A utilização das ferramentas digitais nas camadas mais novas é uma vantagem. Além disso, como estas permitem ver o início e fim, ou seja, o percurso, ficam com a sessão de controle e desta forma permite que cada um faça esse percurso ao seu ritmo.

É a vantagem, mas do nosso lado enquanto formadores ou professores é bem mais trabalhoso.

A escolha da e-atividade mais adequada depende de vários fatores relacionados aos objetivos de aprendizagem, ao perfil dos estudantes, e ao contexto pedagógico.

Como vimos, existem diversas tipologias de e-atividades, que variam desde discussões online, quizzes interativos, até simulações e estudos de caso colaborativos. Para selecionar a e-atividade mais apropriada, é importante considerar:

Objetivos de Aprendizagem: A e-atividade deve alinhar-se aos resultados esperados. Se o objetivo é desenvolver competências colaborativas, atividades como fóruns de discussão ou projetos em grupo podem ser mais eficazes. Para avaliar conhecimento factual, quizzes ou testes de escolha múltipla são mais indicados.

Nível de Competência dos Estudantes: A maturidade digital e o nível de familiaridade dos estudantes com a plataforma e com as e-atividades influenciam a escolha. Estudantes mais experientes podem lidar com atividades complexas, enquanto iniciantes podem precisar de atividades mais estruturadas e guiadas.

Interatividade e Feedback: As e-atividades que permitem interação entre os estudantes e oferecem feedback imediato são eficazes para manter o compromisso e reforçar a aprendizagem.

Tempo e Recursos Disponíveis: A seleção deve também considerar o tempo disponível para a realização da atividade, a sua complexidade, e os recursos tecnológicos necessários.

Resumidamente, a escolha deve ser estratégica, focada em atingir os objetivos de aprendizagem e maximizar o empenho e o desenvolvimento dos estudantes dentro do contexto de ensino online.

A criação das comunidades virtuais e aprendentes, permite o envolvimento dos formandos e dá-lhe mais motivação para participar.

O problema é que na formação das turmas são normalmente muito heterogéneas, o que torna muito difícil encontrar estratégias que sejam eficazes para a totalidade dos formandos.

Penso que apesar de cada vez mais existirem novas plataforma do tipo e-atividades, além de ser ótimo utilizá-los em contexto online, também é uma forma muito interessante de consolidar conhecimento e motivar os nossos alunos em contexto educativo presencial. Principalmente em determinadas faixas etárias. Um bom exemplo de uma ferramenta que cumpre essa função é o QUIZIZZ. https://quizizz.com/

A e-atividade é uma poderosa ferramenta de aprendizagem que promove a interação, a colaboração e o desenvolvimento contínuo de habilidades num ambiente digital dinâmico.

O modelo enfatiza a importância de um processo progressivo, onde os participantes passam por diferentes níveis de compromisso.

Estes estágios vão desde o acesso inicial e a motivação, passando pela socialização, desenvolvimento da informação, até chegar à construção de conhecimentos e, finalmente, à colaboração e integração.

As e-atividades permitem que os alunos interajam de forma prática e reflexiva, facilitando uma aprendizagem colaborativa e significativa.

Leiam com atenção o texto e vamos refletir sobre o que é necessário para desenhar uma "boa" e-atividade de aprendizagem. Boas leituras e boas anotações. Façam anotações e comentem as anotações dos colegas. saudações académicas António Moreira

Whitman embraces "death" with his fresh-picked lilacs and other flowers. He wishes to share the beauty he sees in these flowers with the other coffins in addition to this one.

Associating the darkness with grief

In the line "O singer bashful and tender, I hear your notes, I hear your call" from Walt Whitman's "When Lilacs Last in the Dooryard Bloom'd," the speaker addresses the "singer" as a symbol of both mourning and beauty. The adjectives "bashful" and "tender" evoke a sense of vulnerability and fragility, suggesting that the song of the singer—likely representing Abraham Lincoln or a broader embodiment of loss—is both poignant and gentle. This duality reflects the complexities of grief, where the beauty of memory intertwines with sorrow. The repetition of "I hear" emphasizes the speaker’s deep connection to this voice, suggesting that the act of listening becomes an essential part of processing grief. This line encapsulates the overarching theme of the poem: the interplay between loss and the enduring power of remembrance. In a world marked by violence and tragedy, the tender notes serve as a reminder of the beauty that persists even amidst profound sorrow.

Whitman's imagery of the great star disappearing and the black murk that hides the star is symbolic of grief. His feeling of helplessness is due to his inability to control the death of someone he loved.

Considera se o número anterior é par ou impar? Pois aprendi uma vez que se o antecedente do número 5 (eliminado) for par, mantem; se for impar, arredonda.

Sød lille opgave: Vis at \(S^o\) er åben.

it's a good effort to make a strong connection but I'm not seeing why this image is generalized. Explain further.

Notamos que Jesús utilizó el espíritu, para dirigir a Pablo, después que el llegaba hasta cierto lugar, Entonces desde ahí le indicaba por donde seguir. Éste relato nos enseña que con nosotros puede ser igual, si tenemos una meta o queremos hacer algo, debemos orar por guía y empezar a trabajar, y una vez que nos movemos, en el momento preciso, el espíritu nos indicará el mejor camino, y debemos ser pacientes, decididos para insistir una y otra vez cómo lo hizo Pablo.

Pregunta a ChatGPT y te da foros interesantes para aprender

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors use analysis of existing data, mathematical modelling, and new experiments, to explore the relationship between protein expression noise, translation efficiency, and transcriptional bursting.

Strengths:

The analysis of the old data and the new data presented is interesting and mostly convincing.

Thank you for the constructive suggestions and comments. We address the individual comments below.

Weaknesses:

(1) My main concern is the analysis presented in Figure 4. This is the core of mechanistic analysis that suggests ribosomal demand can explain the observed phenomenon. I am both confused by the assumptions used here and the details of the mathematical modelling used in this section. Firstly, the authors' assumption that the fluctuations of a single gene mRNA levels will significantly affect ribosome demand is puzzling. On average the total level of mRNA across all genes would stay very constant and therefore there are no big fluctuations in the ribosome demand due to the burstiness of transcription of individual genes. Secondly, the analysis uses 19 mathematical functions that are in Table S1, but there are not really enough details for me to understand how this is used, are these included in a TASEP simulation? In what way are mRNA-prev and mRNA-curr used? What is the mechanistic meaning of different terms and exponents? As the authors use this analysis to argue ribosomal demand is at play, I would like this section to be very much clarified.

Thank you for raising two important points. Regarding the first point, we agree that the overall ribosome demand in a cell will remain more or less the same even with fluctuations in mRNA levels of a few genes. However, what we refer to in the manuscript is the demand for ribosomes for translating mRNA molecules of a single gene. This demand will vary with the changes in the number of the mRNA molecules of that gene. When the mRNA copy number of the gene is low, the number of ribosomes required for translation is low. At a subsequent timepoint when the mRNA number of the same gene goes up rapidly due to transcriptional bursting, the number of ribosomes required would also increase rapidly. The process of allocation of ribosomes for translation of these mRNA molecules will vary between cells, and this process can lead to increased expression variation of that gene among cells.

Regarding the second point, each of the 19 mathematical functions was individually tested in the TASEP model and stochastic simulation. The parameters ‘mRNA-curr’ and ‘mRNA-prev’ are the mRNA copy numbers at the current time point and the previous time point in the stochastic simulation, respectively. These numbers were calculated from the rate of production of mRNA, which is influenced by the burst frequency and the burst size, as well as the rate of mRNA removal. We would expand this section with explanation for all parameters and terms in the revised manuscript.

(2) Overall, the paper is very long and as there are analytical expressions for protein noise (e.g. see Paulsson Nature 2004), some of these results do not need to rely on Gillespie simulations. Protein CV (noise) can be written as three terms representing protein noise contribution, mRNA expression contribution, and bursty transcription contribution. For example, the results in panel 1 are fully consistent with the parameter regime, protein noise is negligible compared to transcriptional noise.

Thank you for referring to the paper on analytical expressions for protein noise. We introduced translational bursting and ribosome demand in our model, and these are linked to stochastic fluctuations in mRNA and ribosome numbers. In addition, our model couples transcriptional bursting with translational bursting and ribosome demand. Since these processes are all stochastic in nature, we felt that the stochastic simulation would be able to better capture the fluctuations in mRNA and protein expression levels originating from these processes. For consistency, we used stochastic simulations throughout even when the coupling between transcription and translation were not considered.

Reviewer #2 (Public review):

This work by Pal et al. studied the relationship between protein expression noise and translational efficiency. They proposed a model based on ribosome demand to explain the positive correlation between them, which is new as far as I realize. Nevertheless, I found the evidence of the main idea that it is the ribosome demand generating this correlation is weak. Below are my major and minor comments.

Thank you for your helpful suggestions and comments. We note that the direct experimental support required for the ribosome demand model would need experimental setups that are beyond the currently available methodologies. We address the individual comments below.

Major comments:

(1) Besides a hypothetical numerical model, I did not find any direct experimental evidence supporting the ribosome demand model. Therefore, I think the main conclusions of this work are a bit overstated.

Direct experimental evidence of the hypothesis would require generation of ribosome occupancy maps of mRNA molecules at the level of single cells and at time intervals that closely match the burst frequency of the genes. This is beyond the currently available methodologies. However, there are other evidences that support our model. For example, earlier work in cell-free systems have showed that constraining cellular resources required for transcription or translation can increase expression heterogeneity (Caveney et al., 2017). In addition, genome-wide analysis of expression noise in yeast also revealed that the association between protein noise and translational efficiency was highest in the group of genes with the most bursty transcription (Supplementary fig. S20).

(2) I found that the enhancement of protein noise due to high translational efficiency is quite mild, as shown in Figure 6A-B, which makes the biological significance of this effect unclear.

Although we agree with the reviewer’s comment that the effect of translational efficiency on protein noise may not be as substantial as the effect of transcriptional bursting, it has been observed in studies across bacteria, yeast and Arabidopsis (Ozbudak et al., 2003; Blake et al., 2003; Wu et al., 2022). In addition, the relationship between translational efficiency and protein noise is in contrast with the inverse relationship observed between mean expression and noise (Newman et al., 2006; Silander et al., 2012). We also note that the goal of the manuscript was not to evaluate the strength of the association, but to understand the basis of the influence of translational efficiency on protein noise.

(3) The captions for most of the figures are short and do not provide much explanation, making the figures difficult to read.

We will revise the figure captions to include more details as per the reviewer’s suggestion.

(4) It would be helpful if the authors could define the meanings of noise (e.g., coefficient of variation?) and translational efficiency in the very beginning to avoid any confusion. It is also unclear to me whether the noise from the experimental data is defined according to protein numbers or concentrations, which is presumably important since budding yeasts are growing cells.

For all published datasets where we had measurements from a large number of genes/promoters, we used the measures of adjusted noise (for mRNA noise) and Distance-to-median (DM, for protein noise). For experiments that we performed on a limited number of promoters, we used the measure of coefficient of variation (CV) to quantify noise, as calculation of adjusted noise or DM was not possible. Translational efficiency refers to translation rate which is determined by both the translation initiation rate and the translation elongation rate. The noise at the protein level was quantified from the signal intensity of GFP tagged proteins, which was proportional to protein numbers without considering cell volume. For quantification of noise at the mRNA level, single-cell RNA-seq data was used, which provided mRNA numbers in individual cells.

(5) The conclusions from Figures 1D and 1E are not new. For example, the constant protein noise as a function of mean protein expression is a known result of the two-state model of gene expression, e.g., see Equation (4) in Paulsson, Physics of Life Reviews 2005.

Yes, they are not new, but we included these results for setting the baseline for comparison with simulation results that appear in the later part of the manuscript where we included translational bursting and ribosome demand in our models.

(6) In Figure 4C-D, it is unclear to me how the authors changed the mean protein expression if the translation initiation rate is a function of variation in mRNA number and other random variables.

The translation initiation rate varied from a baseline initiation rate depending on the mRNA numbers and other variables. We changed the baseline initiation rate to alter the mean protein expression levels. We will elaborate this section in the revised manuscript.

(7) If I understand correctly, the authors somehow changed the translation initiation rate to change the mean protein expression in Figures 4C-D. However, the authors changed the protein sequences in the experimental data of Figure 6. I am not sure if the comparison between simulations and experimental data is appropriate.

It is an important observation. Even though we changed the translation initiation rate to change the mean expression (Fig. 4C-D), we noted in the description in the model (Fig. 3D) that the changes in the translation initiation rate was also linked with changes in the translation elongation rate. The translation initiation rate can only increase if the ribosomes already bound to the mRNA traverse quicker through the mRNA. This means that an increase in the translation initiation rate will occur only if the translation elongation rate is also increased, which will lead to lower traversal time of the ribosomes through the mRNA (Fig. 3D). Similarly, an increase in the translation elongation rate will allow more ribosomes to initiate translation. Thus, the parameters translation initiation rate and translation elongation rate are interconnected. This has also been observed in an experimental study by Barrington et al. (2023). Having said that, however, the models can also be expressed in terms of the translation elongation rate, instead of the translation initiation rate, and this modification will not change the results of the simulations due to interconnectedness of the initiation rate and the elongation rate.  

References

C. L. Barrington, G. Galindo, A. L. Koch, E. R. Horton, E. J. Morrison, S. Tisa, T. J. Stasevich, O. S. Rissland. Synonymous codon usage regulates translation initiation. Cell Rep. 42, 113413 (2023).

W. J. Blake, M. Kaern, C. R. Cantor, J. J. Collins, Noise in eukaryotic gene expression. Nature 422, 633-637 (2003).

P. M. Caveney, S. E. Norred, C. W. Chin, J. B. Boreyko, B. S. Razooky, S. T. Retterer, C. P. Collier, M. L. Simpson, Resource Sharing Controls Gene Expression Bursting. ACS Synth Biol. 6, 334-343 (2017)

J. R. Newman, S. Ghaemmaghami, J. Ihmels, D. K. Breslow, M. Noble, J. L. DeRisi, J. S. Weissman, Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature, 441, 840-846 (2006).

E. M. Ozbudak, M. Thattai, I. Kurtser, A. D. Grossman, A. van Oudenaarden, Regulation of noise in the expression of a single gene. Nat Genet. 31, 69-73 (2002).

O. K. Silander, N. Nikolic, A. Zaslaver, A. Bren, I. Kikoin, U. Alon, M. Ackermann, A genome-wide analysis of promoter-mediated phenotypic noise in Escherichia coli. PLoS Genet. 8, e1002443 (2012).

H. W. Wu, E. Fajiculay, J. F. Wu, C. S. Yan, C. P. Hsu, S. H. Wu, Noise reduction by upstream open reading frames. Nat Plants. 8, 474-480 (2022).

O Mustafa Kabha εργαζεται σε Ισραηλινο πανεπιστημιο:

Mustafa Kabha is full Professor in the Department of History, Philosophy and Judaic Studies and the Head of the Middle Eastern studies at the Open University of Israel.

Το μεμαλοποιει, στη λιστα με τις σφαγες στη Wikipedia δεν βρισκω πολλες τετοιες.

• Não é devida a leitura da hospedagem como uma obrigação de dar, porquanto não se depreende dela, para fins tributários, unicamente uma locação da unidade habitacional pelo preço vertido na diária. A interpretação constitucional da materialidade tributária prevista no art. 156, inc. III, da Constituição da República, não se limita à classificação obrigacional derivada da dogmática civilista.

• De acordo com o entendimento do Supremo Tribunal Federal, o ISS incide sobre atividades que representam tanto obrigações de fazer quanto obrigações mistas, que também incluem uma obrigação de dar. É inadequado o decote da base de cálculo do Imposto sobre Serviços de qualquer Natureza com a finalidade de excluir a parcela referente à locação da unidade habitacional, porque a circulação econômica de serviço vertida no contrato de hospedagem tem caráter singular, justificando-se a partir de sua visualização unitária, logo é inviável a cisão apriorística de modo a retirar da base imponível desse imposto municipal a fração relativa à locação da unidade habitacional. Desse modo, é assente a orientação jurisprudencial segundo a qual todas as parcelas que integram o preço do serviço de hotelaria compõem a base de cálculo do ISS.

[ADI 5.764, rel. min. André Mendonça, j. 2-10-2023, P, DJE de 13-10-2023.]

Author response:

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

We would like to thank the reviewers for their interest in our studies. In response to their comments, we have conducted additional experiments and made the necessary revisions to the manuscript. The new studies included to address the reviewers’ comments are shown in Figure 1B, 1F, Figure 2—figure supplement 1, Figure 3, Figure 3—figure supplement 1, Figure 3—figure supplement 2, Figure 3—figure supplement 3, Figure 4E, Figure 4—figure supplement 1, Figure 5, Figure 5—figure supplement 1, Figure 5—figure supplement 2D, and Figure 6. We are grateful for the critiques, which have helped us substantially improve the quality of the manuscript.

Below, we have provided a point-by-point response to the reviewers’ comments.  

Public Reviews:

Reviewer #1 (Public Review):

In this paper, the authors show that disruption of calcineurin, which is encoded by tax-6 in C. elegans, results in increased susceptibility to P. aeruginosa, but extends lifespan. In exploring the mechanisms involved, the authors show that disruption of tax-6 decreases the rate of defecation leading to intestinal accumulation of bacteria and distension of the intestinal lumen. The authors further show that the lifespan extension is dependent on hlh-30, which may be involved in breaking down lipids following deficits in defecation, and nhr-8, whose levels are increased by deficits in defecation. The authors propose a model in which disruption of the defecation motor program is responsible for the effect of calcineurin on pathogen susceptibility and lifespan, but do not exclude the possibility that calcineurin affects these phenotypes independently of defecation.

We thank the reviewer for providing an excellent summary of our work. We have performed additional experiments as suggested by both the reviewers and believe we have thoroughly addressed all the reviewers' concerns.

Reviewer #2 (Public Review):

The manuscript titled "Calcineurin Inhibition Enhances Caenorhabditis elegans Lifespan by Defecation Defects-Mediated Calorie Restriction and Nuclear Hormone Signaling" by Priyanka Das, Alejandro Aballay, and Jogender Singh reveals that inhibiting calcineurin, a conserved protein phosphatase, in C. elegans affects the defecation motor program (DMP), leading to intestinal bloating and increased susceptibility to bacterial infection. This intestinal bloating mimics calorie restriction, ultimately resulting in an enhanced lifespan. The research identifies the involvement of HLH-30 and NHR-8 proteins in this lifespan enhancement, providing new insights into the role of calcineurin in C. elegans DMP and mechanisms for longevity.

The authors present novel findings on the role of calcineurin in regulating the defecation motor program in C. elegans and how its inhibition can lead to lifespan enhancement. The evidence provided is solid with multiple experiments supporting the main claims.

Strengths:

The manuscript's strength lies in the authors' use of genetic and biochemical techniques to investigate the role of calcineurin in regulating the DMP, innate immunity, and lifespan in C. elegans. Moreover, the authors' findings provide a new mechanism for calcineurin inhibitionmediated longevity extension, which could have significant implications for understanding the molecular basis of aging and developing interventions to promote healthy aging.

(1) The study uncovers a new role for calcineurin in the regulation of C. elegans DMP and a potential novel pathway for enhancing lifespan via calorie restriction involving calcineurin, HLH-30, and NHR-8 in C. elegans.

(2) Multiple signaling pathways involved in lifespan enhancement were investigated with fairly strong experimental evidence supporting their claims.

We thank the reviewer for an excellent summary of our work and for highlighting the strengths of the findings.

Weaknesses:

The manuscript's weaknesses include the lack of mechanistic details regarding how calcineurin inhibition leads to defects in the DMP and induces calorie restriction-like effects on lifespan.

The exact site of calcineurin action, i.e., whether in the intestine or enteric muscles (Lee et al., 2005), and the possible molecular mechanisms linking calcineurin inhibition, DMP defects, and lifespan were not adequately explored. Although characterization of the full mechanism is probably beyond the scope of this paper, given the relative simplicity and advantages of using C. elegans as a model organism for this study, some degree of rigor is expected with additional straightforward control experiments as listed below:

The authors state that tax-6 knockdown animals had drastically reduced expulsion events (Figure 2G), leading to irregular DMP (Lines 144-145), but did not describe the nature of DMP irregularity. For example, did the reduced expulsion events still occur with regular intervals but longer cycle lengths? Or was the rhythmicity completely abolished? The former would suggest the intestine clock is still intact, and the latter would indicate that calcineurin is required for the clock to function. Therefore, ethograms of DMP in both wild-type and tax6 mutant animals are warranted to be included in the manuscript. Along the same line, besides the cycle length, the three separable motor steps (aBoc, pBoc, EMC) are easily measurable, with each step indicating where the program goes wrong, hence the site of action, which is precisely the beauty of studying C. elegans DMP. Unfortunately, the authors did not use this opportunity to characterize the exact behavior phenotypes of the tax-6 mutant to guide future investigations. Furthermore, it is interesting that about 64% of tax-6 (p675) animals had normal DMP. The authors attributed this to p675 being a weak allele. It would be informative to further examine tax-6 RNAi as in other experiments or to make a tax-6 null mutant with CRISPR. In addition, in one of the cited papers (Lee et al., 2005), the exact calcineurin loss-of-function strain tax-6(p675) was shown to have normal defecation, including normal EMC, while the gain-of-function mutant of calcineurin tax-6(jh107) had abnormal EMC steps. It wasn't clear from Lee et al., 2005, if the reported "normal defecation" was only referring to the expulsion step or also included the cycle length. Nevertheless, this potential contradiction and calcineurin gain-of-function mutant is highly relevant to the current study and should be further explored as a follow-up to previously reported results. For some of the key experiments, such as tax-6's effects on susceptibility to PA14, DMP, intestinal bloating, and lifespan, additional controls, as the norm of C. elegans studies, including second allele and rescue experiments, would strengthen the authors' claims and conclusions.

We have now included lifespan, survival on P. aeruginosa, and DMP data using an additional knockout allele, tax-6(ok2065). Additionally, we have added ethograms of DMP for both tax-6 RNAi and the tax-6(ok2065) mutant. Our observations indicate that tax-6 inhibition leads to a complete loss of DMP rhythmicity, suggesting that calcineurin is essential for maintaining the DMP clock. While characterizing the DMP, we noticed that expulsion events appeared superficial in the tax-6(ok2065) mutant, with little to no gut content released. Consequently, we examined the movement of gut content and found that both tax-6(ok2065) mutants and tax-6 knockdown animals showed significantly reduced gut content movement. The new findings on the characterization of DMP are presented in Figure 2—figure supplement 1, Figure 3, Figure 3—figure supplement 1, and Figure 3—figure supplement 2. The text in the results section reads (lines 160-176): “Next, we investigated whether the reduced number of expulsion events was due to regular intervals with longer cycle lengths or if rhythmicity was entirely disrupted upon tax-6 knockdown. To assess this, we obtained ethograms of the DMP for N2 animals grown on control and tax-6 RNAi. While animals on control RNAi displayed regular cycles of pBoc, aBoc, and EMC, the tax-6 RNAi animals exhibited disrupted rhythmicity (Figure 3A and Figure 3—figure supplement 1). Most tax-6 knockdown animals lacked the pBoc and aBoc steps and had sporadic expulsion events. Isolated pBoc events were occasionally observed, indicating a complete loss of rhythmicity in tax-6 knockdown animals. Ethograms for tax-6(ok2065) animals also showed disrupted rhythmicity (Figure 3B and Figure 3—figure supplement 2). Although the number of expulsion events appeared higher in tax-6(ok2065) animals compared to tax-6 RNAi animals (Figure 3—figure supplement 1 and 2), these expulsion events seemed superficial, releasing little to no gut content. This suggested slow movement of gut content in tax6(ok2065) animals, leading to constipation and intestinal bloating. We examined gut content movement by measuring the clearance of blue dye (erioglaucine disodium salt) from the gut. The clearance was significantly slower in tax-6(ok2065) animals compared to N2 animals (Figure 3C), indicating impaired gut content movement due to the loss of tax-6. Similarly, tax-6 knockdown animals also showed significantly slowed gut content movement (Figure 3D).”

Moreover, we have added a potential reason for the tax-6(p675) contradictory results from Lee et al., 2005 (lines 154-159): “At the 1-day-old adult stage, about 36% of tax-6(p675) animals showed irregular and slowed DMP, while the remainder had regular DMP (Figure 2H), suggesting that tax-6(p675) is a weak allele. The fraction of the animals with irregular DMP appeared to increase with age, indicating that this phenotype might be agedependent. This may also explain why tax-6(p675) animals were reported to have a normal defecation cycle in an earlier study (Lee et al., 2005).”

The second weakness of this manuscript is the data presentation for all survival rate curves. The authors stated that three independent experiments or biological replicates were performed for each group but only showed one "representative" curve for each plot. Without seeing all individual datasets or the averaged data with error bars, there is no way to evaluate the variability and consistency of the survival rate reported in this study.

We now provide all replicates data in the source data files.

Overall, the authors' claims and conclusions are justified by their data, but further experiments are needed to confirm their findings and establish the detailed mechanisms underlying the observed effects of calcineurin inhibition on the DMP, calorie restriction, and lifespan in C. elegans.

We have conducted additional experiments to elucidate the role of calcineurin in the DMP and to investigate the impact of the DMP on calorie restriction and lifespan in C. elegans, as described in the various responses to the reviewers’ comments. 

Recommendations for the authors:

Our specific comments to guide the authors, should they choose to revise the manuscript:

The RNAi experiments in the eat-2 mutant background are difficult to interpret. If these animals are eating fewer bacteria, it is possible that there is also less tax-6 dsRNA being ingested and therefore less tax-6 inactivation. These experiments should be conducted with a tax-6 null allele.

We have included lifespan experiments with the eat-2(ad465);tax-6(ok2065) double mutant, along with the individual single mutant controls, as shown in Figure 4E. These results demonstrate that the eat-2 mutation does not further extend the lifespan of the tax-6(ok2065) mutant. Additionally, we confirmed that the eat-2(ad465) mutants do not exhibit defects in feeding-based RNAi (Figure 4—figure supplement 1).

While aak-2, hlh-30, and nhr-8 mutants may not have an eat phenotype, the negative tax-6 RNAi results should be confirmed with a tax-6 null mutant to obviate the consideration that these background mutations reduce RNAi efficacy.

The genes hlh-30 and nhr-8 are located very close to tax-6 on chromosome IV (https://wormbase.org//#012-34-5), which made it challenging to generate double mutants. However, we tested the RNAi sensitivity of the hlh-30(tm1978) and nhr-8(ok186) mutants and confirmed that they are not defective in RNAi (Figure 5—figure supplement 1). We also found that tax-6 RNAi disrupted the DMP in both hlh-30(tm1978) and nhr-8(ok186) mutants (Figure 5—figure supplement 2). Furthermore, our results show that hlh-30(tm1978) and nhr-8(ok186) animals have increased susceptibility to P. aeruginosa upon tax-6 knockdown (Figure 6A, B), indicating that tax-6 RNAi was effective in these mutants. Since the phenotype in the aak-2 mutant was only partially observed, we did not conduct further experiments with aak-2 mutants.

Reviewer #1 (Recommendations For The Authors):

The low penetrance of defecation cycle defects in tax-6(p675) worms brings into question the role of the defecation deficits in the phenotypes caused by the disruption of tax6. At the same time, the low penetrance provides a golden opportunity to test this. Do tax6(p675) worms with a normal defecation cycle length have extended longevity? Increased susceptibility to bacterial pathogens? Smaller body size? Distended lumen? Decreased fat accumulation? Increased pha-4 and nhr-8 expression? It would be relatively straightforward to measure defecation cycle length in individual tax-6(p675) worms, bin them into normal defecation and slow defecation groups, and then compare the above-mentioned phenotypes.

We appreciate the reviewer's interesting suggestion. However, the DMP defect phenotype in tax-6(p675) worms appears to be age-dependent, with the number of DMPdefective worms increasing as they age. Additionally, we observed that exposure to P. aeruginosa accelerates the onset of DMP defects in tax-6(p675) worms. As a result, tax6(p675) worms are not suitable for the type of experiments the reviewer suggested. Nevertheless, we believe that the additional data using the tax-6(ok2065) mutant, along with the characterization of ethograms of DMP, firmly establishes the role of calcineurin in maintaining a regular DMP in C. elegans.

Another way to dissect specific effects of calcineurin disruption from phenotypes resulting from defecation motor program deficits would be to further characterize other worms with deficits in defecation (flr-1, nhx-2, pbo-1 RNAi). It is mentioned that they have decreased lifespan. Do they also show increased susceptibility to bacterial pathogens? Do they show decreased fat? Is their lifespan dependent on HLH-30 and NHR-8?

We thank the reviewer for this important suggestion. We have now included data with flr-1, nhx-2, and pbo-1 RNAi, which shows that the knockdown of these genes also enhances susceptibility to P. aeruginosa (Figure 3—figure supplement 3G). Knockdown of these genes is already known to reduce fat levels in N2 worms, and we demonstrate that they similarly reduce fat levels in hlh-30(tm1978) and nhr-8(ok186) animals (Figure 5B, C, F, G). Additionally, we found that the increased lifespan observed upon knockdown of these genes (as well as with tax-6 knockdown) is dependent on HLH-30 and NHR-8 (Figure 5A, D).

To place "enhanced susceptibility to pathogen" within the proposed model, it would be important to examine the effect of HLH-30 and NHR-8 disruption on this phenotype. The proposed model suggests that this phenotype is independent of HLH-30 and NHR-8, but this should be tested experimentally. Similarly, it would be important to test the effect of HLH-30 and NHR-8 disruption on defecation cycle length to determine if defecation deficits are upstream or downstream of deficits in the defecation motor program

We show that the knockdown of tax-6 leads to defects in the DMP in hlh30(tm1978) and nhr-8(ok186) animals (Figure 5—figure supplement 2). Moreover, we show that hlh-30(tm1978) and nhr-8(ok186) animals have increased susceptibility to P. aeruginosa upon tax-6 knockdown (Figure 6A, B). These results are described as (lines 279-285): “Given that HLH-30 and NHR-8 are essential for lifespan extension upon calcineurin inhibition, we investigated whether these pathways also influence survival in response to P. aeruginosa infection following calcineurin knockdown. Both hlh-30(tm1978) and nhr-8(ok186) animals showed significantly reduced survival upon tax-6 RNAi (Figure 6A, B). These findings suggested that the reduced survival on P. aeruginosa following calcineurin inhibition is independent of HLH-30 and NHR-8 and is more likely due to increased gut colonization by P. aeruginosa resulting from DMP defects (Figure 6C).”

Is the lifespan of tax-6(p675) increased? This would be important to measure and include in Figure 1.

Indeed, the lifespan of tax-6(p675) mutants is increased. We have included the lifespan of tax-6(p675) and tax-6(ok2065) in Figure 1F.

In Figure 2, disruption of tax-6 appears to result in a clear decrease in body size. To what extent is the decrease in fat/worm in Figure 3 simply a result of the worms being smaller? Perhaps, a measurement of Oil-Red-O intensity PER AREA would be a more appropriate measure.

The ORO intensity values we had shown per animal were already area normalized. We have now indicated this in the Figure Legends.

There are multiple long-lived mutant strains such as clk-1 and isp-1 that have an increased defecation cycle length. To what extent do these worms exhibit phenotypes similar to tax-6 disruption? isp-1 have increased resistance to bacterial pathogens suggesting that defecation motor program deficits are not sufficient to increase susceptibility to bacterial pathogens.

We have now examined the clk-1 and isp-1 mutants and found that these mutants exhibit reduced gut colonization by P. aeruginosa compared to N2 animals. This reduction in colonization may be attributed to the slowed pharyngeal pumping rates observed in these mutants. These findings suggest that the phenotypes associated with a slow DMP versus a disrupted DMP could be significantly different. The manuscript with the new data on these mutants reads (lines 177-192): “We then explored whether the disruption of DMP rhythmicity due to tax-6 knockdown affected P. aeruginosa responses similarly to longer but regular DMP cycles. To do this, we studied P. aeruginosa colonization in clk-1(qm30) and isp1(qm150) mutants, which have regular but extended DMP cycles (Feng et al., 2001; Wong et al., 1995). Interestingly, both clk-1(qm30) and isp-1(qm150) mutants showed significantly reduced intestinal colonization by P. aeruginosa compared to N2 animals (Figure 3—figure supplement 3A-D). This reduced colonization could be attributed to their significantly decreased pharyngeal pumping rates (Wong et al., 1995; Yee et al., 2014), suggesting a lower intake of bacterial food in these mutants. While the survival of clk-1(qm30) animals on P. aeruginosa was comparable to N2 animals (Figure 3—figure supplement 3E), isp1(qm150) animals exhibited significantly improved survival (Figure 3—figure supplement 3F). Conversely, knockdown of flr-1, nhx-2, and pbo-1 in N2 animals resulted in significantly reduced survival on P. aeruginosa compared to control RNAi (Figure 3—figure supplement 3G). Knockdown of these genes causes complete disruption of DMP rhythmicity, increasing gut colonization by P. aeruginosa (Singh and Aballay, 2019a). Overall, these findings demonstrated that calcineurin is crucial for maintaining the DMP ultradian clock, and its inhibition increases susceptibility to P. aeruginosa by disrupting the DMP.”

Line 192. This statement is speculative. There is no evidence that HLH-30 is mediating lipid depletion in these worms.

We have removed this statement. We observed that the knockdown of flr-1, nhx2, and pbo-1 resulted in significant fat depletion in hlh-30(tm1978) animals (Figure 5B, C). Additionally, tax-6 knockdown also caused a small but significant reduction in fat levels in hlh-30(tm1978) animals. This contrasts with our initial submission, possibly due to the increased number of animals included in the analysis. These findings suggest that the increase in lifespan due to DMP defects requires HLH-30, likely through a mechanism independent of HLH-30’s role in fat depletion. We have updated the manuscript text and model (Figure 6C) accordingly.

In Figure S2, tax-6 RNAi appears to have a more detrimental effect in pmk-1 mutants than the other mutants. The authors should comment on this.

We have added the following sentence in the manuscript (lines 123-125): “The knockdown of tax-6 appeared to have a more pronounced effect in pmk-1(km25) mutants than in other mutants, suggesting that inhibition of tax-6 might exacerbate the adverse effects observed in pmk-1(km25) mutants.”

Reviewer #2 (Recommendations For The Authors):

Line 192-193: The statement is confusing and not accurate because HLH-30 did not enhance lifespan with or without calcineurin (Figure 4A and S4A, also in Lapierre 2023). The takeaway should be along the lines of calcineurin inhibition enhancing lifespan through HLH-30 or HLH-30 being required for lifespan enhancement via calcineurin inhibition.

We have removed this statement. We now state (lines 237-239): “Knockdown of tax-6 did not extend the lifespan of hlh-30(tm1978) animals (Figure 5A), indicating that HLH-30 is required for the increased lifespan observed with calcineurin inhibition.”

Line 261: Similar to the point above. Where is the data showing NHR-8 increases lifespan with or without calcineurin?

We have removed this sentence.

Figure 1 legend line 699: animals per condition per replicate >90, but in the Method section Line 317, it says more than 80 animals per condition per replicate. Could be more accurate.

We have now specified in the Methods section that the exact number of animals per condition is provided in the source data files. Since different lifespan curves within a given figure panel had varying numbers of animals, we have indicated the lower boundary for all curves (including the replicates). The precise number of animals for each lifespan experiment is available in the source data files.

Figures 2F and G, "tax-6" should be labeled as "tax-6 RNAi" to be consistent with other figures.

We thank the reviewer for this suggestion and have updated the label to “tax-6 RNAi”.

In summary, we would like to thank the reviewers again for providing constructive critiques. We believe we have fully addressed all the concerns of the reviewers by carrying out several new experiments and modifying the text. The manuscript has undergone substantial revision and has thereby improved significantly. We do hope that the evidence in support of the conclusions is found to be complete in the revised manuscript.

The belief that humans are meant for greater pursuits intertwines with the inevitability of death. Despite Eliot’s lack of appreciation for Tennyson’s narrative, this idea seems to have been inspired by Tennyson’s poem. The final line of “Ulysses” becomes the most striking and seemingly summarizes its entire purpose as Ulysses states “to strive, to seek, to find, and not to yield.” For Ulysses, life’s meaning lies in this quest for knowledge and purpose, despite the certainty of death.

Dante’s description offers a deeper insight into this theme. When Ulysses describes his story he explains how he encouraged his comrades to embark on a journey, declaring “you were not made to live like brutes or beasts, but to pursue virtue and knowledge.” The comparison to inhumane and violent creatures further signifies a human aspiration to seek meaning beyond mere survival. This pursuit, however, is deemed ineffective: as Ulysses describes, “the whirlwind" stroke the boat “the sea closed over” them, describing how human ambition becomes futile in the face of inescapable fate.

Eliot reiterates the same theme for Phlebas, who is merely a product of Eliot’s imagination. The readers have no idea of the life of the character and it never becomes important: this meaningless life of a made-up character, however, leads to the same outcome, which Eliot underscores by invoking a similar image of a whirlpool. Furthermore, Eliot’s choice to introduce a character without a rich backstory underscores this notion: all lives, regardless of their perceived significance, converge on the same fate, where death, as an unyielding current, eventually claims every life. A change from whirlwind, which is caused by instability of wind can happen anywhere, to whirlpool, which are results of an intersection of two opposing currents, becomes particularly interesting. Instead of focusing on unpredictability, Eliot uses this image to highlight the result of opposing a sort of “life’s current.”

The title of this section, a reiteration of Madame Sosostris’s prophecy to “fear death by water,” adds a layer of irony to this opposition: what is the point of fearing this death if Eliot had already predetermined it, had already written both the readers’ and the characters’ fates within the poem? This pathology serves as a direct analogy to reality, where no matter the fear or attempts to battle the circumstances, the outcome remains unchanged.

In a sense, it appears that Eliot encourages readers to resign to circumstances, as he contrasts the previous Fire Sermon with this Death by Water. The struggles, dissociations, and pleas of the Fire Sermon are juxtaposed with the calm detached description of the Death by Water.

In contrasting these two sections, Eliot presents two different responses to existential challenges. Whether with Philomela, who attempts to voice her pain as a nightingale with “tereu,” however, as a female nightingale is unable to produce sound, the unnamed female who desperately asks her partner “Why do you never speak to me,” or the five burnings that might relate to disconnection from the five senses in response to the character’s final plea: “O lorg thou pluckst me out”, the characters in "The Fire Sermon" grapple with external circumstances. Whether relationships, disconnection, or the chaos of modern life, their attempts to fight against these forces prove ineffective. Instead, their search for meanings only reveals the futility of their resistance.

In contrast, "Death by Water" offers a sense of acceptance of mortality. Phlebas has been dead for two weeks – a “fortnight,” when there is nothing to be done anymore. Instead, external circumstances, like “a current under the sea,” carry his body. Eliot reiterates its overarching nature, drawing a comparison between Phlebas, “Gentile or Jew” and the readers.

The effectiveness of this acceptance is further reflected through form: Death by Water becomes straightforward and concise, while the Fire Sermon constantly grapples with dissonance, contrasting voices, and changes in form.

Whether in life or in death, external circumstances, like a “current under the sea” seem to carry us to the final destination that remains unchanged despite the life pursuits. Death by Water, thus, invokes a question: maybe it is in the acceptance of the certainty of death and refusal to fight these external circumstances that lies a potential for peace amid this chaos of life?

The line, “O you who turn the wheel and look to windward” is a direct reference to the insatiable Ulysses from Tennyson’s poem. Driven by his thirst for knowledge and refusal to be buried by old age, Ulysses heeds the siren call of his ship and departs on another adventure. His death by water is, to him, heroic. It is better than living what remains of his life as a sheltered retiree. For Ulysses, death by water is a reward. Eliot’s second person pronoun use here, namely, the incorporation of the reader so directly, reinforces the authoritative nature of the poem. Yet, the poem is not merely a cautionary tale. The last line of “Ulysses” is “To strive, to seek, to find, and not to yield”; Tennyson is warning against possession, over land, knowledge, people, not adventure. Ulysses yearns for adventure, and on the wild waves, finally gets it. Eliot’s final stanza features a “you” who “turn the wheel”, who actively decides their own fate, and who “look to windward”, gazing off into the distance, at a farawary land, at the future. Yes, death by water deters the average humanist and punishes the Icarus stan, but the gentle and hopeful undertones of just one line in the final stanza suggest that death is not the end, but rather a journey. Ulysses urges his friends, “'T is not too late to seek a newer world”. Dante’s Inferno portrays a trip in the underworld. Eliot’s figure looks windward. None of these sources is concerned with the final destination (not Ithaka, as Tennyson would argue, but the long discovery of her), but rather the direction of it that death by water appoints.

Any reader who has ventured TWL knows death is not terminal. In fact, nothing really is. The fluidity of water, in the mobility of its waves, the omnipresence of its “deep sea swells”, the simultaneous pacificity and force, currents and whirlpools, means there is no rest for the dead (Eliot). Phlebas the Phoenician, referenced initially in the Tarot deck, is a fortnight dead and still rising and falling to the pulse of the sea. The cautioned reader sails animatedly towards inevitable death. So when Eliot tells us to “Consider Phlebas, who was once handsome and tall as you”, never fear. Hoist the anchors and raise your sails.

In Death By Drowning, Eliot draws from Tennyson’s Ulysses and the book of Corintheans, which have some prominent parallels that Eliot seems to bring together in his own writing. Corinthians emphasizes the idea of unity through the Eucharist: “Because there is one bread, we who are many are one body, for we all partake of the one bread.” (Corintheans, 13-14). In Eliot’s original draft of Death By Water, the bread on the boat is rotten and ridden with worms, so the sailors don’t share it: “‘For when you got through digging out the weevils // From every biscuit, there 's no time to eat.’” (Eliot, 34-35). By the logic of Corinthians, the men are not in fact “one body.” Even the sailors on Tennyson’s boat in Ulysses (presumably the inspiration for the sailing metaphor in “The Waste Land”) are described as “one equal temper of heroic hearts,” yet the sailor in “The Waste Land” is completely solitary, isolated from the shipmates that appear in Eliot’s original draft. Perhaps this has to do with the fact that the “heart” of the Phonecian sailor has ceased to beat at all in this passage. Additionally, in the original draft, the beginning of the final section (which made it into the final draft) marks a shift from first person to second person. Interestingly, Corinthians is written in the second person, making this section of the poem feel like a haunted echo of the book in the Bible, deprived of its spirit of unity, making it feel more like a warning than a sermon.

Between Tenison’s Ulyssess and Dante’s Inferno, a rather complete picture forms of Odysseus’ unquenchable thirst for adventure. The need to explore is so strong that it becomes integral to who he is, “And this gray spirit yearning in desire / To follow knowledge like a sinking star, / Beyond the utmost bound of human thought” (Tennyson 2). The invocation of the spirit describes his nearly biological “desire[s]”, truly welding them to his identity. After being away for most of his child’s lifetime, he is fine with leaving Telemachus once more, placing his conquests over his own family (In some ways this quest to go beyond human thought coincides with the desire of major powers of the Great War to expand beyond their means in search of ultimate power). So, it is most fitting that Ulysses meets his end in a whirlpool at the end of what is known. He becomes encompassed with the ocean that transported him through his various conquests, his sense of identity, as “our stern reared up, / the prow went down—as pleased Another— / until the sea closed over us.” Death by water, “until the sea closed over” thus becomes a positive end, enveloped while doing what one loves. But it is also a cautionary tale, as Eliot uses the vocative “O you” to associate the reader with Phlebas and Odysseus’ fate. We all run the risk of being overcome by our passions, burning, burning, burning.

Unrelated observation: the gaps between the lines, the only like it in the poem, form two, wave-like shapes, enveloping the words like the water that “closed over” Ulysses.

As a natural extension of my passion for IR, the history of Carthage has long fascinated me. Carthage is a prime, although far from straightforward, exemplification of the “Thucydides Trap,” a phenomenon where there is an inherent tendency towards conflict when a rising power threatens the dominance of an established hegemony. In Carthage’s case, this conflict manifested in the three Punic Wars where power fluctuated between Rome and Carthage. Most strikingly, it is Carthage’s almost fanciful obsession with controlling minor powers of little relevance–such as Numidia–that ultimately enabled its subjugation by room.

Now, moving from grand IR theories to the perhaps more relevant realm of literary analysis, let us consider how Carthage’s history ties to Augustine’s “Confessions,” naturally on a personal level rather than a state one. Augustine writes of a life obsessed with trivialities and superficialities: “Stage-plays also carried me away, full of images of my miseries, and of fuel to my fire.” He suggests that human desire is somehow distorted beyond a state of natural connection with God and nature, writing of a “cauldron of unholy loves.” Note how the imagery of a “cauldron,” a burning entity, correlates with the previous passage on superficial “Stage-plays” being “of fuel to my fire.” Applying our IR analogy here, Augustine–representing Carthaginian people–is the rising power and nature–or at least some force more fundamental than petty plays and floating whimsies–is the established hegemon. And as in real life, where Carthage was horribly subjugated by Rome–“50,000 Carthaginians were sold into slavery…the city was set ablaze and razed to the ground, leaving only ruins and rubble” according to Wikipedia–one possible interpretation from TWL, made possible by Eliot’s lack of punctuation, is that Carthage is the entity that is “Burning burning burning burning,” destroyed by its vanity and lack of groundedness.

But, burning is a lot more than a simple inducer of destruction. Indeed, the broader realm of Eliot’s allusions features burning as an act that can induce purification or relate to nobility. In Gotterdammerung, Brünnhilde “returns the ring to the Rhine maidens as she commits suicide on Siegfried's funeral pyre.” Through her valiant self-sacrifice, Brünnhilde brings a righteous end to the ring’s cycle of greed and destruction; her shattering of this perverse cycle leaves not even the complicit gods immune. Per Buddha’s “Fire Sermon,” the burning line’s direct source as referenced in Eliot’s notes, everything “All things..are on fire”; burning is all-consuming as it dominates every emotion and stage of life. But “burning” is not inherently negative here as it is only by recognizing one’s being in a state of “burning” and creating “aversion[s]” can one become free. The state of burning, insofar as it is naturally torturous, is necessary as part of the process by which one can break free of perverse constructions and strive towards nobility, or, in the case of Buddha and his disciples, nirvana.

So, bringing this full circle and returning to the analytical foundation of taking Carthage as a “burning” entity in TWL, Eliot’s placement of Buddha’s enlightenment philosophy alongside tales of destructive Carthaginian great power conflict, one sees a call for purification rather than simply catastrophe (potentially even optimistic). After acknowledging his “Burning burning burning burning,” with burning here being most simply defined as destructive, the speaker/Augustine seeks spirituality and nobility, asking that “O Lord” “pluckest” him “out.” As a result of this return to a more noble foundation, he reaches in line 311 a final state of “burning.” Could this burning be one of purification, a sign of moving beyond Carthage’s dark fate? Eliot seems to be suggesting that, just as states break free from the age old security dilemma that the Thucydides Trap truly as by setting aside aggressive expansionism for more moderate and localized policy, humans can find a better reality–an escape from their “Waste Land–by creating an “aversion” to the politics of superficiality and avarice, instead pursuing purification and simplicity.

Here Eliot divulges almost entirely into quoted sources from Buddha's Fire Sermon and St. Augustine's Confessions. The “collocation”, as Eliot puts it, of these two major representations of asceticism from Eastern and Western culture was entirely purposeful, most likely to ensure a blanket statement for the reader. Immediately after reading Eliot's footnote, I was reminded of my annotation from last reading where I asserted that Tiresias becomes synonymous with the reader. The “I” of this stanza is preceded by the detached Tiresias, allowing the reader to embody the pronoun, and inherit the major dogmas of self-discipline from either hemisphere.

Crucial to understanding Tiresias and the reader as passive agents of the poem, one must turn to Confessions which the pronoun invokes, “thou pluckest me.” While describing the many things that lead to sin and a lower state of being, St. Augustine says, “Stage-plays also carried me away, full of images of my miseries, and of fuel to my fire. Why is it, that man desires to be made sad, beholding doleful and tragical things, which yet himself would no means suffer? yet he desires as a spectator to feel sorrow at them, this very sorrow is his pleasure. What is this but a miserable madness?” The success of a play, like most art, lies entirely in invoking the emotion of its viewer. This was the predominant goal of most modernist artists, across all mediums, so is entirely fitting embedded in Eliot’s crowning modernist achievement. The viewer in the poem is Tiresius (“I Tiresias / Perceived the scene”), or the reader if one believes my proof of this fact. In the context of a tragedy, the word “scene” becomes that of a play, and the following assault is of tragic proportions.

Before returning to the analogy of the Fire Sermon as a tragedy, I must first discuss a connection to the Aeneid. St. Augustine and Eliot alike say, “To Carthage then I came/ burning burning burning burning”. Aeneas travels to Carthage from the burning Troy, escaping from a doomed city and the wrath of the gods destroying it (when he is being urged by various divinities and ghosts to flee the “already destroyed” Troy, Aeneas continually gets distracted and tries to stay and defend his home. It is not until Venus lifts the mortal veil concealing his sight to reveal the city actively being destroyed by Olympic Gods, that he realizes he must save the Penates (the gods and essence of the city) and flee), surviving only because Neptune and Zeus intervene. Neptune literally plucks Aeneas’s ship out of a whirlpool, saving him from death. The language “pluckest” from St. Augustine resounds with similar undertones of this Aeneid passage. However, he wishes he would have died, because living with his city having been lost is worse. In many ways, the guilt of surviving and leaving his kingdom behind phantom burns him. So, the “burning” of the Buddha's fire sermon and St. Augustine’s Confessions become united through the Aeneid.

Returning to the tragedy connection of “The Waste Land”, when Aeneas and his men first arrive to the shores of Carthage, the beach that receives them is described as a scaena (146), or the set design of a Greek tragedy. Such a setting foreshadows the impending destruction of Dido, a mini tragedy embedded within the greater epic. Further relating these ideas, Dido also burns, like Aeneas, mankind (according to the Buddha), and the “I” of the Waste Land. When Cupid infects her with the love for Aenas that will be her undoing, she ardescitque tuendo (she burns by gazing), meaning as she looks at Aeneas she is entirely consumed by her passion for him. This flame inevitably consumes her, and she fittingly kills herself atop a massive pyre.

Essentially, the Fire Sermon becomes analogous with an epic tragedy: the scene is set, the audience is established, the character is introduced, tragically assaulted, and the curtains close, leaving the reader “burning” in the “miserable madness” of watching the story unfold. In the Buddha’s Fire Sermon, “while this exposition was being delivered, the minds of the thousand priests became free from attachment and delivered from depravities. Here Endeth the Fire-Sermon.” The Sermon ends vindicating the priests from earthly desires, but Eliot’s reader leaves the Sermon more enveloped in flame than they began.

10.1

The positioning of this French phrase is most curious. It seems to deliver some message about innocence – especially chastity, given the general themes of this section of the poem – but finds itself stuck between colliding episodes of unnatural, purchased, and violent sex. Most notably, the “Sweeney” mentioned a few lines before recurs in several of Eliot’s poems as a client of prostitution; using the clues given by Eliot, the “Mrs. Porter” in the same line seems to refer to a wartime brothel-keeper who owns the bodies of her “daughters”; lastly, in the following stanza, we see another retelling of Philomela’s rape, its now archaic voicing (as the conjugation of “forc’d” suggests) rendering this savagery continuous across time (198-200, 205).

Why would Eliot place the line here? To start, I don’t think sarcasm or optimism are feasible explanations. Eliot would not have used the portrayed sexual innocence of children to satirize contemporary carnal practices; throughout the poem, he has repeatedly illustrated that reproductive virility is beneficial and even necessary to the health of the land and its inhabitants, so this line’s message must not run contrariwise. For the same reason the line is not designed to simply express optimism – for Eliot, a more ideal state of the world would not be one of complete religious abstinence evoked by the voices of choir youths.

Perhaps it would be valuable that we now reflect on the general characterization of children in the literature of the early 20th century. For many, including Conrad, childishness entails traditional qualities of innocence and simplicity; on the other hand was, however, an emerging view of childhood as a pre-adulthood, with all its potential strengths, challenges, and vices. This can be exemplified through the Boy Scout movement prevalent in England at the time – premature masculinity/virility was increasingly welcomed and strived for among the general public.

I believe Eliot tries to capture this sentiment with the excerpted line from Parsifal. The sound of children singing in a distant church no longer constitutes the imagery of a young knight resisting sexual temptations with his natural innocence, but is stranded in – and even possibly contaminated by – a society of sinful sexual morbidity. Recall that for Eliot, morbid reproduction is identified with degeneration and sterilization. Thus, this message is consistent with another depiction of the action of youth in the poem: the departure of the presumably young Hyacinth pair from the Hyacinth garden, a symbol of healthy and cyclical reproductive forces, to a degenerative state (36-7). Even children cannot escape the devitalizing forces of the modern waste land.

...we can inhabit God?

En el apartado de las tareas encontramos actividades enlazadas cuya realización progresiva es de suma importancia para obtenerse el resultado deseado que es siempre una acción en el mundo (diferentemente de las actividades aisladas que representan una acción lingüística o una acción en sala de clase

This highlights how seemingly neutral policies, like self-scheduling, actually privilege students with more knowledge and resources. The "myth" of choice obscures how these systems advantage some students while disadvantaging others, particularly those from marginalized backgrounds.

The deliberate and reactive, understandable and non-understandable are tangled and he doesn't intend to draw hard lines between them in pursuit of meaning.

Russell's paradox (1901) in set theory can be stated as:

If $$P$$ is the set of all sets, one can form the set $$Q = {A \in P | A \notin A}$$ which can lead to the contradiction $$Q \in Q$$ iff $$Q \notin Q$$.

This can be done by dividing P into two non-empty subsets, $$P_1 = {A \in p | A \notin A}$$ and $$P_2={A \in P | A \in A}$$. We then have the contradiction $$P_1 \in P_1$$ iff $$P_1 \notin P_1$$.

The paradox happens when we allow as sets A for which $$A \in A$$. It can be remedied by agreeing that no collection can be a set which would be an element of itself.

Relation to Groucho Marx's quote (earliest 1949) about resigning membership of a club which would have him as a member: https://hypothes.is/a/3_zAfITjEe-H5-PlfOlK8A

rallying americans together to help fight the british and end the kings rein. Oppression has taken over the world, freedom has been expelled from every nation in danger.

Calling the people to revolt and gain the freedom they deserve after being mistreated.

Boa tarde autores!

Como o tema me instigou a curiosidade, resolvi tecer algumas sugestões e apontamentos que acredito que possa ser de relevante contribuição. Na introdução foi apresentado um bom panorama sobre o tema, mas poderia ser mais concisa e clara para facilitar a compreensão do leitor. Há alguns pontos que podem ser ajustados para melhorar o fluxo do texto e reduzir a redundância. O texto poderia ser melhor estruturado com parágrafos mais curtos, cada um focando em uma ideia ou tema específico. Isso ajudaria a melhorar a leitura e interpretação. Poderiam também ser utilizadas palavras de transição para melhorar a fluidez entre as ideias. Por exemplo, ao introduzir novos conceitos ou estudos, utilizasse expressões como "Além disso", "Por outro lado", ou "Em contraste". As referências (como “GALLO; WAITT, 2011”) poderiam ser melhor formatadas de forma consistente ao longo do texto. Isso inclui a padronização na citação de autores e datas. Analisando a gramática e a ortografia, por exemplo, "professoras e professores do" deveria ser corrigido para "professoras e professores do". Também poderia remover redundâncias, para evitar repetições desnecessárias de ideias. Por exemplo, a ideia de que "a ciência cidadã é um processo formativo" e "a ciência cidadã exige conscientização" poderia ser consolidada. Nas considerações finais observa-se um resumo detalhado dos resultados, mas poderia ainda ser otimizada para garantir maior clareza e objetividade.

C'est une petite magazine Il parle de communication, il aide les enterprises C'est un texte informatif

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Vítejte do světa Animatrixu, vizionářské fúze počítačové animace a japonské školy animace, od světově nejuznávanějších tvůrců animovanch filmů. Vychutnejte si snímek, který vznikl před filmem Matrix a dozvíte se o posledních městech lidské rasy, válce mezi stroji a lidmi a o závěrečném úpadku lidstva. Staňte se svědky posledního letu Osirise, který připravil půdu pro film Matrix Reloaded a videohru Enter The Matrix. Doplňte si své povědomí o Matrixu a získejte informace, které jinde nenajdete. Je čas zapojit se... (oficiální text distributora)

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.

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.

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.

Entendo que o título e as palavras chaves estão adequadas por não estarem repetidas, isso aumenta a amplitude de pesquisas que o artigo pode ser localizado nas buscas. Ou seja, deve-se evitar repetir palavras para maximinizar a visibilidade do artigo.

Sugiro trazer outro exemplo do IA generativa. Pois o título do artigo fala sobre Inteligência Artificial generativa, e em alguns pontos o ChatGPT é tratado como o centro do trabalho, como se só existisse ele nessa categoria de IA generativa.

O mesmo relatado anteriormente. Sugiro trazer uma comparação ou um contraste para garantir uma base mais sólida para suas afirmações.

Sugiro mostrar se o estudo concorda ou contrasta com as pesquisas prévias (A análise dos resultados, trás essa informação com mais clareza), porém senti que aqui faltou fazer a ligação dos resultados da pesquisa com a literatura já existente.

Sugiro recomendar futuros aprofundamentos (tópicos a serem estudados) das questões discutidas no trabalho, para deixar em aberto uma lacuna a ser preenchida por futuras pesquisas. Num todo o trabalho está bem escrito e apresenta riqueza de dados. Como sugestão para trabalhos futuros, aplicar o questionário em várias IEFs de todo o pais para poder construir um panorama nacional sobre o uso das ferramentas de IA.

Sugiro troca dessa palavra chave. Essa palavra chave aparece somente 2 vezes ao longo de todo o texto do documento. O tema "Novas tecnologias" é abordado no trabalho, porém o termo "Inteligência Artificial" é mais abordado no texto.

Sugiro referenciar o parágrafo.

tentr relacionar com o eixo de expressao e conteudo

Maurice Richard is portrayed as a symbol of Montreal’s pride of its people during the 1940s and '50s. His presence on the ice resonated deeply with Quebecois. Richard became more than just a hockey player—he embodied the identity and spirit of the city and its people.

Bom resumo dividido entra as parte do do artigo porém com duas ressalvas. Objetivo muito amplo, senti falta dos objetivos específicos. Não fala sobre as discussões e as considerações finais não apresenta exatamente a evolução e sim as consequências como vantagens e desvantagens.

Apresenta conceitos e referências sobre o tema

Apresenta os itens da metodologia de uma revisão bibliográfica mas é pouco explorada, como revisão poderia ter um esquema visão para melhorar o entendimento

Título claro e conciso é exatamente o que eu estava buscando sobre o tema

São apresentadas os pontos de vista das referências, mas pela data de publicação acredito que existem mais referências sobre o mesma tema que pudessem ser comparadas.

Traz boas discussões, porém sobre o ponto de vistas de novas referências que não foram apresentadas anteriormente.

ESTO ES INTERESANTE

Significa adorarlo por puro amor y devoción, y no simplemente por obligación o porque nos parezca lo correcto. También implica mantener vivo ese amor durante toda la vida

te falta terminar la línea con un "verificando:" o un "tal que"

used to r/o rather than to diagnose.

N^o 62 = VA 11 499</br> N^o 67 = VA 11 509</br> N^o 73 = VA 11 508</br>

Esta parte del artículo me pareció muy interesante; intuitivamente se piensa que la fotosíntesis a sido un gran regulador del CO2 y oxígeno en los primeros años de la Tierra, no obstante, este párrafo da brinda otra explicación interesante. Considero que no debe ser descartable esta posibilidad ya que la Tierra y todas las interaciones que hoy conocemos eran totalmente diferentes, hace 500 millones de años, en el Cambrico. Lo importante es no cegarse ante una o dos posibles explicaciones, sino más bien, tomar los mejor de ellas, e intentar dar explicación de estos sucesos para entender procesos actuales.

Meus cumprimentos aos autores, o artigo possui um tema muito relevante, a sífilis congênita é um problema de saúde pública. Trabalho em Laboratório de Análises Clínicas e vejo na prática a alta incidência de sífilis. O título escolhido é claro. O resumo contempla objetivos, método, resultado e conclusão. Os autores utilizaram literatura recente, demostrando como estão as pesquisas atualmente. Achei a leitura de fácil compreensão sendo colocada de forma objetiva. Os autores utilizaram uma referência de 2004 que é justificada pelo fato de ser um informe técnico elaborado por representantes do Ministério da Saúde cujo objetivo principal foi modificar a definição de casos para ajustar a vigilância epidemiológica da sífilis congênita aumentando sua especificidade. A metodologia está clara, resultando em dados epidemiológicos importantes. O uso de dados obtidos por fontes oficiais aumenta a credibilidade da pesquisa. Os resultados da pesquisa podem dar sustentação às políticas públicas e ações preventivas como tratamento de parceiros e melhoria do pré-natal, assim como a importância da adesão ao tratamento. A conclusão está coerente com a proposta da pesquisa. Observei uma generalização restrita, os dados se limitam a Porto Alegre. Outras regiões do Brasil possuem realidade diferente, com relação às condições socioeconômicas e até mesmo de acesso à saúde. Uma sugestão seriam futuros estudos que ampliem a área de pesquisa para regiões com diferentes contextos.

È un'altra tra le librerie più importanti in Python. Utilizza Numpy e permette di gestire dati tabulari, anche con valori vuoti o nulli.

Author response:

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

Reviewer 1：

One major issue arises in Figure 4, the recording of VLPO Ca2+ activity. In Lines 211-215, they stated that they injected AAV2/9-DBH-GCaMP6m into the VLPO, while activating LC NE neurons. As they claimed in line 157, DBH is a specific promoter for NE neurons. This implies an attempt to label NE neurons in the VLPO, which is problematic because NE neurons are not present in the VLPO. This raises concerns about their viral infection strategy since Ca activity was observed in their photometry recording. This means that DBH promoter could randomly label some non-NE neurons. Is DBH promoter widely used? The authors should list references. Additionally, they should quantify the labeling efficiency of both DBH and TH-cre throughout the paper.

In Figure 5, we found that the VLPO received the noradrenergic projection from LC, indicating the recorded Ca2+ activity may come from the axon fibers corresponding to the projection. Similarly, Gunaydin et al. (2014) demonstrated that fiber photometry can be used to selectively record from neuronal projection.

We appreciate the reviewer's insightful suggestion to elaborate on the DBH promoter, we have now expanded our discussion to address the DBH (pg. 18): “DBH (Dopamine-beta-hydroxylase), located in the inner membrane of noradrenergic and adrenergic neurons, is an enzyme that catalyzes the conversion of dopamine to norepinephrine, and therefore plays an important role in noradrenergic neurotransmission. DBH is a marker of noradrenergic neurons. Zhou et al. (2020) clarified the probe specifically labeled noradrenergic neurons by immunolabeling for DBH. Recently, DBH promoter have been used in several studies (e.g., Han et al., 2024; Lian et al., 2023). The DBH-Cre mice are widely used to specifically labeled noradrenergic neurons (e.g., Li et al., 2023; Breton-Provencher et al., 2022; Liu et al., 2024). It is difficult to distinguish the role of NE or DA neurons when using the TH promoter in VLPO. Therefore, we used DBH promoter with more specific labeling. LC is the main noradrenergic nucleus of the central nervous system. In our study, we injected rAAV-DBH-GCaMP6m-WPRE (Figure 2 and 8) and rAAV-DBH-EGFP-S'miR-30a-shRNA GABAA receptor)-3’-miR30a-WPRES (Figure 9) into the LC. The results showed that DBH promoter could specifically label noradrenergic neurons in the LC, while non-specific markers outside the LC were almost absent.”

As suggested, we have quantified the labeling efficiency of both DBH and TH-cre throughout the revised manuscript (Fig.2D; Fig.3D, N-O; Fig.4E-F, J, L; Fig.5E, L; Fig.6L, S, X; Fig.7G).

A similar issue arises with chemogenetic activation in Fig. 5 L-R, the authors used TH-cre and DIO-Gq virus to label VLPO neurons. Were they labelling VLPO NE or DA neurons for recording? The authors have to clarify this.

As previously addressed in response to Comment #1, we agree that it is difficult to distinguish the role of NE or DA neurons when using the TH promoter in the VLPO. Therefore, we injected the mixture of DBH-Cre-AAV and AAV-EF1a-DIO-hChR2(H134R)-eYFP/AAV-Ef1a-DIO-hM3Dq-mCherry viruses into bilateral LC and AAV-EF1a-DIO-hChR2(H134R)-eYFP/AAV-Ef1a-DIO-hM3Dq-mCherry virus into bilateral VLPO. Moreover, we quantified the labeling efficiency of DBH in the LC to demonstrate that this promoter can specifically label NE neurons (Fig. 5). Importantly, these corrections did not alter the outcomes of our results. Both photogenetic and chemogenetic activation of LC-NE terminals in the VLPO can effectively promote midazolam recovery (Fig. 5G, N).

Another related question pertains to the specificity of LC NE downstream neurons in the VLPO. For example, do they preferentially modulate GABAergic or glutamatergic neurons?

Our study primarily aimed to explore the role of the LC-VLPO NEergic neural circuit in modulating midazolam recovery. We acknowledge that our evidence for the role of LC NE downstream neurons in the VLPO, derived from activation of LC-NE terminals and pharmacological intervention in the VLPO (Fig.5, Fig.6, Fig.8, Fig.9) is limited. Accordingly, we now present the VLPO’s role as a promising direction for future research in the limitation section of our revised manuscript: “This study shows that the LC-VLPO NEergic neural circuit plays an important role in modulating midazolam recovery. However, the specificity of LC NE downstream neurons in the VLPO is not explained in this paper, which is our next research direction, VLPO neurons and their downstream regulatory mechanisms may be involved in other nervous systems except the NE nervous system, and the deeper and more complex mechanisms need to be further investigated.”

In Figure 1A-D, in the measurement of the dosage-dependent effect of Mida in LORR, were they only performed one batch of testing? If more than one batch of mice were used, error bar should be presented in 1B. Also, the rationale of testing TH expression levels after Mid is not clear. Is TH expression level change related to NE activation specifically? If so, they should cite references.

As recommended, we have supplemented error bar and modified the graph of LORR’s rate in the revised manuscript. (Fig. 1A-B; Fig. 9G-H).

We agree that the use of TH as a marker of NE activation is controversial, so in the revised manuscript, we directly determined central norepinephrine content to reflect the change of NE activity after midazolam administration (Fig. 1D).

Regarding the photometry recording of LC NE neurons during the entire process of midazolam injection in Fig. 2 and Fig. 4, it is unclear what time=0 stands for. If I understand correctly, the authors were comparing spontaneous activity during the four phases. Additionally, they only show traces lasting for 20s in Fig. 2F and Fig. 4L. How did the authors select data for analysis, and what criteria were used? The authors should also quantify the average Ca2+ activity and Ca2+ transient frequency during each stage instead of only quantifying Ca2+ peaks. In line 919, the legend for Figure 2D, they stated that it is the signal at the BLA; were they also recorded from the BLA?

In this study, we used optical fiber calcium signal recording, which is a fluorescence imaging based on changes in calcium. The fluorescence signal is usually divided into different segments according to the behavior, and the corresponding segments are orderly according to the specific behavior event as the time=0. The mean calcium fluorescence signal in the time window 1.5s or 1s before the event behavior is taken as the baseline fluorescence intensity (F0), and the difference between the fluorescence intensity of the occurrence of the behavior and the baseline fluorescence intensity is divided by the difference between the baseline fluorescence intensity and the offset value. That is, the value ΔF/F0 represents the change of calcium fluorescence intensity when the event occurs. The results of the analysis are commonly represented by two kinds of graphs, namely heat map and event-related peri-event plot (e.g., Cheng et al., 2022; Gan-Or et al., 2023; Wei et al., 2018). In Fig. 2, the time points for awake, midazolam injection, LORR and RORR in mice were respectively selected as time=0, while in Fig. 4, RORR in mice was selected as time=0. The selected traces lasting for 20s was based on the length of a complete Ca2+ signal. We have explained the Ca2+ recording experiment more specifically in the figure legends and methods sections of our revised manuscript.

To the BLA, we sincerely apologize for our carelessness, the signal we recorded were from the LC rather than the BLA. We have carefully checked and corrected similar problems in the revised manuscript.

Reviewer 2：

In figure legends, abbreviations in figure should be supplemented as much as possible. For example, "LORR" in Figure 1.

As suggested, we have supplemented abbreviations in figure as much as possible in the revised manuscript.

Additional recommendations:

The main conceptual issue in the paper is the inflation of the conclusion regarding the mechanism of sedation induced by midazolam. The authors did not reveal the full mechanism of this but rather the relative contribution of NE system. Several conclusions in the text should be edited to take into account this starting from the title. I think the following examples are more appropriate: "NE contribution to rebooting unconsciousness caused by midazolam' or 'NE contribution to reverse the sedation induced by midazolam'.

As suggested, we have moderated the assertions about the mechanism of sedation induced by midazolam in several conclusions starting from the title (Line 1,125,150,169,202,237,482), to present a more measured interpretation in the manuscript.

Line 178-179, the authors state 'these suggest that intranuclear ... suppresses recovery from midazolam administration'. In fact, this intervention prolonged or postponed recovery from midazolam.

In our revised manuscript, we have corrected this inappropriate term (Line 178).

Pharmacology part (page 12) that aimed to pinpoint which NE receptor is implicated would suffer from specificity issues.

In relation to the specificity issue, the focus on VLPO might be rational but again other areas are most likely involved given the pharmacological actions of midazolam.

In the revised manuscript, we have discussed those specificity issues of NE receptor and areas involved throughout the midazolam-induced altered consciousness: “In addition, given the pharmacological actions of midazolam, other areas may also be involved. Current studies suggest that the neural network involved in the recovery of consciousness consists of the prefrontal cortex, basal forebrain, brain stem, hypothalamus and thalamus. The role of these regions in midazolam recovery remains to be further investigated. Therefore, we will apply more specific experimental methods to determine the importance of LC-VLPO NEergic neural circuit and related NE receptors in the midazolam recovery, and conduct further studies on other relevant brain neural regions, hoping to more fully elucidate the mechanism of midazolam recovery in the future”.

Line 274, the authors used 'inhibitory EEG activity'. what does it mean? a description of which rhythm-related power density is affected would be more objective.

Example of conclusion inflation: in line 477, the word 'contributes' is better than 'mediates' if the specificity issue is taken into account.

As suggested, we have improved our expression of words in our revised manuscript (pg. 13-14).

References

Gunaydin LA, Grosenick L, Finkelstein JC, et al. Natural neural projection dynamics underlying social behavior. Cell. 2014;157(7):1535-1551. doi:10.1016/j.cell.2014.05.017

Zhou N, Huo F, Yue Y, Yin C. Specific Fluorescent Probe Based on "Protect-Deprotect" To Visualize the Norepinephrine Signaling Pathway and Drug Intervention Tracers. J Am Chem Soc. 2020;142(41):17751-17755. doi:10.1021/jacs.0c08956

Han S, Jiang B, Ren J, et al. Impaired Lactate Release in Dorsal CA1 Astrocytes Contributed to Nociceptive Sensitization and Comorbid Memory Deficits in Rodents. Anesthesiology. 2024;140(3):538-557. doi:10.1097/ALN.0000000000004756

Lian X, Xu Q, Wang Y, et al. Noradrenergic pathway from the locus coeruleus to heart is implicated in modulating SUDEP. iScience. 2023;26(4):106284. Published 2023 Feb 27. doi:10.1016/j.isci.2023.106284

Li C, Sun T, Zhang Y, et al. A neural circuit for regulating a behavioral switch in response to prolonged uncontrollability in mice. Neuron. 2023;111(17):2727-2741.e7. doi:10.1016/j.neuron.2023.05.023

Breton-Provencher V, Drummond GT, Feng J, Li Y, Sur M. Spatiotemporal dynamics of noradrenaline during learned behaviour. Nature. 2022;606(7915):732-738. doi:10.1038/s41586-022-04782-2

Liu Q, Luo X, Liang Z, et al. Coordination between circadian neural circuit and intracellular molecular clock ensures rhythmic activation of adult neural stem cells. Proc Natl Acad Sci U S A. 2024;121(8):e2318030121. doi:10.1073/pnas.2318030121

Cheng J, Ma X, Li C, et al. Diet-induced inflammation in the anterior paraventricular thalamus induces compulsive sucrose-seeking. Nat Neurosci. 2022;25(8):1009-1013. doi:10.1038/s41593-022-01129-y

Gan-Or B, London M. Cortical circuits modulate mouse social vocalizations. Sci Adv. 2023;9(39):eade6992. doi:10.1126/sciadv.ade6992

Wei YC, Wang SR, Jiao ZL, et al. Medial preoptic area in mice is capable of mediating sexually dimorphic behaviors regardless of gender. Nat Commun. 2018;9(1):279. Published 2018 Jan 18. doi:10.1038/s41467-017-02648-0

This function is an algorithmic policy that determines the minimum throttle time for a process between consecutive writeback operations for dirty pages based on heuristics. It is used for balancing the load of the I/O subsystems so that there will not be excessive I/O operations that impact the performance of the system.

When enabled via lru_gen_enabled(), it alters how the kernel tracks page access patterns and manages evictions. This policy introduces the concept of generation-based tracking, where pages are grouped into generations based on their access time. It uses functions like lru_gen_eviction() and lru_gen_refault() to handle page evictions and refaults respectively. This approach allows for more nuanced decisions in page reclaim, potentially improving memory utilization and reducing I/O operations by better identifying truly cold pages and protecting hot pages from premature eviction.

acá daría a conocer las preguntas en cuestión, puede ser una imagen del cuestionario, o redactarlas bien. Me refiero a las de conflicto y percepción de desigualdad

When I first read this I was in agreement, especially in places such as California, and as I thought about it more I remember when teachers would talk about subject like these they're intellectual vocabulary would double and they're speaking speed would halve as to not say something controversial. While I know they can get introuble in a legal aspect It makes sense that on a smaller scale the classroom could certainly get rowdy.

I think that in primary education, these topics are very much avoided or talked about minimally. In universities, there is a healthy amount of discourse and discussion about these topics. Many people challenge these ideas and I believe that this is a good thing. We need to challenge the ideas and not fear talking about them

I really connect with this sentence because, as a teacher, I’ve noticed that challenging students' established ways of thinking can be uncomfortable for them, and I always try to acknowledge that discomfort. When we ask students to rethink long-held beliefs, especially around sensitive topics like race and racism, it can be unsettling. I feel it's my responsibility to guide them through this discomfort, helping them see that growth often comes from questioning old ideas and embracing new perspectives, even when it’s difficult.

This passage emphasizes the importance of hearing different voices in the classroom, not only as a basis for communication, but also as an act of mutual recognition and understanding. By ensuring that every student has the opportunity to speak, teachers can help students feel seen and valued.

In my opinion, I believe that educators should be more open-minded to change since we live in a rapidly changing society and we need to be able to provide kids with different forms of learning.

What’s worse is how entrenched this inequality is in our system. The disparities in school funding, based on property taxes, only widen the gap, creating a cycle that’s hard to break. If we truly want to address this, we need to think about deeper reforms—equitable school funding, accessible college pathways, and community support systems. Every student deserves the same chance to thrive, regardless of where they come from, and until we start focusing on those solutions, we’ll keep losing talented individuals to a system that doesn’t see their full potential.

Educational inequality has a ripple effect that goes far beyond the classroom, shaping the entire course of a student's life. The fact that success in today’s world is so closely tied to a college degree highlights just how deep this problem runs. It’s frustrating to think that a student's potential is often dictated by the resources their family can provide, rather than their talents or drive. Wealthier students have the advantage of tutors, better schools, extracurricular activities, and financial stability, while students from lower-income families may be just as capable but are held back by factors beyond their control.

I love seeing openminded people. The fact that these professors are able to recognize that they need to change shows how dedicated they are to their teaching. They are able to change their perspectives and teaching style in order to benefit their students/audience. Love the self growth. Holding themselves accountable and willing to grow from it!!

Yes!!! Some people have unrecognized biases and that is completely normal. Some people may grow up hearing things and just keep those thoughts in their unconscious mind, it may not be intentionally hurtful in their mind, but to others their may be a problem. Anyone can say something that seems completely normal to them, but their audiences can all interpret them completely different. I think that through these discussions with the professors will allow them to release their unconscious biases and learn from one another and transform their teaching/curriculum to better fit a multicultural classroom.

education should serve its original purpose, on this point I totally agree with this passage. We hope to see a world that everyone is free, and fullfilled with hope.

It is extremely difficult to transform paradigms when it involves beliefs and practices. There’s resistance to change, not because people are reluctant, but because the effects of change can be uncertain and overwhelming. And that's what works for the method I just described: collaborative and dialogical. When you enable a community where faculty can speak candidly about their concerns and strategies, you enable learning and development. I think this is particularly helpful if more traditionalists and conservationists can relate their experience with change. That inclusion invites others to think about their own practices, to think critically.

it is really the doors to many new possibilities for teachers and students to consider multiculturalism as part of the curriculum. If educators recognise and respect these strict limits, they make room for alternative viewpoints. This recognition drives us to work on breaking down prejudice and create a more equitable and welcoming classroom.

This last sentence was beautifully said. Consciousness within education is so important because it will allow us detect what needs fixing in our world today and learn how to respect one another. Free expression is also so crucial in our classrooms so that students can fully express themselves without feeling as if they're wrong and makes them feel like they're in a safe place to do so.

Embracing new ideas and learning can be challenging, but it’s a necessary part of growth. How can educators help students navigate this discomfort while learning new approaches?

This paragrahy explores the impact of multiculturalism on education. It emphasizes that educators should rethink their approaches to sharing knowledge and acknowledge their own biases. Students are eager to overcome learning obstacles, and when teachers incorporate multicultural methods, they can offer a more meaningful educational experience. The conversation between Watkins and bell hooks underscores the significant influence of Paulo Freire on their thinking, stressing the importance of critical thinking and the liberation of education.

It explains that some teachers avoid discussing race, gender, and class because they fear chaos and strong emotions. This can stop important conversations. Creating a safe space for students to express themselves might help teachers feel more comfortable with these topics.

I think this aspect of teachers/professors making racist comments needs to be more emphasized because in our modern system entitles every single person o an equal educational opportunity and shouldn't be discriminated. By which racist comment, picking on certain students, or generally just making student discriminated. By which affects their academic performs.

• Informativo 1151
• ADI 3837 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. NUNES MARQUES
• Julgamento: 20/09/2024 (Virtual)
• Ramo do Direito: Tributário, Financeiro
• Matéria: ICMS; Crédito Tributário; Modalidades de Extinção do Crédito Tributário; Compensação; Transação/Federalismo Fiscal; Repartição das Receitas Tributárias; ICMS

ICMS: extinção de créditos tributários estaduais por meio de operações de compensação ou transação

Resumo - Os valores dos créditos tributários extintos que decorram de compensação ou de transação (CTN/1966, arts. 170 e 171) devem integrar o cálculo do percentual de transferência da quota pertencente às municipalidades sobre o produto da arrecadação do ICMS relativo à repartição constitucional das receitas tributárias, na medida em que é desnecessário, para esse cômputo, o efetivo recolhimento do imposto.

• A Constituição Federal de 1988 — com a finalidade de promover o federalismo fiscal cooperativo de equilíbrio e dar efetividade aos pilares do “Estado Fiscal” — instituiu um regime de partilha dos recursos tributários. Nesse contexto, ela conferiu aos entes municipais o repasse de 25% (vinte e cinco por cento) do produto da arrecadação do ICMS (CF/1988, art. 158, IV, “a”), cuja competência tributária é dos estados-membros e do Distrito Federal (1). Conforme jurisprudência desta Corte, na hipótese de parcela do produto de ICMS já arrecadado, é de pleno direito dos próprios municípios o repasse que lhes é devido na repartição constitucional de receitas, sendo incabível qualquer forma de condicionamento ou retenção pelos estados (2). Ademais — à luz do conceito técnico de arrecadação —, não se pode exigir o repasse, aos municípios, de parcela deduzida, renunciada ou extinta de ICMS resultantes de benefícios fiscais, pois esses recursos sequer ingressaram nos cofres estaduais (3). Na espécie, a compensação de um crédito tributário pelo estado com uma dívida por ele adquirida configura evidente proveito econômico, mesmo sem recolhimento por parte do contribuinte, visto que representa um aumento na sua disponibilidade de recursos pela redução do seu passivo. Já no instituto da transação, a vantagem financeira obtida com o acordo equivale, na prática, à arrecadação do tributo. Portanto, seja por compensação, pagamento em espécie de tributos ou transação — modalidades de extinção do crédito tributário — os municípios têm direito à repartição de receitas tributárias, já que a receita pública é um fenômeno que antecede o recolhimento do imposto.
• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, julgou improcedente a ação para assentar a constitucionalidade do art. 4º, § 1º, da Lei Complementar nº 63/1990 (4).

(1) CF/1988: “Art. 158. Pertencem aos Municípios: (...) IV - 25% (vinte e cinco por cento): a) do produto da arrecadação do imposto do Estado sobre operações relativas à circulação de mercadorias e sobre prestações de serviços de transporte interestadual e intermunicipal e de comunicação.”

(2) Precedente citado: RE 572.762 (Tema 42 RG).

(3) Precedente citado: RE 705.423 (Tema 653 RG).

(4) Lei Complementar nº 63/1990: “Art. 4º Do produto da arrecadação do imposto de que trata o artigo anterior, 25% (vinte e cinco por cento) serão depositados ou remetidos no momento em que a arrecadação estiver sendo realizada à ‘conta de participação dos Municípios no Imposto sobre Operações relativas à Circulação de Mercadorias e sobre Prestações de Serviços de Transporte Interestadual e Intermunicipal e de Comunicações’, aberta em estabelecimento oficial de crédito e de que são titulares, conjuntos, todos os Municípios do Estado. § 1º Na hipótese de ser o crédito relativo ao Imposto sobre Operações relativas à Circulação de Mercadorias e sobre Prestações de Serviços de Transporte Interestadual e Interestadual e Intermunicipal e de Comunicação extinto por compensação ou transação, a repartição estadual deverá, no mesmo ato, efetuar o depósito ou a remessa dos 25% (vinte e cinco por cento) pertencentes aos Municípios na conta de que trata este artigo.”

Legislação: CF/1998: art. 158, IV, "a". CTN/1966: arts. 170 e 171. Lei Complementar 63/1990: art. 4º, §1º.

Precedentes: RE 572.762 (Tema 42 RG) e RE 705.423 (Tema 653 RG).

Os dados obtidos poderiam ser apresentados em tabelas e/ou gráficos para facilitar o entendimento do leitor?

A entrevista, apesar de estar justificada, estava fora do "planejamento" e não consta na metodologia do estudo. O que concede um ar informal ao estudo.

No Resumo, faltou deixar claro o objetivo do estudo.

Senhores autores, em primeiro lugar gostaria de parabenizá-los pela escolha da temática e pelo desenvolvimento do presente estudo. Como servidora atuando nos últimos cinco anos na secretaria do curso de Nutrição em uma Universidade Federal, vivenciei de perto os desafios enfrentados durante o ensino remoto emergencial e reconheço a importância da presente discussão. Gostaria de deixar minhas observações e contribuições para o artigo.

O título do artigo é claro e conciso, refletindo o conteúdo do trabalho e sendo coerente com a proposta. Minha sugestão seria apenas apontar de forma mais específica que os docentes mencionados fazem parte de instituições federais de ensino superior.

O resumo sintetiza bem o conteúdo do artigo, contemplando todos os passos do planejamento da pesquisa. A sugestão nesse ponto seria detalhar o tamanho da amostra estudada, e o número de docentes que responderam a pesquisa.

As palavras-chave estão bem escolhidas e são termos relevantes para o tema abordado. Contudo, poderiam ser incluídos mais termos relacionados às metodologias de ensino e Tecnologias de Informação e Comunicação (TICs), substituindo, por exemplo, "ensino online" por "ensino remoto", expressão mais utilizada na área.

A introdução foi muito bem escrita. Contextualiza o cenário do Ensino Remoto Emergencial no contexto da pandemia da COVID-19, os desafios e oportunidades do ensino de Saúde Pública para os cursos da área da Saúde, em especial ao curso de Nutrição, seguindo a estrutura clássica da introdução de artigos científicos de se apresentar um panorama geral e partir para o contexto específico. Na introdução são utilizadas referências importantes e atuais, e fica bastante claro os objetivos e contribuições do estudo.

O método está descrito de forma clara e coerente com a questão de pesquisa. Senti falta, entretanto, de mais referências nos parágrafos iniciais da metodologia. O desenho do estudo foi explicitado e está adequado, descrevendo a população e amostra, e os critérios de inclusão e exclusão foram mencionados. O processo de coleta de dados está bem explicado, minha sugestão seria incluir o período da coleta de dados e detalhar um pouco mais como se deu a utilização do software de apoio à análise. A aprovação do projeto de pesquisa pelo Comitê de Ética está referida, assim como as precauções tomadas para se preservar a confidencialidade dos dados.

Os resultados estão apresentados de forma clara e bem articulados com os objetivos do estudo. As citações diretas dos entrevistados enriquecem a narrativa e a compreensão dos desafios do ensino remoto. A apresentação de tabelas, figuras ou quadros seria bem-vinda para sintetizar as informações e facilitar a visualização dos principais achados.

A discussão está bem fundamentada nos resultados apresentados, apresentando os desafios e oportunidades encontrados no estudo. No entanto, faltou uma discussão mais aprofundada das limitações do estudo, como a possibilidade de generalização dos achados. Essas limitações são apresentadas apenas mais adiante, na seção de considerações finais. A literatura utilizada é atual e bem escolhida, mas a inclusão de mais estudos recentes poderia fortalecer ainda mais a argumentação.

As conclusões resumem bem os principais achados, destacando os desafios enfrentados no ensino remoto e a importância da interação presencial para o ensino de Saúde Coletiva. No entanto, seria interessante destacar de forma mais enfática as inovações e contribuições deste estudo, especialmente no que se refere às recomendações para futuras práticas de ensino

As referências são pertinentes e abrangentes, e estão em quantidade adequada. Entretanto, por se tratar de um artigo publicado em 2024, acredito que seria interessante utilizar também referências ainda mais atuais, de 2022 e 2023, já do período pós-pandêmico, que poderiam refletir ainda mais os impactos do período de ensino remoto na saúde coletiva e as adaptações já realizadas após o ápice do período pandêmico.

No geral, trata-se de um artigo interessante, bem estruturado, e que aborda um tema relevante e atual. Novamente parabenizo os autores e espero que as contribuições possam oferecer novas perspectivas para fortalecer ainda mais a relevância deste trabalho.

Resumo sintetiza bem o que será abordado bem como os métodos que serão empregados.

Detalha e embasa todos os métodos usados na pesquisa. Mai a frente destro dessa seção há uma oportunidade de melhoria identificada. Além disso o autor estrutura e descreve muito bem e justifica os métodos usados,

map also needs updating

El mostrar favoritismo, va en contra de la ley de Jehová y de Jesús. Por ese motivo debemos tener cuidado de no mostrar la misma clase de maldad en nuestros juicios o prejuicios.

Advertencia a - ganancias deshonestas, - derramar sangre inocente - cometer fraude y extorsión’. Hay que Defender al inocente sea pobre o rico, con la justicia de Jehová.

Que debían juzgar con imparcialidad, al grande y al pequeño, y si el asunto es muy díficil o delicado, se lo debían presentar a Jehová, y ese es el modelo del cual aprenden los ancianos

Los ancianos tienen que tomar muchas decisiones pero regularmente ellos se reúnen para recomendar hermanos para siervos ministeriales o ancianos y estas recomendaciones tienen que estar libre de prejuicios 1 Timoteo 5:21

DOI: 10.1101/2024.09.22.614347

Resource: Nipype (RRID:SCR_002502)

Curator: @scibot

SciCrunch record: RRID:SCR_002502

What is this?

DOI: 10.1101/2024.09.23.614448

Resource: Addgene_81020

Curator: @scibot

SciCrunch record: RRID:Addgene_81020

What is this?

Theme: Water Snakes: The mariner realizes them that all of God's creatures are beautiful and made in His name. Thus, releasing him from this curse. SK

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The study "Endogenous oligomer formation underlies DVL2 condensates and promotes Wnt/βcatenin signaling" by Senem Ntourmas et al. contributes to the understanding of phase separation in Dishevelled (DVL) proteins, specifically focusing on DVL2. It builds upon existing research by investigating the endogenous complexes of DVL2 using ultracentrifugation and contrasting them with DVL1 and DVL3 behavior. The study identifies a DVL2-specific region involved in condensate formation and introduces the "two-step" concept of DVL2 condensate formation, enriching the field's knowledge. 

Strengths: 

A notable strength of this study is the validation of endogenous DVL2 complexes, providing insights into its behavior compared to DVL1 and DVL3. The functional validation of the DVL C-terminus (here termed conserved domain 2 (CD2) and the identification of DVL2-specific regions (here termed LCR4) involved in condensate formation are significant contributions that complement the current knowledge on the importance of DVL DIX domain, DEP domain and intrinsically disordered regions between DIX and PDZ domains. Additionally, the introduction of the concept where oligomerization (step 1) precedes condensate formation (step 2) is an interesting hypothesis, which can be further experimentally challenged in the future.

We thank the reviewer for her/his interest in our work and for acknowledging our significant contributions to the understanding of DVL2 phase separation.   

Weaknesses: 

However, the applicability of the findings to full-length DVL2 protein, hence the physiological relevance, is limited. This is mostly due to the fact that the authors almost completely depend on the set of DVL2 mutants, which lack the (i) DEP domain and (ii) nuclear export signal (NES). These variants fail to establish DEP domain-mediated interactions, including those with FZD receptors. Of note, the DEP domain itself represents a dimerization/tetramerization interface, which could affect the protein condensate formation of these mutants. Possibly even more importantly, the used mutants localize into the nucleus, which has different biochemical & biophysical properties than a cytoplasm, where DVL typically reside, which in turn affects the condensate formation. On top, in the nucleus, most of the DVL binding partners, including relevant kinases, which were reported to affect protein condensate formation, are missing.

The most convincing way to address this valid concern and to support a physiological relevant role of our findings is to extend our experiments with full-length DVL2, which we did alongside the suggestion in point two (please see below). In addition, we address the specific issues as follows:

We completely agree that interaction through the DEP domain contributes to condensate formation, which was thoroughly demonstrated in great studies by Melissa Gammons and Mariann Bienz, and complex formation (Fig. 2B, C). We deleted this domain on purpose for our mapping experiments, since we obtained more consistent results without any additional contribution of the DEP domain. Once we mapped CFR and identified crucial amino acids within CFR (VV, FF), we demonstrated that CFR-mediated interaction contributes to complex formation, condensate formation and pathway activation in the context of full-length DVL2 (Fig. 7A-G). 

We also agree that the nuclear localization may affect condensate formation because of the reasons mentioned by the reviewer or others, such as differences in DVL2 protein concentration. However, later proof-of-concept experiments in full-length DVL2 confirmed that CFR and its identified crucial amino acids (VV, FF), which were mapped in this rather artificial nuclear context, contribute to the typical cytosolic condensate formation of DVL2 (Fig. 7C, D). Moreover, we also observed cells with cytosolic condensates for the NES-lacking DVL2 constructs, although to a lower extent as compared to cells with nuclear condensates. A new analysis of NES-lacking key constructs focusing exclusively on cells with cytosolic condensates revealed similar differences between the DVL2 mutants as were observed before when investigating cells with nuclear (and cytosolic) condensates (new Fig. S3E, F), suggesting that the detected differences are not due to nuclear localization but reflect the overall condensation capacity. 

In addition, our condensate-challenging experiments (osmotic shock, 1,6-hexandiol) suggested that cytosolic condensates of full-length DVL2 and nuclear CFR-mediated condensates of deletion proteins lacking the DEP domain behave quite similar (Fig. 6A-C).

Second, the use of an overexpression system, while suitable for comparing DVL2 protein condensate features, falls short in functional assays. The study could benefit from employing established "rescue systems" using DVL1/2/3 knockout cells and re-expression of DVL variants for more robust functional assessments. 

We used the suggested established rescue system of DVL1/2/3 knockout cells (T-REx DVL1/2/3 triple knockout cells and T-REx DVL1/2/3 RNF43 ZNRF3 penta knockout cells, which are even more sensitive towards DVL re-expression as they lack RNF43/ZNRF3-mediated degradation of DVL activating receptors; both cell lines from the Bryja lab). Upon overexpression, our key mutants DVL2 VV-AA FF-AA and ∆CFR showed markedly reduced pathway activation compared to WT DVL2 (new Figs. 7F and S5J), as we observed before. Especially in the DVL1/2/3 triple knockout cells, DVL2 VV-AA FF-AA hardly activated the pathway and was as inactive as the established M2 mutant (new Fig. 7F). Most importantly, while re-expression of WT DVL2 at close to endogenous expression levels fully rescued Wnt3a-induced pathway activation in DVL1/2/3 knockout cells, DVL2 VV-AA FF-AA revealed significantly reduced rescue capacity and was almost as inactive as DVL2 M2 (new Figs. 7G and S5K). 

Furthermore, the discussion and introduction overlook some essential aspects of DVL biology. One such example is the importance of the open/close conformation of DVL and its effects on DVL phase separation and activity. In the context of this study, it is important to say that this conformational plasticity is mediated by DVL C-terminus (CD2 in this study). The second example is the reported roles of DVL1 and DVL3, which can both mediate the Wnt3a signal. How this can be interpreted when DVL1 and DVL3 lack LCR4 and still form condensates? 

We included the open/close conformation of DVL in our manuscript (introduction p. 3 and new discussion paragraph p. 10) and discussed it in the context of our findings. It is intriguing to speculate that Wnt-induced opening of DVL2 increases the accessibility of LCR4 and CD2, thereby triggering pre-oligomerization and subsequent phase separation of DVL2 (see discussion).

We extended the last paragraph of the discussion to interpret the roles of DVL1 and DVL3 lacking LCR4 (see p. 10). In short, the general ability of DVL1 and DVL3 to form condensates and to activate the Wnt pathway can be potentially explained through the other interaction sites (DIX, DEP, intrinsically disordered region). However, previous studies suggest that the DVL paralogs exhibit (quantitative) differences in Wnt pathway activation and that all three paralogs have to interact at a certain ratio for optimal pathway activation. In this context, a physiologic role for DVL2 LCR4 may be to promote the formation of these DVL1/2/3 assemblies and/or to enhance the stability of these assemblies.

In order to increase the physiological relevance of the study, I would recommend analyzing several key mutants in the context of the full-length DVL2 protein using the rescue/complementation system. Further, a more thorough discussion and connections with the existing literature on DVL protein condensates/puncta/LLPS can improve the impact of the study. 

We thank the reviewer for her/his suggestions to improve our study, which we addressed as detailed above.

Reviewer #2 (Public Review): 

Summary: 

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

Strengths: 

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

We thank the reviewer for her/his interest in our work and the constructive suggestions to improve our study.

Weaknesses: 

I think that several of the approaches of the manuscript are subpar to achieve the goals and/or support several of the conclusions. For example: 

(1) Although endogenous DVL2 indeed seems to form complexes (Figure 1A), neither the number of proteins involved nor whether those are homo-complexes can be determined with a density gradient. Super-resolution imaging or structural analyses are needed to support these claims. 

We agree that it will be very interesting to study the nature of the detected endogenous complexes in detail and we will consider this for any follow-up study, as structural analyses were out of scope for the revision of the presented manuscript. To address the issue, we mentioned that the calculation of about eight DVL2 molecules per complex is based on the assumption of homotypic complexes (results p. 4) and we discussed, why we think that homotypic complexes are the most likely assumption based on the currently available (limited) data (discussion p. 8).

(2) Follow-up analyses of the relevance of the DVL2 domains solely rely on overexpressed proteins. However, there were previous questions arising from o/e studies that prompted the focus on endogenous, physiologically relevant DVL interactions, clustering, and condensate formation.

Although the title, conclusions, and relevance all point to the importance of this study for understanding endogenous complexes, only Figures 1A and B deal with endogenous DVL2. 

We think that the biochemical detection of endogenous DVL2 complexes itself represents a valuable contribution to the understanding of endogenous DVL clustering, especially (i) since it is still lively discussed in the field whether and to which extent endogenous DVL assemblies exist (see introduction) and (ii) since recent studies addressing this issue rely on fluorescent tagging of the endogenous protein, which, among all benefits, harbors the risk to artificially affect DVL assembly. The follow-up analysis predominantly strengthens this key finding through (i) associating the detected complexes with established (DEP domain) and newly mapped (LCR4) DVL2 interaction sites, which we think is crucial to validate our biochemical approach, and (ii) linking the complexes with condensate formation and pathway activation for functional insights.

In addition, we performed new experiments with re-expression of DVL2 and our key mutants at close to endogenous expression levels in DVL1/2/3 knockout cells, supporting a physiological relevant role of our findings (new Figs. 7G and S5K, please also see point (5) below).

(3) Mutants lacking activity/complex formation, e.g. DVL2_1-418, may need further validation. For instance, DVL2_1-506 (same mutant but with DEP) seems to form condensates and it is functional in WNT signalling (King et al., 20223). These differences could be caused by the lack of DEP domain in this particular construct and/or folding differences. 

We would definitely expect that DVL2 1-506 exhibits increased condensate formation and pathway activation as compared to DVL2 1-418, since the DEP domain was thoroughly characterized as interaction domain in the Bienz lab and the Gammons lab (see references), which we confirmed in our assays (Fig. 2B-D). However, as the DEP domain is an established DVL2 interaction site, we were not interested to further characterize the DEP domain but to explain the marked difference in complex formation between DVL2 ∆DEP and 1-418 (Fig. 2A-C), which could not be associated with any known DVL2 interaction site and which we finally mapped to CFR (Fig. 4A-D). 

Since fusion of the newly-characterized interaction site CFR to DVL2 1-418 (1-418+CFR) rescued complex formation, condensate formation and signaling activity (Fig. 3B-E and Fig. 4C, D), we think that the lacking activity/complex formation of DVL2 1-418 is more likely due to missing interaction sites than due to folding problems. However, as it is hard to exclude folding differences of deletion mutants, we confirmed the CFR activity through loss-of-function experiments in the context of fulllength DVL2 with minimal point mutations (Fig. 7A-G, VV,FF). 

(4) The key mutants, DeltaCFR and VV/FF only show mild phenotypes. The authors' results suggest that these regions contribute but are not necessary for 1) complex formation (Density gradient Figures 7A and B), condensate formation (Figures 7C and D), and WNT activity (Figure 7E). Of note Figure 7C shows examples for the mutants with no condensates while the qualification indicates that 50% of the cells do have condensates. 

Condensate formation and Wnt pathway activation by DVL VV-AA FF-AA were reduced by more than 50% as compared to WT (Fig. 7D, E). We consider these marked differences, since loss of function always ranges between 0% and 100%. In newly performed experiments in DVL1/2/3 knockout cells, the differences were even more pronounced, see point (5) below.

Yes, Fig. 7C shows an example to qualitatively visualize the change in condensate formation, while Fig. 7D provides the corresponding quantification allowing quantitative assessment of the differences.

(5) Most of the o/e analyses (including all reporter assays) should be performed in DVL1-3 KO cells in order to explore specifically the behaviour of the investigated mutants. 

As suggested, we employed DVL1/2/3 knockout cells for performing reporter assays (T-REx DVL1/2/3 triple knockout cells and T-REx DVL1/2/3 RNF43 ZNRF3 penta knockout cells, which are even more sensitive towards DVL re-expression as they lack RNF43/ZNRF3-mediated degradation of DVL activating receptors; both cell lines from the Bryja lab). Here, we focused on key mutants in the context of full-length DVL2, as they are closest to the physiologic situation. Upon overexpression, DVL2 VV-AA FF-AA and DVL2 ∆CFR showed markedly reduced pathway activation as compared to WT DVL2 (new Figs. 7F and S5J). Especially in the DVL1/2/3 triple knockout cells, DVL2 VV-AA FF-AA hardly activated the pathway and was as inactive as the established M2 mutant (new Fig. 7F). Moreover, re-expression at close to endogenous expression levels revealed that DVL2 VV-AA FF-AA less efficiently rescued Wnt3a-induced pathway activation as compared to WT (Figs. 7G and S5K).

(6) How comparable are condensates found in the cytoplasm (usually for wt DVL) with those located in the nucleus (DEP mutants)? 

In principal, cytosolic condensates could differ from nuclear condensates due to various reasons, such as e.g. different protein concentration, different availability of interaction partners or different biochemical/biophysical properties (please also see point 1 of reviewer 1). In our condensatechallenging experiments (osmotic shock, 1,6-hexandiol), cytosolic condensates of full-length DVL2 and nuclear condensates of DVL2 mutants behaved quite similar (Fig. 6A-C).

We are confident that the differences between different DEP mutants in our mapping experiments are not due to nuclear localization but reflect the overall condensation capacity because later proofof-concept experiments demonstrated that CFR, which was identified in these mapping experiments, contributes to cytosolic condensate formation in the context of full-length DVL2 (Fig. 7C, D). Moreover, a new analysis focusing only on cells with cytosolic condensates, which can also be observed for DEP mutants to a low extent, revealed similar differences between key DEP mutants as observed before (Fig. S3E, F; for details please also see point 1 of reviewer 1).

Several studies in the last two decades have analysed the relevance of DVL homo - and heteroclustering by relying on overexpressed proteins. Recent studies also explored the possibility of DVL undergoing liquid-liquid phase separation following similar principles. As highlighted by the authors in the introduction, there is a need to understand DVL dynamics under endogenous/physiological conditions. Recent super-resolution studies aimed at that question by characterising endogenously edited DVL2. The authors seemed to aim in the same direction with their initial findings (Figure 1A) but quickly moved to o/e proteins without going back to the initial question. This reviewer thinks that to support their conclusions and advance in this important question, the authors should introduce the relevant mutations in the endogenous locus (e.g. by Cas9+ donor template encoding the required 3' exons, as done by others before for WNT components, including DVL2) and determine their impact in the above-indicated processes.

We agree that genomic editing of the DVL2 locus would be the cleanest system to study the relevance of CFR at endogenous expression levels. As we did not have the resources to generate the suggested cells, we, as an alternative, transiently re-expressed DVL2 and the respective mutants at low levels that were really close to the endogenous expression levels in DVL1/2/3 triple knockout cells (Fig. S5K). These experiments revealed that DVL2 VV-AA FF-AA less efficiently rescued Wnt3ainduced pathway activation as compared to DVL2 WT (Fig. 7G).

Reviewer #2 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

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

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

The statement critiques the practice of tracking in education, asserting that it has significant negative implications. The speaker, drawing from their experience supervising student teachers in various contexts, highlights the widespread nature of ability grouping and its detrimental effects on students, suggesting a need for greater awareness and change in educational practices.

I personally have experienced this myself. In second grade, my elementary teacher used to pair students up for a math activity based off of their academic prowess. While this made competition even, it ensure that the smart students were getting smarter, while those that were behind didn't improve as much.

This quote exposes the deep systemic nature of class replication through education. It's frustrating to see that the same cycle of privilege continues unchecked. The way schools are funded, how access to good teachers and resources depends on wealth—these all ensure that education keeps reinforcing class divides. It feels like we are trapped in a cycle that we are unwilling to break because doing so would threaten the privileges of the wealthy.

The passage begins by acknowledging that ability grouping may seem harmless, even logical, in educational settings. However, it quickly becomes clear that the practice has negative consequences, particularly for poor children and children of color, who are disproportionately labeled as less capable.

Algorithmic policy for memory allocation. Checks if there are enough free pages for an allocation request based on a watermark-based allocation policy criteria. Is is tone by checking the number of free pages in the zone to a threshold "mark".

DOI: 10.1002/brb3.70057

Resource: Parkinson's Progression Markers Initiative (RRID:SCR_006431)

Curator: @scibot

SciCrunch record: RRID:SCR_006431

What is this?

DOI: 10.1080/19420862.2024.2406788

Resource: (NIH Nonhuman Primate Reagent Resource Cat# PR-1230, RRID:AB_2819287)

Curator: @scibot

SciCrunch record: RRID:AB_2819287

What is this?

DOI: 10.1158/0008-5472.CAN-23-3553

Resource: AB_2921382

Curator: @scibot

SciCrunch record: RRID:AB_2921382

What is this?

what do 'o' and 'b' indicate here?

Me interesa la manera de como esta pensado en personas que no son expertas en este tecnología o que no tienen un acercamiento a ella a diferencia de otros.

Es interesante el terma de que no solo se puede corregir o se le pueden añadir cosas al texto una vez este hecho, sino que se puede hacer en conjunto y prácticamente al instante en que surjan nuevas ideas o comentarios.

Ninguna decisión, no solo las financieras.

en polos fríos de agua o en el océano cerca de respiraderos en aguas profundas

Si eres propietario de tus acciones y el resultado de éstas (tu trabajo) que alguien te lo expropie para repartirla entre aquellos a quien les venga en gana (lo necesiten o no) es un robo. La solidaridad, la caridad, no se puede imponer.

Dependiendo de la decisión que tomes, el suceso que va a pasar sí o sí te afectará de una u otra manera.

We see our lives as non fiction, as real but we question that as there’s nothing o back that up. I really like how Plato speaks on these reading because it shows how easy it is to change someone’s belifs and perspective, wethers it’s religion or just general beliefs

For any collection of non-empty sets, one can create a set by choosing one element from each set in the given collection.

There are a variety of other equivalent ways to state this as well as names. One variation is Zorn's lemma.

Reviewer #2 (Public review):

Summary / Strengths:

In this manuscript, Klemm et al., build on past published findings (Klemm et al., 2021) to characterize caspase activation in distal cells following necrotic tissue damage within the Drosophila wing imaginal disc. Previously in Klemm et al., 2021, the authors describe necrosis-induced-apoptosis (NiA) following the development of a genetic system to study necrosis that is caused by the expression of a constitutive active GluR1 (Glutamate/Ca2+ channel), and they discovered that the appearance of NiA cells were important for promoting regeneration.

In this manuscript, the authors aim to investigate how tissues regenerate following necrotic cell death. They find that:<br /> (1) the cells of the wing pouch are more likely to have non-autonomous caspase activation than other regions within the wing imaginal disc (hinge and notum),<br /> (2) two signaling pathways that are known to be upregulated during regeneration, Wnt (wingless) and JAK/Stat signaling, act to prevent additional NiA in pouch cells, and may explain the region specificity,<br /> (3) the presence of NiA cells promotes regenerative proliferation in late stages of regeneration,<br /> (4) not all caspase-positive cells are cleared from the epithelium (these cells are then referred to as Necrosis-induced Caspase Positive (NiCP) cells), these NiCP cells continue to live and promote proliferation in adjacent cells,<br /> (5) the caspase Dronc is important for creating NiA/NiCP cells and for these cells to promote proliferation. Animals heterozygous for a Dronc null allele show a decrease in regeneration following necrotic tissue damage.

The study has the potential to be broadly interesting due to the insights into how tissues differentially respond to necrosis as compared to apoptosis to promote regeneration.

Weaknesses:

However, here are some of my current concerns for the manuscript in its current version:

(1) The presence of cells with activated caspase that don't die (NiCP cells) is an interesting biological phenomenon but is not described until Figure 5. How does the existence of NiCP cells impact the earlier findings presented? Is late proliferation due to NiA, NiCP, or both? Does Wg and JAK/STAT signaling act to prevent the formation of both NiA and NiCP cells or only NiA cells? Moreover, the authors are able to specifically manipulate the wound edge (WE) and lateral pouch cells (LP), but don't show how these manipulations within these distinct populations impact regeneration. The authors provide evidence that driving UAS-mir(RHG) throughout the pouch, in the LP or the WE all decrease the amount of NiA/NiCP in Figure 3G-O, but no data on final regenerative outcomes for these manipulations is presented (such as those presented for Dronc-/+ in Fig 7M). The manuscript would be greatly enhanced by quantification of more of the findings, especially in describing if the specific manipulations that impacted NiA /NiCP cells disrupt end-point regeneration phenotypes.

(2) How fast does apoptosis take within the wing disc epithelium? How many of the caspase(+) cells are present for the whole 48 hours of regeneration? Are new cells also induced to activate caspase during this time window? The author presented a number of interesting experiments characterizing the NiCP cells. For the caspase sensor GC3Ai experiments in Figure 5, is there a way to differentiate between cells that have maintained fluorescent CG3Ai from cells that have newly activated caspase? What is the timeline for when NiA and NiCP are specified? In addition, what fraction of NiCP cells contribute to the regenerated epithelium? Additional information about the temporal dynamics of NiA and NiCP specification/commitment would be greatly appreciated.

(3) The notum also does not express developmental JAK/STAT, yet little NiA was observed within the notum. Do the authors have any additional insights into the differential response between the pouch and notum? What makes the pouch unique? Are NiA/NiCP cells created within other imaginal discs and other tissues? Are they similarly important for regenerative responses in other contexts?

Author response:

Reviewer #1 (Public Review):

Summary:

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

Strengths:

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

Weaknesses:

No weaknesses were identified.

We thank the reviewer for their positive evaluation and kind words.

Reviewer #2 (Public Review):

Summary / Strengths:

In this manuscript, Klemm et al., build on past published findings (Klemm et al., 2021) to characterize caspase activation in distal cells following necrotic tissue damage within the Drosophila wing imaginal disc. Previously in Klemm et al., 2021, the authors describe necrosis-induced-apoptosis (NiA) following the development of a genetic system to study necrosis that is caused by the expression of a constitutive active GluR1 (Glutamate/Ca2+ channel), and they discovered that the appearance of NiA cells were important for promoting regeneration.

In this manuscript, the authors aim to investigate how tissues regenerate following necrotic cell death. They find that the cells of the wing pouch are more likely to have non-autonomous caspase activation than other regions within the wing imaginal disc (hinge and notum),two signaling pathways that are known to be upregulated during regeneration, Wnt (wingless) and JAK/Stat signaling, act to prevent additional NiA in pouch cells, and may explain the region specificity, the presence of NiA cells promotes regenerative proliferation in late stages of regeneration, not all caspase-positive cells are cleared from the epithelium (these cells are then referred to as Necrosis-induced Caspase Positive (NiCP) cells), these NiCP cells continue to live and promote proliferation in adjacent cells, the caspase Dronc is important for creating NiA/NiCP cells and for these cells to promote proliferation. Animals heterozygous for a Dronc null allele show a decrease in regeneration following necrotic tissue damage.

The study has the potential to be broadly interesting due to the insights into how tissues differentially respond to necrosis as compared to apoptosis to promote regeneration.

Weaknesses:

However, here are some of my current concerns for the manuscript in its current version:

The presence of cells with activated caspase that don't die (NiCP cells) is an interesting biological phenomenon but is not described until Figure 5. How does the existence of NiCP cells impact the earlier findings presented? Is late proliferation due to NiA, NiCP, or both? Does Wg and JAK/STAT signaling act to prevent the formation of both NiA and NiCP cells or only NiA cells? Moreover, the authors are able to specifically manipulate the wound edge (WE) and lateral pouch cells (LP), but don't show how these manipulations within these distinct populations impact regeneration. The authors provide evidence that driving UAS-mir(RHG) throughout the pouch, in the LP or the WE all decrease the amount of NiA/NiCP in Figure 3G-O, but no data on final regenerative outcomes for these manipulations is presented (such as those presented for Dronc-/+ in Fig 7M). The manuscript would be greatly enhanced by quantification of more of the findings, especially in describing if the specific manipulations that impacted NiA /NiCP cells disrupt end-point regeneration phenotypes.

We thank the reviewer for their assessment and helpful suggestions to improve the manuscript. Regarding the presence of NiA and NiCP cells, and the proportion of each within a regenerating wing disc, unfortunately we are currently limited in our ability to distinguish each type of cell using available tools. This applies to both visualizing these cells via anti-cDcp-1 staining or the activity of GC3Ai, DBS-GFP and CasExpress, and detecting their function via their influence on proliferation. As such, although the identification of NiCP does not change any of the conclusions prior to Figure 5 in which NiCP are described, we are currently unable to comment on the contribution of NiA versus NiCP to late proliferation, or whether they are differently affected by Wg and JAK/STAT signaling. This issue is touched on in the discussion, but we will expand upon our commentary to better highlight these issues.

With respect to the reviewer’s suggestion to include evidence on whether blocking NiA/NiCP influences final regenerative outcomes, these data were published in our first paper on this work (Klemm et al., 2021, PMID: 34740246), which we will gladly reiterate in this work.

Finally, we agree that further quantification of our findings will strengthen the work, which is also suggested by Reviewer 3, and plan to add it where possible in a revised manuscript.

How fast does apoptosis take within the wing disc epithelium? How many of the caspase(+) cells are present for the whole 48 hours of regeneration? Are new cells also induced to activate caspase during this time window? The author presented a number of interesting experiments characterizing the NiCP cells. For the caspase sensor GC3Ai experiments in Figure 5, is there a way to differentiate between cells that have maintained fluorescent CG3Ai from cells that have newly activated caspase? What is the timeline for when NiA and NiCP are specified? In addition, what fraction of NiCP cells contribute to the regenerated epithelium? Additional information about the temporal dynamics of NiA and NiCP specification/commitment would be greatly appreciated.

Regarding the timing of apoptosis, Schott et al., 2017 (PMID:28870988) demonstrated that apoptotic GC3Ai-labeled cells in imaginal discs are extruded within 1 hr of labeling, the kinetics of which agree with previously published work on the temporal dynamics of apoptotic cell extrusion by Monier et al., 2015 (PMID:25607361). This is much faster than the continued labeling that we observe up to 64 hr post necrosis. We will include this information alongside a quantification of the percent of the wing pouch with GC3Ai-positive cells over time to better address whether the GC3Ai signal is maintained over time or if newly activated caspases account for the signal in late regenerating discs. We plan to include PH3 staining to distinguish between cells that have activated GC3Ai and are proliferating versus new caspase activity. Additionally, we plan to include new experimental evidence to evaluate the timing of GC3Ai-labelled apoptotic cell loss in our system.

The question of when NiA/NiCP are specified is difficult to address due to the issue of not being able to easily distinguish between these cell types. We previously attempted to answer this particular question, and also to determine what fraction of these cells contribute to the regenerated epithelium, using caspase-based lineage tracing with CasExpress. However, as shown in the paper, we are unable to label NiA/NiCP with CasExpress, either due to the lack of caspase activity level or subcellular localization. We are currently attempting to combine other caspase reporters with lineage tracing tools and examine late-stage wing discs to address these questions.

The notum also does not express developmental JAK/STAT, yet little NiA was observed within the notum. Do the authors have any additional insights into the differential response between the pouch and notum? What makes the pouch unique? Are NiA/NiCP cells created within other imaginal discs and other tissues? Are they similarly important for regenerative responses in other contexts?

As noted by Martin et al., 2017 (PMID:28935711), Martin & Morata, 2018 (PMID:29938762), and our own observations in Harris et al., 2016 (PMID:26840050), the notum does not respond to damage in a way that leads to regeneration, while the pouch does. As NiA/NiCP are a pro-regenerative response, we speculate that this intrinsic difference in regenerative capacity that is potentially caused by a different proliferative and genetic response to injury may account for the disparity in NiA/NiCP occurrence in the pouch vs the notum. A difference in the presence of the (currently unidentified) DAMPs or PRRs in notum vs pouch cells may also be responsible. Alternatively, since the hinge tissue is also refractory to NiA/NiCP due to the presence of genetic factors such as Wg and JAK/STAT, there may be an analogous pathway present in notum cells that acts to protect against the induction of pro-apoptotic factors. Indeed, caspase 3 activation does not seem to occur upon ablation of the notum (Bergantinos et al. 2010, PMID:20215351). We plan to add these points to the discussion.

Regarding the existence of NiA/NiCP in other contexts, we have additional data stemming from our clonal patch experiments (Figure S1) that demonstrates this phenomenon occurs in other imaginal discs, which we plan to include in the revised manuscript.

Reviewer #3 (Public Review):

The manuscript "Regeneration following tissue necrosis is mediated by non- apoptotic caspase activity" by Klemm et al. is an exploration of what happens to a group of cells that experience caspase activation after necrosis occurs some distance away from the cells of interest. These experiments have been conducted in the Drosophila wing imaginal disc, which has been used extensively to study the response of a developing epithelium to damage and stress. The authors revise and refine their earlier discovery of apoptosis initiated by necrosis, here showing that many of those presumed apoptotic cells do not complete apoptosis. Thus, the most interesting aspect of the paper is the characterization of a group of cells that experience mild caspase activation in response to an unknown signal, followed by some effector caspase activation and DNA damage, but that then recover from the DNA damage, avoid apoptosis, and proliferate instead. Many questions remain unanswered, including the signal that stimulates the mild caspase activation, and the mechanism through which this activation stimulates enhanced proliferation.

The authors should consider answering additional questions, clarifying some points, and making some minor corrections:

Major concerns affecting the interpretation of experimental results:

Expression of STAT92E RNAi had no apparent effect on the ability of hinge cells to undergo NiA, leading the authors to conclude that other protective signals must exist. However, the authors have not shown that this STAT92E RNAi is capable of eliminating JAK/STAT signaling in the hinge under these experimental conditions. Using a reporter for JAK/STAT signaling, such as the STAT-GFP, as a readout would confirm the reduction or elimination of signaling. This confirmation would be necessary to support the negative result as presented.

We thank the reviewer for their assessment and helpful suggestions to improve the manuscript. Although the knockdown of Stat92E using this RNAi line has been shown to produce phenotypes associated with loss of JAK/STAT signaling in previous papers (Monahan and Starz-Gaiano, 2014, 2016 PMID:26277564, 26993259), we agree it would be useful to demonstrate this in our hands and therefore plan to include these data.

Similarly, the authors should confirm that the Zfh2 RNAi is reducing or eliminating Zfh2 levels in the hinge under these experimental conditions, before concluding that Zfh2 does not play a role in stopping hinge cells from undergoing NiA.

We attempted to demonstrate the loss of Zfh2 using this RNAi line, but as noted by the reviewer the antibody staining appears mostly unchanged. A reduction in Zfh2 protein levels by this RNAi has previously been demonstrated in cells of the gut (Rojas Villa et al., 2019, PMID: 31841513), suggesting that the persistent Zfh2 staining we see could be due to perdurance of the Zfh2 protein, high levels of expression or high sensitivity of the Zfh2 antibody (or a combination of these factors). We plan to repeat the experiment using a longer knockdown duration prior to ablation to show a change in Zfh2, and/or test alternative RNAi lines. In the absence of these data, we will alter our conclusions to state that Zfh2 cannot be ruled out as playing a role in preventing NiA/NiCP formation in the hinge.

EdU incorporation was quantified by measuring the fluorescence intensity of the pouch and normalizing it to the fluorescence intensity of the whole disc. However, the images show that EdU fluorescence intensity of other regions of the disc, especially the notum, varied substantially when comparing the different genetic backgrounds (for example, note the substantially reduced EdU in the notum of Figure 3 B' and B'). Indeed, it has been shown that tissue damage can lead to suppression of proliferation in the notum and elsewhere in the disc, unless the signaling that induces the suppression is altered. Therefore, the normalization may be skewing the results because the notum EdU is not consistent across samples, possibly because the damage-induced suppression of proliferation in the notum is different across the different genetic backgrounds.

We agree with the reviewer that the use of EdU cannot distinguish between an increase in proliferation in the pouch versus a decrease in proliferation of the notum (or a combination of the two), since EdU incorporation by its nature is a relative rather than absolute measure of proliferation. However, we believe that the important finding is that a localized change in proliferation is observed late in necrosis, which is dependent on NiA/NiCP since blocking the formation of these cells prevents this change. While it is possible that this observed change is caused by a reduction in proliferation of the notum, with little or even no alteration in the pouch, this would imply that NiA/NiCP act to non-autonomously limit the proliferation of cells far from where they appear in the pouch, rather than causing localized proliferation in the immediately surrounding tissue that is representative of a blastema. Although we cannot rule this possibility out, our use of a different marker for proliferation in this work (fluorescent E2F) and a more objective proliferation marker, PH3, (Klemm et al., 2021, PMID: 34740246) agree with our observations made using EdU and suggest the formation of a localized blastema in the pouch. To attempt to address this issue, due to the variability of EdU staining between samples, we aim to quantify changes in EdU that are normalized to undamaged discs stained and mounted in the same sample, thus allowing a more objective background level of proliferation to be used for comparison.

The authors expressed p35 to attempt to generate "undead cells". They take an absence of mitogen secretion or increased proliferation as evidence that undead cells were not generated. However, there could be undead cells that do not stimulate proliferation non-autonomously, which could be detected by the persistence of caspase activity in cells that do not complete apoptosis. Indeed, expressing p35 and observing sustained effector caspase activation could help answer the later question of what percentage of this cell population would otherwise complete apoptosis (NiA, rescued by p35) vs reverse course and proliferate (NiCP, unaffected by p35).

While it is very possible that expression of P35 in NiA/NiCP could induce a previously uncharacterized type of undead cell that persists but does not secrete known AiP-related factors, the way in which P35 blocks activity of effector caspases (Drice and Dcp-1) precludes our ability to reliably detect and assay NiA/NiCP over time: P35 inactivates caspases by binding to their catalytic site, which causes cDcp-1 labeling to become weak and diffuse (Klemm et al 2021, PMID: 34740246), likely because the detectable epitope is in the catalytic site (Florentin & Arama, 2012. PMID: 22351928). Similarly, the GC3Ai reporter acts as a substrate for caspases and must be cleaved for fluorescence to occur (Zhang et al., 2013 PMID: 23857461). Thus, co-expressing P35 with GC3Ai actually reduces the number of NiA/NiCP cells labeled by GC3Ai and weakens cDcp-1 staining, preventing us from assaying their persistence or association with proliferative markers.

It is unclear if the authors' model is that the NiCP cells lead to autonomous or non-autonomous cell proliferation, or both. Could the lineage-tracing experiments and/or the experiments marking mitosis relative to caspase activity answer this question?

While we see GC3Ai-labeled NiA/NiCP in the same area of the pouch with high levels of proliferation (PH3), we observe a mixture of GC3Ai cells that overlapped the PH3 cells and GC3Ai cells that were adjacent to PH3(+) cells. Thus, we are unable to conclusively say whether proliferation is induced autonomously or non-autonomously. We have attempted to answer this question with lineage tracing, however NiA/NiCP are not labeled by the CasExpress tool, and we were unable to define a relationship between NiA/NiCP and proliferation through lineage tracing. However, we add further explanation of our findings to better clarify the proposed model of NiA/NiCP-induced proliferation.

Many of the conclusions rely on single images. Quantification of many samples should be included wherever possible.

As suggested by Reviewers 2 and 3 we plan to strengthen our findings by adding quantification of phenotypes where possible, in particular in Figure 2 as mentioned in the “Recommendations for the authors”.

Why does the reduction of Dronc appear to affect regenerative growth in females but not males?

We note that the effect on regenerative growth does appear to be present in males, but that the effect is not significant. We suspect that the lower n for this experiment is the cause, and are addressing this by repeating the experiment to increase the n.

No se si es una idea muy extendida entre el liberalismo. Pero si fuera así el desarrollo lógico y la defensa argumentativa que muchos hacen no sería tan buena como es. Entiendo que esta postura será la típica de los menos formados o más conformistas que no se preocupan por defender públicamente sus ideas.

Las creencias están interrelacionadas y se siguen unas a otras. En otro caso no habría coherencia y en ciertos puntos se llega a contradicciones, lo cual es un error.

El escepticismo debería ser un estado intermedio temporal. O un estado que se toma respecto a ciertos temas sobre los que uno no ha investigado o analizado a fondo para formarse una opinión con base.

¿Buscamos estas fuentes para nuestra bibiografia individualmente, o las encontramos en una lista que usted nos da?

Focus on education? Social change?

• porque pueden debilitar nuestra relación con Jehová
• y también pueden ser el primer paso que nos lleve a cometer pecados más graves

La realidad es que todos tenemos nuestros puntos débiles o tentaciones. Quizá sea comer o beber con exceso, el entrenamiento, el dinero, o quizá la inmoralidad sexual. El párrafo también menciona, espíritu de independencia, el temor al hombre o el mal carácter. “cada uno es probado al ser atraído y seducido por su propio deseo” (Sant. 1:14).

Porque el ejemplo de Pedro nos hizo meditar en que si bajamos la guardia, puede sorprendernos una tentación que ya creíamos controlada o vencida.

• Primero vigilar lo que pensamos para no ser tentados por nuestro propio deseo

• debemos tener cuidado con quienes nos asociamos, que lugares frequentamos ya sea lugares físicos o virtuales en internet

debemos mantenernos alejados de todos los pasos que nos puedan llevar a cometerlo.b Esto puede incluir evitar ciertas situaciones o actividades que para los cristianos en general no son malas pero que sabemos que a nosotros podrían llevarnos a caer en una tentación (Mat. 5:29, 30).

Que terminemos cometiendo un pecado grave, de repente, cómo sin saber cómo sucedió. Por No haber tenido cuidado con los pasos que se dieron antes como por ejemplo: - Aceptar malas compañías - Elegir entretenimiento inapropiado - Querer ver sitios peligrosos, sean físicos o de internet. - descuidando quizá la actividad espiritual, oración Estudio Reuniones Predicación

Nuestras debilidades,

Es cómo en las ciudades amurallada, los puntos más débiles, son las puertas, y por eso se ponía mayor énfasis en su protección, en nuestro caso es igual, nuestras debilidades, son la puerta o el punto más vulnerable que nos conduzca a cometer algún pecado.

“EL ESPÍRITU está dispuesto, pero la carne es débil” (Mat. 26:41b).a

Jesús tiene claro que somos imperfectos y aunque tengamos buenas intenciones, podemos caer en el error de pensar o actuar con exceso de confianza, cómo decir que a nosotros o a mi, nada nada me apartará de hacer siempre lo correcto. Porque amo a Jehová y Jesús.

Edward Carpenter’s “Towards Democracy” evidently offers an utopian vision for humanity’s future, one where there is “a great land poised as in a dream—waiting for the kiss and the re-awakening…a great land waiting for its own people to come and take possession of it.” In parallel, Carpenter critiques industrialization, contrasting the “sob and gasp of pumps and the solid beat of steam and tilt-hammers” with “beautiful centuries-grown villages and farmhouses.” Interestingly, Carpenter seems to be suggesting that humanity has the potency to “take possession” of a better future with a “kiss” that leads to “re-awakening”; his utopia is paradoxically a realizable one, within human reach. In questioning the feasibility of Carpenter’s vision in the context of Eliot’s work, Verlaine’s “Parsifal” offers striking parallels.

The King in need of curing, like the land in Carpenter’s poem, needs the “kiss” of The Holy Spear to cure his ailments and return to health. To attain this “kiss,” the King needs Parsifal, “a young man who knows nothing about the evil of the world and who can resist the beauty of the flower-maidens.” But the issue with this poem is that it places excess strain on the inherently morally frail human race. Even assuming that a human has the moral fortitude to overcome “inclination…towards the Flesh,” there is no human who “conquered Hell” with its endless vice and infernal tribulations. And thus, we realize a critical tension present in the opening of “The Fire Sermon.”

In and of itself, drawn from a deeply optimistic poem, Eliot's use of the line “Et O ces voix d’enfants, chantant dans la coupole” (And, O those children’s voices singing in the dome!) is deeply optimistic. But contrasting the preceding lines of “Parsifal,” full of a “virgin boy” virtuously overcoming temptation left and right, and TWL, where “rats” with “slimy bell[ies]” and “bodies naked on the white damp ground” alongside the “sound of horns and motors” paint a grim picture of urbanization and decay, reveals that this line's significance in TWL is vastly different. “Tak[ing] possession” of a better future is a noble goal. However, there seems to be no room for such pure nobility in the TWL as the kiss of “Parsifal,” a figment of inherently unattainable utopia, is infeasible in a waste land.

Eliot offers a motivation for the deep tension between delusional optimism and pragmatic realities by alluding to Marvell’s work: “at my back from time to time I hear/The sound of horns and motors.” The speaker in Marvell’s poem desires that his “vegetable love should grow Vaster than empires and more slow.” As an aside, there is a contradiction within Marvell’s use of “empires” because empires are typically relatively frail entities which “grow Vaster” with great speed but struggle to be “more slow” in sustaining themselves. But more to the point, the line Eliot specifically borrows from Marvell deals less with delusional, prolonged love and more with “Time’s wingèd chariot hurrying near,” bound for a point where “beauty shall no more be found" and “quaint honour turn[s] to dust.” Perhaps if humans had endless time to relish life’s pleasantries and fulfill their desires with “coy mistress[es],” it would be possible to retain nobility. But when one seeks to realize infinite desire within the confines of limited time on Earth, an ignoble–perhaps even evilly hedonistic–method of existence is bound to prevail. For if humans doggedly pursue ambition, subconsciously driven by their mortality and finite existence, how will the virtue necessary for Carpenter’s delusional atavism ever come to fruition? If we are to regard TWL as a somewhat optimistic poem, some answer to this question should be identified.

Me parece que debemos tener en cuenta las fases del proceso.

La importancia de resaltar el contenido estratégico

Es necesario tener claro estos elementos: Las estrategias de comunicación son planes integrales diseñados para transmitir mensajes clave a un público específico de manera efectiva.

DOI: 10.1101/2024.08.29.610322

Resource: Addgene_26931

Curator: @olekpark

SciCrunch record: RRID:Addgene_26931

What is this?

DOI: 10.1186/s13287-024-03921-y

Resource: RRID:Addgene_80945

Curator: @scibot

SciCrunch record: RRID:Addgene_80945

What is this?

DOI: 10.1101/2024.09.19.613771

Resource: (Thermo Fisher Scientific Cat# H3569, RRID:AB_2651133)

Curator: @scibot

SciCrunch record: RRID:AB_2651133

What is this?

Experience/education shows us we are more alike than we are different

Love the notion of teaching and the distribution of knowledge is sacred.

COMENTARIO

DOI: 10.1016/j.str.2024.08.022

Resource: Change-O (RRID:SCR_023986)

Curator: @scibot

SciCrunch record: RRID:SCR_023986

What is this?

DOI: 10.1084/jem.20231835

Resource: (Molecular Probes Cat# A-21244, RRID:AB_2535812)

Curator: @scibot

SciCrunch record: RRID:AB_2535812

What is this?

DOI: 10.1371/journal.pgen.1007154

Resource: RRID:BDSC_7326

Curator: @scibot

SciCrunch record: RRID:BDSC_7326

What is this?

La relevancia de los datos determinan el uso o no de JSON

Author response

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

We thank the editors and reviewers for their thoughtful comments on our manuscript. We greatly appreciated the suggestions and recommendations that helped us to improve the study. With adaptations, and inclusion of novel data and analyses, we have addressed all points raised, and hope that by these improvements the study further meets the standards for eLife. 

Reviewer #1 (Recommendations For The Authors):

Minor text edits should be made.

(1.1) As a recent study from the Wong lab also showed sebaceous gland regeneration following complete ablation (Veniaminova et al., 2023), this finding should be mentioned in the text, and the abstract ("Most strikingly...") should be toned down.

We thank the reviewer for the positive feedback, and for highlighting this part of the study from the Wong lab. Although we cited this study study in a different context, we had not discussed the sebaceous gland regeneration finding. We have now added this to the discussion section of the manuscript.

(1.2) Introduction: In lines 31-33 discussing the connection of sebaceous glands with skin disorders, the 5 references cited seem to replicate the citations from a similar sentence in Veniaminova et al., 2019. The authors should vary their citations, as there are likely other publications that can be cited here.

Additional references have been added.

Reviewer #2 (Recommendations For The Authors):

The manuscript is well written and the data are well presented in the figures.

We thank the reviewer for the positive feedback.

(2.1) Here are some points that could be taken into consideration to improve the manuscript:

- Row 75 "the primary" regulator could be changed to "a crucial".

We appreciate this suggestion and have made the text edit.

- Row 86 could be added: ...is the dominant ligand of the Notch signalling.

We have made the text edit as suggested.

(2.2) Row 107-109 from the quantification of Figure 1G and Figure 2 it seems that only the aJ2 treatment has an SG phenotype. Why aJ1 doesn't have any effect? (same is true in other figures). If the data on aJ1 are maintained in the manuscript, this should be argued in the discussion section.

The reviewer is correct in noting that the aJ1 treatment does not cause the phenotype, and this is indeed one of the key findings of the study. This is maintained throughout the manuscript. We have also cited references showing that embryonic and adult deletions of Jag1 do not cause any sebaceous gland defects. All these data argue that Jag1 is not the relevant Notch signaling ligand in sebocyte differentiation. We have further clarified this in the manuscript.

(2.3) Related to Figure 3G. As the Lrig1 stem cells can go towards both the sebocyte differentiation, or the sebaceous duct differentiation, it would be interesting to evaluate if the differentiation impairment caused by the antibody treatment affects in a similar manner (or not) the sebaceous duct differentiation. This could be tested through immunofluorescence, selecting markers of sebaceous duct.

We thank the reviewer for this thoughtful question. We are unable to find any unique markers of the sebaceous ducts (that are not expressed in other parts of the sebaceous gland, especially sebocytes) in the literature, thus, any analysis of markers would be confounded by its change of expression due to the loss of sebocytes.

However, we have evaluated the histology using bursting sebocytes releasing sebum as a proxy of a functional sebaceous duct. We have not found any significant differences between treatments using this metric (Fig. S1).

(2.4) As the word "therapeutic" is often underlined in the manuscript, maybe a few sentences on the transnational aspects of the results could be added to the discussion.

We thank the reviewer for highlighting this point. We have added this to the discussion.

(2.5) Figure 3 suggests that Jag2 is produced by basal sebocytes and used by these cells to induce sebocyte differentiation. I'm wondering if in an in vitro cell system (with a mixture of marked Jag2-expressing cells and marked Jag2-negative cells), it would be possible to understand if this mechanism of differentiation is a cell-autonomous mechanism or a mechanism based on cell competition (for instance, it would be possible that the progenitors compete for their niche on the basal layer by pushing neighbouring basal cells to differentiate presenting them Jag2).

We thank the reviewer for the insightful suggestion. The mechanistic underpinning of how Notch signaling induces sebocyte differentiation is still unclear, and we find the reviewer’s suggestion very interesting. However, establishing an in vitro model that captures the aspects mentioned, would require a lot of optimization and validation. To help rapid dissemination of our findings we elected to keep this out of the manuscript, but we will certainly consider it for future studies.

Reviewer #3 (Recommendations For The Authors):

(3.1) The authors focussed on mouse back skin sebaceous glands to analyse the phenotype. Are the effects also reproducible in the sebaceous glands of the mouse ears and tail epidermis? If so, the data should be strengthened by quantifying the phenotype using tail epidermal whole mounts (Braun et al., 2003; Development, PMID: 12954714), ideally by co-staining sebaceous glands for differentiation markers (e.g. FASN, Adipophilin) or lipid deposits (e.g., Oil red O). Also, the authors need to clarify how many sebaceous glands were scored per mouse. If not, please provide a rationale explaining the location restriction.

We thank the reviewer for pointing this out. Indeed, we have only incorporated data from the telogen dorsal skin of the animals. We have now more accurately reflected this in the revised manuscript. Additionally, we have added the number of sebaceous glands quantified in each figure per the reviewer’s suggestion.

Since the stage of hair growth cycle can affect the sebaceous glands, we chose the resting (telogen) phase of the hair cycle to reliably study the sebaceous glands. At 8 weeks of age, hair follicles have uniformly entered the telogen phase. As subsequent re-entry into the anagen phase is asynchronous in the adult skin, the color of the dorsal skin of C57BL/6 mice can be used to determine whether the hair follicles are in the telogen phase or not. These reasons led us to choose this location, allowing us to study only telogen phase hair follicles.

We also point out that previously reported data (Estrach et al., 2006) did not show differences between dorsal and tail skin, so we assume the mechanisms must largely be conserved. However, as the reviewer rightfully points out, we cannot be sure and have, therefore, indicated the dorsal location throughout the manuscript.

(3.2) The micrographs in Figure 2 suggest that expression of both Jagged2 and Notch1 (intercellular domain) is not restricted to the sebaceous glands, as both molecules appear to be detected also in the isthmus and lower hair follicle. Of note, the online tool provided by the Kasper and Linnarsson labs (http://linnarssonlab.org/epidermis/) shows that both molecules are more widely expressed in mouse back skin. Please provide some analysis of the overall expression of these molecules in mouse skin. In line, is the observed effect of using the antagonising antibodies restricted to the sebaceous glands? Please provide additional data on proliferation and differentiation in the interfollicular epidermis, hair follicle cycling, and other skin compartments. For instance, the data published in the cited paper by Lafkas et al. (2005) suggest a thickening of the dermal adipocyte layer upon Jagged2 inhibition using monoclonal therapeutic antibodies.

The reviewer is correct in noting that expression of both Jag2 and Notch1 is not restricted to the sebaceous gland. The Notch signaling pathway is a well-known regulator for epidermal differentiation, and members of the pathway are expressed in various locations of the skin, including the interfollicular epidermis and the hair follicle. The expression and function of Notch signaling in these locations has been reviewed in (Hsu et al., 2014; Nowell and Radtke, 2013; Watt et al., 2008). We have also added zoomed out images showing expression of Jag2 and Notch1 in the skin (Figure S2e,f).

The effect of the antagonizing antibodies is not restricted to sebaceous glands, as we already noted in our discussion section: “While injections of the Notch blocking antibodies are systemic, we only observed a reduction in the number of Notch-active cells in the IFE, but not a complete loss.” The functional impact of the antibodies is likely beyond the sebaceous gland, as the reviewer points out, but understanding the full effect in other compartments, we consider beyond the scope of the current study.

In our previous study (Lafkas et al., 2015), the skin was examined at different animal ages/gender and using different antibody dosing regimens, which is the likely explanation for the differences observed. We have now quantified the width of the adipocyte layer and the IFE and show that there are no significant differences between treatments (Figure S1g-j). This together with the histology suggest that there are no significant differences in the differentiation and proliferation of these compartments.

(3.3) Since Jagged1 is a Wnt/beta-catenin target gene that is essential for (ectopic) hair follicle formation and differentiation (Estrach et al., 2006, Development, PMID: 17035290) and the sebaceous gland is widely considered as an epidermal compartment with absent/low Wnt/beta-catenin pathway activity during normal homeostasis (Lim & Nusse, 2013, Cold Spring Habor Perspectives in Biology, PMID: 23209129), how is the expression of Notch1 and Jagged2 regulated upstream in sebocyte progenitors? It would be important to bring some more mechanistic insights into the upstream regulation of Notch activity. In line with comment 2, how are the compartment-specific effects molecularly regulated if the effects are not restricted to the sebaceous glands?

The reviewer is correct in noting that the Wnt pathway does not seem to be a likely candidate for driving sebocyte differentiation through Notch signaling. Indeed, Wnt inhibition is required for sebocyte differentiation (Merrill et al., 2001; Niemann et al., 2002), and the Jag2 promoter region also does not contain TCF binding sites (Katoh and Katoh, 2006).

We speculate that Myc might regulate Notch signaling in the sebaceous gland. It is expressed in the sebaceous gland basal stem cells and has been reported to positively regulate sebocyte differentiation (Cottle et al., 2013). In addition, studies have shown that Jag2 is a Myc target gene (Fiaschetti et al., 2014; Yustein et al., 2010). However, evaluating which upstream pathway potentially regulates Notch signaling, and resolving the regulatory network of sebocyte differentiation beyond the direct Notch ligands and receptors would require extensive in vivo modeling using KO and transgenic animals, which we consider to be beyond the scope of the current manuscript.

References

Cottle DL, Kretzschmar K, Schweiger PJ, Quist SR, Gollnick HP, Natsuga K, Aoyagi S, Watt FM. 2013. c-MYC-Induced Sebaceous Gland Differentiation Is Controlled by an Androgen Receptor/p53 Axis. Cell Rep 3:427–441. doi:10.1016/j.celrep.2013.01.013

Estrach S, Ambler CA, Celso CLL, Hozumi K, Watt FM. 2006. Jagged 1 is a β-catenin target gene required for ectopic hair follicle formation in adult epidermis. Development 133:4427–4438. doi:10.1242/dev.02644

Fiaschetti G, Schroeder C, Castelletti D, Arcaro A, Westermann F, Baumgartner M, Shalaby T, Grotzer MA. 2014. NOTCH ligands JAG1 and JAG2 as critical pro-survival factors in childhood medulloblastoma. Acta Neuropathol Commun 2:39. doi:10.1186/2051-5960-2-39

Hsu Y-C, Li L, Fuchs E. 2014. Emerging interactions between skin stem cells and their niches. Nat Med 20:847–856. doi:10.1038/nm.3643

Katoh Masuko, Katoh Masaru. 2006. Notch ligand, JAG1, is evolutionarily conserved target of canonical WNT signaling pathway in progenitor cells. Int J Mol Med. doi:10.3892/ijmm.17.4.681

Lafkas D, Shelton A, Chiu C, Boenig G de L, Chen Y, Stawicki SS, Siltanen C, Reichelt M, Zhou M, Wu X, Eastham-Anderson J, Moore H, Roose-Girma M, Chinn Y, Hang JQ, Warming S, Egen J, Lee WP, Austin C, Wu Y, Payandeh J, Lowe JB, Siebel CW. 2015. Therapeutic antibodies reveal Notch control of transdifferentiation in the adult lung. Nature 528:127–131. doi:10.1038/nature15715

Merrill BJ, Gat U, DasGupta R, Fuchs E. 2001. Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin. Genes Dev 15:1688–1705. doi:10.1101/gad.891401

Niemann C, Owens DM, Hülsken J, Birchmeier W, Watt FM. 2002. Expression of ΔNLef1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours. Development 129:95–109. doi:10.1242/dev.129.1.95

Nowell C, Radtke F. 2013. Cutaneous Notch Signaling in Health and Disease. Cold Spring Harb Perspect Med 3:a017772. doi:10.1101/cshperspect.a017772

Watt FM, Estrach S, Ambler CA. 2008. Epidermal Notch signalling: differentiation, cancer and adhesion. Curr Opin Cell Biol 20:171–179. doi:10.1016/j.ceb.2008.01.010

Yustein JT, Liu Y-C, Gao P, Jie C, Le A, Vuica-Ross M, Chng WJ, Eberhart CG, Bergsagel PL, Dang CV. 2010. Induction of ectopic Myc target gene JAG2 augments hypoxic growth and tumorigenesis in a human B-cell model. Proc Natl Acad Sci 107:3534–3539. doi:10.1073/pnas.0901230107

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