population derived from classical monocytes. They also show that long-term trained immunity protection in the spleen is dependent on resident cells. Through sequencing, drug and recombinant inhibition of IFNg pathways, the authors reveal STAT1-dependent responses are required for changes in the myeloid population upon training, and recruitment of trained monocytes.

Weaknesses:

A significant amount of work has already been performed for this study. No significant weaknesses found.

testing

Nice coffin

eLife Assessment

This useful study reports that the exogenous expression of the microRNA miR-195 can partially compensate in early B cell development for the loss of EBF1, one of the key transcription factors in B cells. While this finding will be of interest to those studying lymphocyte development, the evidence, particularly with regard to the molecular mechanisms that underpin the effect of miR-195, is currently incomplete.

Reviewer #1 (Public review):

Summary:

Here, the authors are proposing a role for miR-196, a microRNA that has been shown to bind and enhance the degradation of mRNA targets in the regulation of cell processes, and has a novel role in allowing the emergence of CD19+ cells in cells in which Ebf1, a critical B-cell transcription factor, has been genetically removed.

Strengths:

That over-expression of mR-195 can allow the emergence of CD19+ cells missing Ebf1 is somewhat novel.

Their data does perhaps support to a degree the emergence of a transcriptional network that may bypass the absence of Ebf1, including the FOXO1 transcription factor, but this data is not strong or definitive.

Weaknesses:

It is unclear whether this observation is in fact physiological. When the authors analyse a knockout model of miR-195, there is not much of a change in the B-cell phenotype. Their findings may therefore be an artefact of an overexpression system.

The authors have provided insufficient data to allow a thorough appraisal of the step-wise molecular changes that could account for their observed phenotype.

Reviewer #2 (Public review):

Summary:

The authors investigate miRNA miR-195 in the context of B-cell development. They demonstrate that ectopic expression of miR-195 in hematopoietic progenitor cells can, to a considerable extent, override the consequences of deletion of Ebf1, a central B-lineage defining transcription factor, in vitro and upon short-term transplantation into immunodeficient mice in vivo. In addition, the authors demonstrate that the reverse experiment, genetic deletion of miR-195, has virtually no effect on B-cell development. Mechanistically, the authors identify Foxo1 phosphorylation as one pathway partially contributing to the rescue effect of miR-195. An additional analysis of epigenetics by ATACseq adds potential additional factors that might also contribute to the effect of ectopic expression of miR-195.

Strengths:

The authors employ a robust assay system, Ebf1-KO HPC, to test for B-lineage promoting factors. The manuscript overall takes on an interesting perspective rarely employed for the analysis of miRNA by overexpressing the miRNA of interest. Ideally, this approach may reveal, if not the physiological function of this miRNA, the role of distinct pathways in developmental processes.

Weaknesses:

At the same time, this approach constitutes a major weakness: It does not reveal information on the physiological role of miR-195. In fact, the authors themselves demonstrate in their KO approach, that miR-195 has virtually no role in B-cell development, as has been demonstrated already in 2020 by Hutter and colleagues. While the authors cite this paper, unfortunately, they do so in a different context, hence omitting that their findings are not original.

Conceptually, the authors stress that a predominant function of miRNA (in contrast to transcription factors, as the authors suggest) lies in fine-tuning. However, there appears to be a misconception. Misregulation of fine-tuning of gene expression may result in substantial biological effects, especially in developmental processes. The authors want to highlight that miR-195 is somewhat of an exception in that regard, but this is clearly not the case. In addition to miR-150, as referenced by the authors, also the miR-17-92 or miR-221/222 families play a significant role in B-cell development, their absence resulting in stage-specific developmental blocks, and other miRNAs, such as miR-155, miR-142, miR-181, and miR-223 are critical regulators of leukocyte development and function. Thus, while in many instances a single miRNA moderately affects gene expression at the level of an individual target, quite frequently targets converge in common pathways, hence controlling critical biological processes.

The paper has some methodological weaknesses as well: For the most part, it lacks thorough statistical analysis, and only representative FACS plots are provided. Many bar graphs are based on heavy normalization making the T-tests employed inapplicable. No details are provided regarding the statistical analysis of microarrays. Generation of the miR-195-KO mice is insufficiently described and no validation of deletion is provided. Important controls are missing as well, the most important one being a direct rescue of Ebf1-KO cells by re-expression of Ebf1. This control is critical to quantify the extent of override of Ebf1-deficiency elicited by miR-195 and should essentially be included in all experiments. A quantitative comparison is essential to support the authors' main conclusion highlighted in the title of the manuscript. As the manuscript currently stands, only negative controls are provided, which, given the profound role of Ebf1, are insufficient, because many experiments, such as assessment of V(D)J recombination, IgM surface expression, or class-switch recombination, are completely negative in controls. In addition, the authors should also perform long-term reconstitution experiments. While it is somewhat surprising that the authors obtained splenic IgM+ B cells after just 10 days, these experiments would be certainly much more informative after longer periods of time. Using "classical" mixed bone marrow chimeras using a combination of B-cell defective (such as mb1/mb1) bone marrow and reconstituted Ebf1-KO progenitors would permit much more refined analyses.

With regard to mechanism, the authors show that the Foxo1 phosphorylation pathway accounts for the rescue of CD19 expression, but not for other factors, as mentioned in the discussion. The authors then resort to epigenetics analysis, but their rationale remains somewhat vague. It remains unclear how miR-195 is linked to epigenetic changes.

Reviewer #3 (Public review):

Summary:

In this study, Miyatake et al. present the interesting finding that ectopic expression of miR-195 in EBF1-deficient hematopoietic progenitor cells can partially rescue their developmental block and allow B cells to progress to a B220+ CD19+ cells stage. Notably, this is accompanied by an upregulation of B-cell-specific genes and, correspondingly, a downregulation of T, myeloid, and NK lineage-related genes, suggesting that miR-195 expression is at least in part equivalent to EBF1 activity in orchestrating the complex gene regulatory network underlying B cell development. Strengthening this point, ATAC sequencing of miR-195-expressing EBF1-deficient B220+CD19+ cells and a comparison of these data to public datasets of EBF1-deficient and -proficient cells suggest that miR-195 indirectly regulates gene expression and chromatin accessibility of some, but not all regions regulated by EBF1.

Mechanistically, the authors identify a subset of potential target genes of miR-195 involved in MAPK and PI3K signalling. Dampening of these pathways has previously been demonstrated to activate FOXO1, a key transcription factor for early B cells downstream of EBF1. Accordingly, the authors hypothesize that miR-195 exerts its function through FOXO1. Supporting this claim, also exogenous FOXO1 expression is able to promote the development of EBF1-deficient cells to the B220+CD19+ stage and thus recapitulates the miR-195 phenotype.

Strengths:

The strength of the presented study is the detailed assessment of the altered chromatin accessibility in response to ectopic miR-195 expression. This provides insight into how miR-195 impacts the gene regulatory network that governs B-cell development and allows the formation of mechanistic hypotheses.

Weaknesses:

The key weakness of this study is that its findings are based on the artificial and ectopic expression of a miRNA out of its normal context, which in my opinion strongly limits the biological relevance of the presented work.

While the authors performed qPCRs for miR-195 on different B cell populations and show that its relative expression peaks in early B cells, it remains unclear whether the absolute miR-195 expression is sufficiently high to have any meaningful biological activity. In fact, other miRNA expression data from immune cells (e.g. DOI 10.1182/blood-2010-10-316034 and DOI 10.1016/j.immuni.2010.05.009) suggest that miR-195 is only weakly, if at all, expressed in the hematopoietic system.

The authors support their finding by a CRISPR-derived miR-195 knockout mouse model which displays mild, but significant differences in the hematopoietic stem cell compartment and in B cell development. However, they fail to acknowledge and discuss a lymphocyte-specific miR-195 knockout mouse that does not show any B cell defects in the bone marrow or spleen and thus contradicts the authors' findings (DOI 10.1111/febs.15493). Of note, B-1 B cells in particular have been shown to be elevated upon loss of miR-15-16-1 and/or miR-15b-16-2, which contradicts the data presented here for loss of the family member miR-195.

A second weakness is that some claims by the authors appear overstated or at least not fully backed up by the presented data. In particular, the findings that miR-195-expressing cells can undergo VDJ recombination, express the pre-BCR/BCR and class switch needs to be strengthened. It would be beneficial to include additional controls to these experiments, e.g. a RAG-deficient mouse as a reference/negative control for the ddPCR and the surface IgM staining, and cells deficient in class switching for the IgG1 flow cytometric staining.

Moreover, the manuscript would be strengthened by a more thorough investigation of the hypothesis that miR-195 promotes the stabilization and activity of FOXO1, e.g. by comparing the authors' ATACseq data to the FOXO1 signature.

eLife Assessment

This important study presents a new method for generating cell-type restricted knockouts in zebrafish and it reports several interesting applications of this method to study pigmentation and melanomagenesis. The evidence supporting the conclusions is convincing, with rigorous characterization of several knock out mutations that provide a proof of principle. The work will be of broad interest to cell, skin, and cancer biologists.

Reviewer #1 (Public review):

Summary:

Perlee et al. sought to generate a zebrafish line where CRISPR-based gene editing is exclusively limited to the melanocyte lineage, allowing assessment of cell-type restricted gene knockouts. To achieve this, they knocked in Cas9 to the endogenous mitfa locus, as mitfa is a master regulator of melanocyte development. The authors use multiple candidate genes - albino, sox10, tuba1a, ptena/ptenb, tp53 - to demonstrate their system induces lineage-restricted gene editing. This method allows researchers to bypass embryonic lethal and non-cell autonomous phenotypes emerging from whole body knockout (sox10, tuba1a), drive directed phenotypes, such as depigmentation (albino), and induce lineage-specific tumors, such as melanomas (ptena/ptenb, tp53, when accompanied with expression of BRAFV600E). While the genetic approaches are solid, the argued increase in efficiency of this model compared to current tools was untested, and therefore unable to be assessed. Furthermore, the mechanistic explanations proposed to underlie their phenotypes are mostly unfounded, as discussed further in the Weaknesses section. Despite these concerns, there is still a clear use for this genetic methodology and its implementation will be of value to many in vivo researchers.

Strengths:

The strongest component of this manuscript is the genetic control offered by the mitfa:Cas9 system and the ability to make stable, lineage-specific knockouts in zebrafish. This is exemplified by the studies of tuba1a, where the authors nicely show non-cell autonomous mechanisms have obfuscated the role of this gene in melanocyte development. In addition, the mitfa:Cas9 system is elegantly straightforward and can be easily implemented in many labs. Mostly, the figures are clean, controls are appropriate, and phenotypes are reproducible. The invented method is a welcomed addition to the arsenal of genetic tools used in zebrafish.

Weaknesses:

The major weaknesses of the manuscript include the overly bold descriptions of the value of the model and the superficial mechanistic explanations for each biological vignette.

The authors argue that a major advantage of this system is its high efficiency. However, no direct comparison is made with other tools that achieve the same genetic control, such as MAZERATI. This is a missed opportunity to provide researchers the ability to evaluate these two similar genetic approaches. In addition, Fig.1 shows that not all melanocytes express Cas9. This is a major caveat that goes unaddressed. It is of paramount importance to understand the percentage of mitfa+ cells that express Cas9. The histology shown is unclear and too zoomed out of a scale to make any insightful conclusions, especially in Fig.S1. It would also be beneficial to see data regarding Cas9 expression in adult melanocytes, which are distinct from embryonic melanocytes in zebrafish. Moreover, this system still requires the injection of a plasmid encoding gRNAs of interest, which will yield mosaicism. A prime example of this discrepancy is in Fig.6, where sox10 is clearly still present in "sox10 KO" tumors.

The authors argue that their model allows rapid manipulation of melanocyte gene expression. Enthusiasm for the speed of this model is diminished by minimal phenotypes in the F0, as exemplified in Fig.2. Although the authors say >90% of fish have loss of pigmentation, this is misleading as the phenotype is a very weak, partial loss. Only in the F1 generation do robust phenotypes emerge, which takes >6 months to generate. How this is more efficient than other tools that currently exist is unclear and should be discussed in more detail.

In Figure 3, the authors find that melanocyte-specific knockout of sox10 leads to only a 25% reduction in melanocytes in the F1 generation. This is in contradiction to prior literature cited describing sox10 as indispensable for melanocyte development. In addition, the authors argue that sox10 is required for melanocyte regeneration. This claim is not accurate, as >50% of melanocytes killed upon neocuproine treatment can regenerate. This data would indicate that sox10 is required for only a subset of melanocytes to develop (Fig.3C) and for only a subset to regenerate (Fig.3G). This is an interesting finding that is not discussed or interrogated further.

Tumor induction by this model is weak, as indicated by the tumor curves in Figs.5,6. This might be because these fish are mitfa heterozygous. Whereas the avoidance of mitfa overexpression driven by other models including MAZERATI is a benefit of this system, the effect of mitfa heterozygosity on tumor incidence was untested. This is an essential question unaddressed in the manuscript.

In Fig.6, the authors recapitulate previous findings with their model, showing sox10 KO inhibits tumor onset. The tumors that do develop are argued to be highly invasive, have mesenchymal morphology, and undergo phenotypic switching from sox10 to sox9 expression. The data presented do not sufficiently support these claims. The histology is not readily suggestive of invasive, mesenchymal melanomas. Sox10 is still present in many cells and sox9 expression is only found in a small subset (<20%). Whether sox10-null cells are the ones expressing sox9 is untested. If sox9-mediated phenotypic switching is the major driver of these tumors, the authors would need to knockout sox9 and sox10 simultaneously and test whether these "rare" types of tumors still emerge. Additional histological and genetic evaluation is required to make the conclusions presented in Fig.6. It feels like a missed opportunity that the authors did not attempt to study genes of unknown contribution to melanoma with their system.

Overall, this manuscript introduces a solid method to the arsenal of zebrafish genetic tools but falls short of justifying itself as a more efficient and robust approach than what currently exists. The mechanisms provided to explain observed phenotypes are tenuous. Nonetheless, the mitfa:Cas9 approach will certainly be of value to many in vivo biologists and lays the foundation to generate similar methods using other tissue-specific regulators and other Cas proteins.

Reviewer #2 (Public review):

Summary:

This manuscript describes a genetic tool utilizing mutant mitfa-Cas9 expressing zebrafish to knockout genes to analyze their function in melanocytes in a range of assays from developmental biology to tumorigenesis. Overall, the data are convincing and the authors cover potential caveats from their model that might impact its utility for future work.

Strengths:

The authors do an excellent job of characterizing several gene deletions that show the specificity and applicability of the genetic mitfa-Cas9 zebrafish to studying melanocytes.

Weaknesses:

Variability across animals not fully analyzed.

Reviewer #3 (Public review):

Summary:

Perlee et al. present a method for generating cell-type restricted knockouts in zebrafish, focusing on melanocytes. For this method, the authors knock-in a Cas9 encoding sequence into the mitfa locus. This mitfaCas9 line has restricted Cas9 expression, allowing the authors to generate melanocyte-specific knockouts rapidly by follow-up injection of sgRNA expressing transposon vectors.

The paper presents some interesting vignettes to illustrate the utility of their approach. These include 1) a derivation of albino mutant fish as a demonstration of the method's efficiency, 2) an interrogation and novel description of tuba1a as a potential non-autonomous contributor to melanocyte dispersion, and 3) the generation of sox10 deficient melanoma tumors that show "escape" of sox10 loss through upregulation of sox9. The latter two examples highlight the usefulness of cell-type targeted knockouts (Body-wide sox10 and tuba1a loss elicit developmental defects). Additionally, the tumor models involve highly multiplexed sgRNAs for tumor initiation which is nicely facilitated by the stable Cas9.

Strengths:

The approach is clever and could prove very useful for studying melanocytes and other cell types. As the authors hint at in their discussion, this approach would become even more powerful with the generation of other Cas9-restricted lineages so a single sgRNA construct can be screened across many lineages rapidly (or many sgRNA and fish lines screened combinatorially).

The biological findings used to demonstrate the power of the approach are interesting in their own right. If it proves true, tuba1a's non-autonomous effects on melanosome dispersion are striking, and this example demonstrates very nicely how one could use Perlee et al.'s approach to search for other non-autonomous mechanisms systematically. Similarly, the observation of the sox9 escape mechanism with sox10 loss is a beautiful demonstration of the relevance of SOX10/SOX9's reciprocal regulation in vivo. This system would be a very nice model for further interrogating mechanisms/interventions surrounding Sox10 in melanoma.

Finally, the figure presentation is very nice. This work involves complex genetic approaches including multiple fish generations and multiplexed construct injections. The vector diagrams and breeding schemes in the paper make everything very clear/"grok-able," and the paper was enjoyable to read.

Weaknesses:

The mitfa-driven GFP on their sgRNA-expressing cassette is elegant, but it makes one wonder why the endogenous knock-in is necessary. It would strengthen the motivation of the work if the authors could detail the potential advantages and disadvantages of their system compared to expressing Cas9 with a lineage-specific promoter from a transposon in their introduction or discussion.

Related to the above - is mitfa haplosufficient? If the mitfaCas9/+ fish have any notable phenotypes, it would be worth noting for others interested in using this approach to study melanoma and pigmentation.

A core weakness (and also potential strength) of the system is that introduced edits will always be non-clonal (Fig 2H/I). The activity of individual sgRNAs should always be validated in the absence of any noticeable phenotype to interpret a negative result. Additionally, caution should be taken when interpreting results from rare events involving positive outgrowth (like tumorogenesis) to account for the fact many cells in the population might not have biallelic null alleles (i.e., 100% of the gene product removed).

Along those lines: in my opinion, the tuba1a results are the most provocative finding in the paper, but they lack key validation. With respect to cutting activity, the Alt-R and transgenic sgRNA expression approaches are not directly comparable. Since there is no phenotype in the melanocyte specific tuba1a knockouts, the authors must confirm high knockout efficiency with this set of reagents before making the claim there is a non-autonomous phenotype. This can be achieved with GFP+ sorting and NGS like they performed with their albino melanocytes.

The whole-body tuba1a knockout phenotype is expected to be pleiotropic, and this expectation might mask off-target effects. Controls for knockout specificity should be included. For instance, confidence in the claims would greatly increase if the dispersed melanosome phenotype could be recovered with guide-resistant tuba1a re-expression and if melanocyte-restricted tuba1a re-expression failed to rescue. As a less definitive but adequate alternative, the authors could also test if another guide or a morpholino against tuba1a phenocopies the described Alt-R edited fish.

I have similar questions about the sox10 escapers, but these suggestions are less critical for supporting the authors claims (especially given the nice staining). Are the sox10 tumors relatively clonal with respect to sox10 mutations? And are the sox10 tumor mutations mostly biallelic frameshifts or potential missense mutations/single mutations that might not completely remove activity? I am particularly curious as SOX10 doesn't seem to be completely absent (and is still very high in some nuclei) in the immunohistochemistry.

Poverty is a big issue that affects opportunities but like stated in someone else anootation, it affects physical health. It takes a toll when it comes down to burning yourself out because there is so much financial stress that if you have a job, we can not afford missing work because that day will affect us. It makes us contemplate and think about so many things before actually putting ourselves first. It is sad to say that it is the hard reality even if we do not like it.

This is an example of literally reality today and i can put it in my perspective. I would be first generation graduating knowing that both of my parents did not complete school. It is harder because they had no other option but to work and find different ways to find income. I didn't have the opportunity to be fully raised by my parents because they. would always be working but i'm happy to say that i am able to see what they did for me. Seeing others have a different luxury does emphasize that ones circumstances shape pnes future on how they decide to manage situations.

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Statistics is always something that can be used to mislead or trick readers. In this example, it mentions only 5% of users being bots, but doesn't mention what counts as a user. There could be many inactive accounts lowering the overall number of bots, but not lowering the percentage of bots that users see.

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Résumé de la vidéo [00:00:00][^1^][1] - [00:16:30][^2^][2]:

Cette vidéo explore le dilemme moral de diffuser ou non une interview controversée avec un leader du Jihad islamique à Gaza. Elle aborde les thèmes de la responsabilité des créateurs de contenu, de la manipulation des écritures religieuses et de la quête de la confiance et du respect mutuel dans les discussions.

Temps forts: + [00:00:00][^3^][3] Introduction et dilemme * Présentation du dilemme moral * Rôle et responsabilité en tant que créateur de contenu * Impact potentiel de la diffusion de l'interview + [00:01:02][^4^][4] Développement personnel * Importance de la guérison sociale * Comparaison avec d'autres justifications de la violence * Importance de la confiance et du respect + [00:05:00][^5^][5] Expérience personnelle * Réflexion sur le passé de l'intervieweur * Impact des personnages publics sur la perception * Transition vers un rôle de guérisseur social + [00:10:00][^6^][6] Débat et discours * Différence entre débat et discours * Importance de l'écoute et du respect * Techniques pour des discussions constructives + [00:14:00][^7^][7] Conclusion et résolution * Décision de ne pas diffuser l'interview * Importance de créer un terrain d'entente * Réflexion sur les méthodes de communication efficaces

I've always felt weird seeing these kinds of posts on my feed. It feels weird morally to post about something like helping the less fortunate. The posts make the good deed feel disingenuous.

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Dealing with stripped screws

I found this to be the main reason against grading. I understand that grading makes students study hard in order to get the grade but I often wonder if does effect the desire to learn in a lot of students.

This is probably actually true of a lot of criminal activity. None of it occurs in a vacuum due to "naturally bad people," and none of it is actually solved by our current punitive justice system.

This article was written in 2015. The narrative of gambling addition is much more common nowadays, especially in the sphere of online gaming.

This study has actually come under much controversy over the years for some flaws in methodology and the fact that its results have not been replicated (see "Environment Is Not the Most Important Variable in Determining Oral Morphine Consumption in Wistar Rats" by Bruce Petrie)

The real stories of addiction and treatment never make for as good TV as the damaging "radical" stories in shows like that. Obviously every story is different, but it often only perpetuates the cycle of harm further to be cold, cruel, and isolating to an addict, like Hari observes.

A lot of issues that hurt communities have their blame placed on individual failures, and ignore the need for societal change to benefit all. I appreciate this focus in the article.

I see where he's coming from with this; as much as the internet does to foster community, it is completely unmatched compared to the experience of actually going out in the real world and connecting with said communities in a tangible way.

Consistently, housing is the key for lowering recidivism and guaranteeing those in poverty are healthy and connected to their community.

Again, the author goes to great lengths to establish their credibility on the subject

It's interesting to see the author comparing soldiers to rats, especially considering they're specifically Vietnam War soldiers, probably most morally dubious war of its time.

Several appeals to logos made here, constructing the foundation for the upcoming argument on a solid base of evidence.

This passage and the previous one work to display the author's personal connection to the subject, establishing their credibility to discuss it.

I've heard lots of stories about people who start with a prescription and then turn to street drugs when their doctor stops prescribing. Did they stop using because the pain was cured or because the drugs were labeled medical? If the pain was cured through medical intervention, it makes sense they would be able to stop. But for people with chronic pain, would this be different?

preach

again this is a crazy leap to make without more details

good example of how use of language can greatly impact perspective

But it's not the 'same drug,' the author just said it's a more pure version that hasn't been tampered with.

And fighting what exactly? People who need help? I really wonder who/where this concept of "war" came from. Was it Reagan/federal government? The police? Wouldn't be surprised. The concept seems like it most likely did not come from any recovered addict.

The language that the author chose to use here is interesting. Their argument is that we can't criminalize addicts, but they go on to frame this phrase like this

This makes sense, and lines up with my previous knowledge about addiction. People with trauma or who live in difficult environments are much more likely to become addicted. There have been many studies about the relationship between ACE scores and addiction, physical/mental health issues, and early death.

this is so cool

Very interesting way to describe addiction, definitely agree and appreciate the new insight

The internet is not quite a travesty, but there is some truth in his point. Immersing myself in the internet as a kid did the opposite of motivating or cultivating friendships, so it wasn't able to be directly beneficial in that way. Still, it was a way of feeling that I was closer to understanding and having a chance to participate in a more reasonable and accepting social environment than could be found outside of it with as much abuse I got back then

I don't like the structure of this sentence/paragraph. When I read a paragraph where most sentences start with "From", I expect the last sentence to start with "To". I had to re-read in order to grasp the meaning, structure feels stilted

arguably either good for or damaging to the author's credibility

This sets up a tone for the author, and gives the reader an idea of what themes the author is going to discuss

Starting by introducing rat experiments then shifting to something similar with humans helped both pieces of evidence feel cohesive

That is true. When people think of addiction they will always think of alcohol or drug addictions when addiction can be more than just physical but also bad habits or methods

That's really impressive and interesting. However, I also heard that people still developed addiction even if the drugs are "safer" and "cleaner" than the ones you would find on the streets.

This is the first time I heard that there was some sort of drug addiction during the Vietnam War

That impressive the rats knew right away what was wrong with the water and would avoid drinking the water.

The American dream is held accountable for each student as they go to school. I think seeing the difference back then in where it was more implemented to be college for certain and now they don't really push you to that extent. Like stated in the last paragraph it all comes down to individual success and how one decides to take initiative in education and goods.

BOOM.... THESE ARE COVID MICRO VESSICLES.. THEY COULD CONTAIN ANY COMPONENT OR COMPLETE VIRIONS OF COVID

13页--Q

while sports do move people to pursue things i dont think it can be compared to religion

i think it would be interesting to write an essay on that question

i dont think sports should be compared to religion

Sacrifice is not just a Christian thing, Aztecs and Greeks also had sacrifices to their gods.

Would there be a way to break down the text? I feel like it would be hard to understand.

I agree. This would attract many as it would me. It would be so interesting to learn about this new belief and perspective.

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Cor was not organized, caused enemy to go agaisnt them even stronger

commons wanted to keep some Athenian ships and send some of their people as proof of peace, when Athenians refused they took it as bad intention and armed themselves, Athenians tried to get peace again

Athenian attempt at peace

didn't think tear gas was needed at a sports game

There area many athletes that embody what it is like to love a sport such as babe ruth

sports are what keep us entertained and for some keep us going as it is their passion

An instance of completive rivalry going to far.

Many pieces of art were created based on this historical event. It influenced the world.

Maurice Richard became a symbol. He fought for the resistance of English dominance.

i find it interesting that the critics underestimated a determined man

teammates are all usually there to help defend and support one another

hockey games are usually full of violence but never so bad that the crowd can sense something bad

People will do crazy things for people they love, even if it is a fan celebrity relationship

If I read this 4 years ago I would wonder how could people do this, now how ever, I can easily see this happen.

Honestly that is not all that surprising since that can be true with a lot of things with fandoms, weather it be movies, video games, comics, and especially with sports judging how popular foot ball is.

This is why his influence was so heavy.

It's interesting hearing the breakdown of events. It was more intense than I imagined.

crazy to think there was that many arrests

He didn't think that he did anything fully wrong and only took partial responsibly for his actions

He continued to persevere through it all. Shows the cultural significant impact he had.

Was it the beginning? It definitely triggered something...

This sentence shows that since 1980, Los Angeles has begun to increase a lot of immigrants.

This statement emphasizes the impact of the local economy on local schools. Although the percentage of funding each state provides to schools is similar, the amount of funding each state receives varies greatly because of the difference in revenue. This is very unfair to poor areas, whose schools end up being the same or worse anyway. This is one of the important reasons leading to the educational inequality in the country.

This statement shows the unequal treatment of different schools by the local legislature. Although they give money to poor school areas, but this only helps them maintain the status. In reality it doesn't work for them. It also reflects economic inequality that has led to schools being divided into "poor districts" and "rich districts." I can also see that poor and rich schools are divided into different districts, rather than randomly having poor and rich campuses in one place, which proves that the local economy also has a great impact on schools.

This is my favorite, I like how straightforward it is, I will probably use it on my own essay.

I agree with this point of view, it's important to be open minded and consider what we can gain, even from arguments we disagree with. This reminds me of a class discussion we had about critical thinking in my last English class, where we talked about how every argument, no matter how flawed, can teach us something.

I like how the text highlights the importance of considering different perspectives. It shows a good effort to include different views which makes a better and more balanced discussion.

Looking for strengths in a flawed argument helps you understand it better. Even if it's flawed, there could be good points worth considering. This balanced approach makes your critique more constructive, helps you find ways to improve the argument, and shows empathy for other perspectives.

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I think once you get to a point where you can communicate in a language, you can go out into a lot more places where that language takes center stage, and so you can iron out any quirks or imperfections. I feel like this type of learning primes you for immersion and so lets the rest of the learning be done on the fly, or in action.

This sounds like it would work great as an exercise that you would do in order to learn a language, I don't think that it would be the greatest method if it was your primary tool for learning a new language. I personally find the most trouble with grammar structures when learning a new language. If I were to try this I feel like I might have a lot of success on that front, but I don't think it should be someones only method.

I find that when I am learning a language, even when I am taught specific rules of structure and so on, I only truly grasp the concept when I repeatedly try and fail to apply it correctly. Ultimately I do think we have our own indiviual ways through which we understand the world, and by learning, but focusing on the remembering and the application of what we've learned we can come to better understandings.

I think this is great, I'm gonna start giving myself a reward whenever I learn a word in a separate language. Just jokes, but reward is a great motivator. When it comes to language learning I think it goes much farther than punishment, I mean, what would that look like? lol

I highly agree with this statement, as I have experienced it being used. some individuals rather win the argument by making a statement or question that baits the listener onto agreeing with the individual, it can be seen as a form of manipulation.

it guarantees the right of free speech but it doesn't free the person from the potential of consequences. an example of this could be yelling fire at a theater.

I highly agree with this statement, strong arguments need well researched facts. recently I saw a lot of arguments that are not backed up by facts are often misleading, I see a lot of arguments that obtain personal bias rather than facts.

Decline of classical knowledge in Europe due to the rise of Christianity.

Islamic scholars preserved and enhanced Greek knowledge.

I agree with Wynter that western philosophy is seen as the measure of humanity, and I would go as far as to say that the "Western Men" lifestyle is seen as the standard. Which is very disheartening when considering not everyone is from the west or shares the same experiences. Securing the western man as an ethnoclass has served a great disservice to the human species as a whole. How can we say we care about all humans as a whole and make western philosophy and ideology the standard? 

This is is something you see in Hispanic/latine discourse. The adherence that one's speaking of Spanish in a particular accent, dialect, or mode is more 'proper' or 'ideal' , alongside a host of other signifiers, serves to differentiate a people with a shared history of colonization. Someone from Mexico may not speak or look like someone from Puerto Rico or Haiti but that's not because there is some platonic form of each. The colonialist distinction of 'mestizos' and 'mulatto' is rooted into the ideas which these nations were founded on.

I think Chantal hit on an important point that often diasporic discourse ( for any peoples) can in attempting to reclaim some degree of identity ,stolen by colonization, can fit itself back into the same hegemonic orderings.

In ethnic studies and just in general discourse about 'diasporas', it is often the case that national and by necessity historically contingent differences between a peoples are treated as innate features to a particular group within the diaspora.

Rewording for myself: Mutu's and Butler's works do offer an alternative to environmental ethics in the form of "vital material ethics" (emphasizing the importance of all things? or just living things? kinda unclear to me), however the works are entirely centered around the difficulties that come up with this alternative. Hierarchies are still existent, even if in different forms.

These harmful stereotypes are what Octavia Butler adds into her novel "The Parable of the Sower" to forecast the effects these ideas have on society. An example of this is the way mixed couples are seen in her novel. People tend to stay away from them. Another example of this in the novel is that Lauren was seen as "odd" because, as a woman in her society, she wasn't willing to do what other women were expected to do with their futures. Frazier here is pointing out how mainstream and research have played a role in furthering this narrative against black figures. Butler and Mutu are actively providing their perspectives on what these stereotypes do to society. 

Reminding me of Haraway! An emphasis on connections with and to The World and all it may contain (including people of course) that is not interwoven with all this stuff that had a hand in us getting us into trouble in the first place.

And she is right- these roles do not serve anyone now, and definitely will not if the world comes undone. So interesting to read her logical internal monologue against the backdrop of this world she lives- in which rape is such a prevalent danger and women are not always told they should learn to protect themselves as well as form themselves as individuals (rather than as connected to men: husbands, fathers, etc.). I did not think about this so hard when I was reading it but having my attention on it now makes me realize how much this juxtaposition emphasizes Lauren as a leader and individual.

The invention of paper technology occurred through trade routes like the Silk Road.

The invention of paper technology occurred through trade routes like the Silk Road.

How long would this process take? It's a very interesting process and I'd love to know more.

Joseph Needham argued that science and technology evolved through cultural exchanges, which is something I can agree with.

There was significant collaboration between families and guilds, this highlights the community aspect of the production of goods.

Time was tracked by natural events like sunrise and sunset. Through the invention of clocks, time could be easily measured.

This resource delves deeper into the ways guilds balanced power between merchants and local authorities.

I think it is interesting how Gawain's belt (which is green) is reflective of nature, and also his defence against nature. There is a clear divide between Gawain's "safe" and comfortable experience in the castle, and the harshness and cruel nature that can often be associated with nature or the outside world. The natural world also goes against the idea of the idealized lifestyle that largely deals with courtly love, and everything that comes with that.

This is a very neat point. It is very interesting that the structures and natural elements that compliment this scene actually directly reflect the characterization of Gawain. His chivalry IS demonstrated through the grandness of the castle and the other buildings appearance. As we read on, this is contrasted quite beautifully with the delicateness of Guinevere's character that is reflected through the silks and more "soft" elements of her surroundings. I just think this is so cool and I have not put this much thought into setting's of stories previously, but it is wonderfully effective.

I think this is a very interesting way that Guinevere's "visibility" is perceived. Through her setting, she can be characterized and this is unlike any other character within the text. I find the little juxtapositions that Woods points out here to be fascinating as they're not blatant until you read deeper and understand what the setting around her represents for her. Prior to reading this, I was unaware just how much her setting amplifies her character as a whole.

I absolutley agree with this. Everyone has a different schedule at home and it is important to help them with their academic goals by adjusting to their schedule. The blatant school schedule that everyone abides to cannot fully help every single student equally. Their futures can be highly affected by just this simple fix. There needs to be a stronger relations with students and their teachers.

I feel like the hidden cirriculum is something that is just in general hard to relate to when the teachers are not inbursed in what some of these students go through. When it comes to solutions from this I believe that it is important to have people that the students to relate to. As the book states there could be a lot of people that can benefit from induvidual mentorship as this can help personalize education to help with maximizing results.

"boring" still, and as the "gravitrons" disappear from memory and the county fairs too; the days we had remembered things like "banks that used to have hyperloops in every teller drive through"

or the difference between what was "i can see the whole universe" and ... http://opensea.io/adobrin/created

The human mind is a powerful thing when it comes up against a problem it knows it needs to confront. This is why immersion can be a great way to learn. If you put up walls you can only scale with language you're bound to learn.

Social scientists have access to a layer of motivations for human behavior that we can't get in natural science. We can ask people why they do things. Therefore, sociology is an interpretative understanding of social behavior

lack of objectivity means natural science isn't some "objective" science while social science is subjective

YUUUUPPP

no "objective" as the scientists chose what to observe

whole pg- natural science differs from social science becaus the aims are different. Both require systems of conceptualization.

bars?!

researchers are problem-bound (abstract and distinctly human) but methods can remain systematic.

Weber stood by idea of underlying motivations (people are not just acted or influenced upon?) but also though of science and social science similar in they start with abstractions then move to the emprical.

systems and state understood as extensions of human action

didnt subscribe to idea that natural and social sciences parallel nor idea that human action has no similar patterns to natural world

Why is Modern Western Civilization so driven by rationality? As opposed to tradition, affect, or value-oriented rationality.

Weber combined systematic observations with historical context?

4 types of action: 1. rational- rational end goal is pursued with rational means 2. Value-oriented- end goal not perfectly rational but pursued through rational means 3. Affective- emotional motives, not rational 4. Traditional action- habitual

Weber obsessed with social actors and human behavior as basis for sociology- differs from Durkheim's "social conscience" and Marx's class tension.

hellloooooo????

I'd like to make the point that this myth probably comes from when kids are immersed in a completely different culture, like when an immigrant family moves to the us, and the first to become proficient is the child in school. I do think children have a slight advantage due to their brains being more malleable, but they also have no choice but to learn the language to get by, where as an adult can say, use google translate, or ask for a translator in specific situations.

A fixed mindset will definitely get you no where. By definition being fixed is to be set in position, to not move. In order to learn a language at all you need to be willing to grow. I've learned to acquire more of a growth mindset since it was introduced to me in middleschool.

It would have been interesting to hear about examples of said animal studies and how they compare to these findings.

Is it possible that this is due to confirmation bias and the idea that the mothers want to believe their infant is enjoying the flavored cereal?

What qualifies someone as a "trained rater"?

This is extremely important in many studies as it eliminates any possible bias from skewing the data.

It is interesting to see the breakdown of race within this study, especially because its preference for white pregnant mothers over non-white pregnant mothers is clear and most likely created biased data.

How could a child's preference for their mother's voice be affected if the mother is abusive/mainly uses negative tones? Could this deter the child from preferring their mother's voice to other individuals?

When I was a studnet I also had to switch into the honors cirruculum and I always felt behind becasue I wasnt part of the previous honors classes group of studnets. It is very tough to make this transtion and the ability to succeed is much harder.

I absolutley agree with this as I observed this in highschool. It seemed that everyone that were taking the more academically rigourous classes would seem more inclined to be thinking about college. It seems like people who are maximizing their present schedule are trying to help with their future. There seems to be a need as stated here of people that are encoruaging students to maximize a scheule that is able to help challenge their minds.

he's referring to the recursive example 4.5.1 !

My main thing when i am finding sources for papers is to automatically create a works cited page and pasting them to keep track of websites i find useful.

I was never really sure about reusing old papers for different classes and i honestly have done it before, my teacher let me use my old paper because i was doing a powerpoint on the topic i had wrote bout but i never thought about how it could be flagged as plagiarism

What: A callback function in JavaScript is a function passed as an argument to another function, which is then executed after the completion of that function.

Why: Asynchronous Operations: JavaScript uses callbacks to handle asynchronous tasks like API calls, timers, or file reading. Code Modularity: Callbacks allow for cleaner, reusable, and more maintainable code by delegating operations to specific functions. How: You define a callback function and pass it as a parameter:

javascript Copy code function greet(name, callback) { console.log('Hello, ' + name); callback(); }

function sayGoodbye() { console.log('Goodbye!'); }

greet('Alice', sayGoodbye); Here, sayGoodbye is passed as a callback to greet, and gets executed after the greeting.

You said: output ChatGPT said: ChatGPT The output of the provided code would be:

Copy code Hello, Alice Goodbye! Explanation: The greet function is called with "Alice" and the sayGoodbye function as arguments. Inside greet, it first logs "Hello, Alice". After that, it calls callback(), which refers to sayGoodbye, and the sayGoodbye function logs "Goodbye!".

O conceito de IA Generativa é muio relevante para o trabalho. Entendo que deve ser melhor trabalhado, aprofundando a discussão e detalhando seu funcionamento.

Perde-se a oportunidade de acrescentar importância ao artigo, explicitando modelos de usos adequados das IA's. Além do mais as mesmas refletem intimamente a sociedade, de maneira que um contraponto inserido neste momento do texto citando como poderiam ser realizados as buscas para fugir do viés que as IA's, assim como a internet como um todo possuem. Sugiro assistir ao vídeo abaixo para enriquecer o debate: https://www.youtube.com/watch?v=UuukUKMdXAk

Parte rica da discussão.

Como foi aplicado um questionário, poderia ser mensurado quantos docentes utilizam o ChatGPT, quantos utilizam outros, se conhecem outros. Evitaria a expressão na maioria das vezes sem o dado que ampare. Dos 21 participantes, quantos utilizam ChatGPT? Quantos outras IA's?

Um pouco óbvio o resultado em que discentes de áreas de tecnologias conhecem mais sobre o assunto. Não seria interessante explorar mais a falta de conhecimento dos demais professores? Outro ponto seria caracterizar melhor o perfil dos docentes que responderam o formulário. Perdeu-se a oportunidade de saber se a utilização das IA's tem relação com idade e sexo, por exemplo.

Este modelo de gráfico é de compreensão mais dificil que outros disponíveis. Sugiro colocar outra opção.

A utilização do "mais" na expressão entendo ter ficado inadequada. Sugiro: "cerca de 30%". Entendo ser necessário analisar sob o viés da estatística se este percentual equivale a amostra significativa dentro da boa técnica.

Entendo não ser adequado começar a frase com número.

Acho que existe espaço para um parágrafo falando como se dá o treinamento das IA's Generativas, levando o leitor a entender melhor este processo suas possibilidades e limitações. Ao falar como funciona o leitor entenderá que a IA Generativa tem várias limitações e melhorará seus argumentos nas várias discussões que este assunto gera.

Could you argue that body language or gesture is a language? A side eye could communicate plenty where a sentence might not be useful. The same sentence with a different expression could mean the literal opposite. Define language as exclusively words then no.

I count two, but okay

tentr relacionar com o eixo de expressao e conteudo

I thought it was interesting how the sentence structure drew parallels between wealth in the world and health in medicine, perhaps implying an initial scarcity of the former in the latter but the potential for more of each through the philosopher's stone, which is in this case portrayed by the lions.

If one's email service did not require TLS, would one be unable to email with another who's service did not support TLS?

I was interested in how this passage speaks to Early Modern European narratives surrounding gold, painting gold as something that exists in foreign places but is usually unobtainable so has to be acquired through alchemical knowledge. It also reveals thinking about imperialism and explicitly ties imperialism to attempts, whether successful or unsuccessful to extract gold.

trial x 2?

triallllll

The lilac is a powerful symbol of beauty and the brief nature of life. The “great star” represents Lincoln, who was a guiding light for the nation. The drooping star show feelings of sorrow and loss. The past tense "mourn'd" in conjunction with "shall mourn" expresses a constant state of sadness.

adjective (of a story or statement) of doubtful authenticity, although widely circulated as being true.