DOI: 10.1016/j.celrep.2023.112655

Resource: (Cell Signaling Technology Cat# 3504, RRID:AB_2255663)

Curator: @scibot

SciCrunch record: RRID:AB_2255663

What is this?

crítica ao resultante do processo de algoritmização constante e demasiado

estes surgiram como alternativa de assegurar dados dos usuários de forma mais rápida e eficaz, a fim de otimizar os processos biométricos e ainda evitar que pessoas não autorizadas façam uso indevido de dados pessoais, por exemplo: impressão digital e reconhecimento facial.

Pode-se elencar a obsolescência programada como uma das principais estratégias para a indústria produzir e vender cada vez mais, pois os produtos, sobretudo eletrônicos, são vendidos com "data de validade" pré-determinada, afim de alimentar as catracas da produção, fazendo com que o consumidor sinta necessidade constante pelo mais novo aparelho do mercado, pois o que tem em mãos já não satisfaz suas demandas (mesmo que a compra tenha sido recente).

Esse é um fenômeno contemporâneo quase que invisível, pois é cada vez mais automatizado para que nos seja imperceptível e então, não tenhamos conta da invasão velada, sobretudo por parte das BigTechs as quais convivemos em constante contato e dependência, por exemplo, ao adquirir um aplicativo é solicitada permissão de acesso a alguns dados pessoais, do contrário, o usuário terá dificuldade ou não poderá usar o aplicativo. Levando-o a cada vez mais permitir acesso aos seus dados sem racionalizar o significado disso, tornando natural esse cenário de controle.

A acessibilidade às obras significa uma maior adesão ao cinema? Podemos dizer que há uma democratização de acesso, mas também há uma democratização do cinema em si? A linguagem consegue também atingir uma grande massa, no mesmo escopo que sua divulgação alcança? Questiono-me se essa difusão ampliada significa uma democratização de conhecimento. Penso que as obras promovem difusos atravessamentos em espectadores diversos, mas não sei dimensionar a qualidade deles.

É interessante pensar como o antigo não se torna obsoleto. Pelo contrário, é uma mais uma ferramenta que pode fortalecer a linguagem do cinema. Assim, pensa-se que a película é um símbolo de resistência e permanência do cinema.

Levanta-se a questão dos canais de difusão do cinema, especificamente entre as salas físicas e as plataformas eletrônicas. É interessante que o cinema sempre passou por abalos sísmicos e metamorfoses tecnológicas, mas sua grandeza artística nunca esteve em jogo nessas transformações. Mas, essa dicotomia entre os canais de exibição pode gerar confrontos entre os cinéfilos. Essa reflexão nos convida a considerar os desafios e as mudanças enfrentadas pelo cinema na era digital, onde a forma de distribuição e acesso aos filmes desempenham um papel significativo na experiência cinematográfica.

O cinema nos faz pensar questionamentos intrigantes sobre sua relevância e singularidade. Em um mundo onde a tecnologia e a produção audiovisual estão em constante evolução, é interessante que a gente possa pensar sobre o lugar do cinema nesse cenário.

Seria o cinema a expressão mais elaborada e sofisticada dentre as mídias disponíveis? Ou estaria ele relegado a um mero resíduo de ambição estética em comparação com outras formas de narrativa visual? Essas são indagações que me intrigam a compreender a essência do cinema em nossa era.

O cabimento da ANPP em função do crime praticado pelo infrator mede-se pela pena mínima cominada, isto é, o crime perpetrado não poderá ter pena igual ou superior a 4 anos.

Para o ANPP, o crime não poderá ter sido cometido com violência ou grave ameaça.

Ademais, é necessário, ao revés da Transação Penal e da Suspensão condicional do processo, a confissão formal do acusado.

• Na suspensão condicional do processo, o critério de cabimento quanto ao crime praticado se dá a partir da pena mínima, que deverá ser cominada por tempo igual ou inferior a um 1 ano.

• Aqui difere da transação penal, cujo critério em razão do crime é o da pena máxima para ser considerado crime de menor potencial ofensivo. Isto é, a transação penal mede-se a sua viabilidade pela penalidade máxima de 2 anos.

Informação sobre o processo de secagem das tintas e os métodos que podem ser aplicados.

Aborda praticamente todo tipo de couro e animal que produz o couro.

Beaucoup de petits changement de formulation pour le bibtex

This makes me think of how we view everyday religion. We know it's there, and it's a huge part of everyday life for a lot of people while not for others. Socially, both are acceptable depending on which social circle you surround yourself.

That those should be capable to provide an opinion on the subject matter so long they have tools available to aid them. A sound idea to say the least.

Considero que la familia de investigación que más me conviene conforme a mis objetivos es el método cualitativo, ya que mi investigación está centrada en el análisis entre el empaque y el consumidor, por lo que me podría ayudar en examinar los datos de una manera menos general y más íntima, para priorizar centrarse en cómo percibe el sujeto el soporte tridimensional, así también ayudara a valorar los diseños dentro de un contexto en específico. Además, me parece adecuado aplicar el método de trabajo de gabinete, porque pienso recolectar información por medio de internet y desarrollar mi trabajo a base de bibliografías y datos reunidos por otras personas y en este caso también por parte del sitio web de la empresa. En cuanto al enfoque de investigación, decidí escoger el de las encuestas, para así poder evaluar de mejor manera información proporcionada de un grupo de personas representativas como lo son compradores de cannabis. Por último, la técnica de investigación que realizaré son los cuestionarios y entrevistas.

DOI: 10.7554/eLife.83856

Resource: (Molecular Probes Cat# A-21424, RRID:AB_141780)

Curator: @scibot

SciCrunch record: RRID:AB_141780

What is this?

Reviewer #2 (Public Review):

In this manuscript, Castanera et al. investigated how transposable elements (TEs) altered gene expression in rice and how these changes were selected during the domestication of rice. Using GWAS, the authors found many TE polymorphisms in the proximity of genes to be correlated to distinct gene expression patterns between O. sativa ssp. japonica and O. sativa ssp. indica and between two different growing conditions (wet and drought). Thereby, the authors found some evidence of positive selection on some TE polymorphisms that could have contributed to the evolution of the different rice subspecies. These findings are underlined by some examples, which illustrate how changes in the expression of some specific genes could have been advantageous under different conditions. In this work, the authors manage to show that TEs should not be ignored when investigating the domestication of rise as they could have played an important role in contributing to the genetic diversity that was selected. However, this study stops short of identifying causations as the used method, GWAS, can only identify promising correlations. Nevertheless, this study contributes interesting insights into the role TEs played during the evolution of rice and will be of interest to a broader audience interested in the role TEs played during the evolution of plants in general.

Author Response:

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

Reviewer #1 (Public Review):

[…] Overall, the authors build a convincing case for TEs being an important source of regulatory information. I don't have any issues with the analysis, but I am concerned about the sweeping claims made in the title. Once you get rid of eQTLs that could be altered by either SNPs or TIPs and include only those insertions that show strong evidence of selection, the number of genes is reduced to only 30. And even in those cases, the observed linkage is just that, not definitive evidence for the involvement of TEs. Although clearly beyond the scope of this analysis, transgenic constructs with the TEs present or removed, or even segregating families, would have been far more convincing. 

We notice that the referee thinks that we "built a convincing case for TEs being an important source of regulatory information". This is what we wanted to convey in the title, were we were cautious to not claiming that TEs are the most important contributor to gene expression variability in rice populations. However, we agree with the referee that the title may be improved to better describe the results presented. We have therefore changed the title to "Transposons are an important contributor to gene expression variability under selection in rice populations".

With respect to demonstrating causality by removing or introducing the TEs, this is indeed a work we plant to do but that, as stated by the referee, is beyond the scope of this analysis.

The fact that many of the eQTL-TIPs were relatively old is interesting because it suggests that selection in domesticated rice was on pre-existing variation rather than new insertions. This may strengthen the argument because those older insertions are less likely to be purged due to negative effects on gene expression. Given that the sequence of these TEs is likely to have diverged from others in the same family, it would have been interesting to see if selection in favor of a regulatory function had caused these particular insertions to move away from more typical examples of the family. 

The TIP-eQTL are from different classes, superfamilies and families and the number of TIP-eQTLs of the same family is too small to deduce sequence communalities (4.6 TIP-eQTLs/family in indica and 3.6 TIP-eQTLs/family in japonica). On the other hand the effect of TIPs on expression can be positive or negative (we show actually that it is often negative). In the later case, a plausible scenario would be of the insertion inactivating a promoter element, and in this case it would be the insertion itself, and not the actual sequence of the TE what would be selected.

Also, previous work done in our lab has shown that TEs can amplify and mobilize transcription factor binding sites that are bound by the TF even when they are not close to a gene and therefore probably not directly affecting gene expression (Hénaff et al.,2014. The Plant Journal). In that case, the sequence of the eQTL TEs and those that are far away from genes will not necessarily differ. 

Reviewer #2 (Public Review):

In this manuscript, Castanera et al. investigated how transposable elements (TEs) altered gene expression in rice and how these changes were selected during the domestication of rice. Using GWAS, the authors found many TE polymorphisms in the proximity of genes to be correlated to distinct gene expression patterns between O. sativa ssp. japonica and O. sativa ssp. indica and between two different growing conditions (wet and drought). Thereby, the authors found some evidence of positive selection on some TE polymorphisms that could have contributed to the evolution of the different rice subspecies. These findings are underlined by some examples, which illustrate how changes in the expression of some specific genes could have been advantageous under different conditions. In this work, the authors manage to show that TEs should not be ignored when investigating the domestication of rise as they could have played an important role in contributing to the genetic diversity that was selected. However, this study stops short of identifying causations as the used method, GWAS, can only identify promising correlations. Nevertheless, this study contributes interesting insights into the role TEs played during the evolution of rice and will be of interest to a broader audience interested in the role TEs played during the evolution of plants in general. 

We agree with the referee that the results presented do not allow concluding on causality, and we have been careful not to pretend they would in the manuscript. We plan to perform analysis of adding or removing TEs by CRIPR/Cas 9 approaches to address this, but, in line with referee's 1 comment, we think this is beyond the scope of this analysis.

---------- 

Reviewer #1 (Recommendations For The Authors): 

Everything that I need to say is provided in the public portion of my review. 

Reviewer #2 (Recommendations For The Authors): 

Major concerns:

1. The authors compare the proportion of the variance explained by the most significant TIP and SNP on the observed eQLTs associated with TIPs and SNPs. Thereby the authors conclude that TIPs explain more variance than SNPs. If I am not mistaken the GWAS was run separately for TIPs and SNPs, however, I am wondering if running the GWAS on the combined TIP and SNP dataset might be the better way to compare the variance explained by TIPs and SNPs on gene expression differences. It would be nice to see if these results also hold true if a TIP and SNP combined dataset is used as the most significant marker in a GWAS might not be the causal mutation but might just be linked to the causal mutation. Further in the TIP dataset, the number of markers is only 45k and in the SNP dataset, it is 1 000k, which could bias the GWAS toward finding markers that explain more of the variation in the dataset with fewer markers. 

We addressed the reviewer concern by using two complementary approaches, whose results are described in the text (lines 119-121) and in the new Figure 1-figure supplement 1.

First, we addressed the concern regarding the independent GWAS for TIPs and SNPs vs a combined strategy. For this, we built new japonica/indica genotype matrices containing all TIP and SNP matrix together and ran eQTL mapping again. Using the same strategy (association + FDR adjust), we found 100% of the previous TIP-eQTLs and 99% of the previous SNP-eQTLs. We repeated the same analysis (proportion of expression variance), and the results were mostly the same (Figure 1-figure supplement 1A).

Second, we addressed the two concerns (combined genotypes and different amount of TIP and SNP markers) using a single approach. SNP matrices were LD pruned using a r2 = 0.9 and later subsampled to the exact number of TIPs (Indica = 30,396, Japonica = 25,168). We verified that these SNPs covered well the 12 rice chromosomes. SNP and TIP genotypes were later merged into a single matrix, and eQTL mapping was repeated for each of the subspecies and conditions using the same parameters as in the previous version of the manuscript. 100 % of the previously reported TIP-eQTL associations were found using this new approach. Nevertheless, we found a very important drop of sensitivity in the SNP-eQTLs (only 15-20% of the previous associations were detected), possibly due to the strong reduction in the number of SNPs (> 95 %), which results in much lower number of markers at < 5Kb from genes). We repeated the analysis of Figure 1D, and observed very similar results (Figure 1-figure supplement 1D). There is a very important number of TIP-eQTL associations that do not coincide with SNP-eQTLs, (74% in indica, 83% in japonica) indicating that TIP-eQTL mapping is complementary to SNP-eQTL mapping as it uncovers additional associations (note that in this case the overlap between TIP-eQTLs and SNP-eQTLs is lower than in the previous analysis due to the lower sensitivity of SNP-eQTL mapping using less markers). In the cases were both a TIP and a SNP coincide as eQTL, TIPs explained slightly more variance than SNPs in both indica and japonica (in 54% of the cases TIP variance > SNP variance).

2. Line 146 to 152: in this section, the authors describe overlaps between TIP-eQTLs in two different growth conditions, however, in the text it is not mentioned if the TIPs have the same effect on gene expression in the two conditions or if the gene expression is up-regulated in one condition but down-regulated in the other. This information would be interesting to have here, especially as the authors go on to say that only a small number of TIP-eQTLs are stress-specific. The same comment also goes for the eQTL overlap described on lines 167 to 170. 

We checked the effect type (positive or negative) of TIP-eQTLs in both scenarios (associations shared between wet/dry conditions, and associations shared between subspecies). In both cases, 100 % of the shared TIP-eQTLs have the same effect type in the two conditions or subspecies. We have updated the text accordingly (Lines 55-157 and Lines 179-181)

3. Lines 192 to 196: the authors mention that the frequency of non-eQTL-TIPs was at the same frequency in indica and japonica, which is in contrast to eQTL-TIPs. However, on line 132 it is mentioned that eQTL-TIPs were overrepresented in 1 kb regions upstream of genes. Hence, is the pattern of the frequency of non-eQTL-TIPs being at the same frequency in indica and japonica also observed in the 1 kb regions upstream of genes and/or if the distribution of non-eQTL-TIPs is matched to one of the eQTL-TIPs? Or is this pattern driven by non-eQTL-TIPs far away from genes?

We checked the frequencies of TIPs at 1Kb upstream genes and found that the general pattern is maintained, with the frequencies of TIP no-eQTLs being more correlated than that of TIP-eQTLs. We have included this information (lines 204-206) an added a new supplementary file (Figure 2-figure supplement 2)

4. In the discussion, the authors could briefly discuss how linked selection affecting TIPs could contribute to the observed results. After reading the second example in the result section where one of the example TIPs (TIP_50059) is found on the Hap B which contains "some additional structural differences" (line 290), I was left wondering how much of the increase in TIP frequency can be attributed to genetic hitchhiking? And how much of the results could be caused by linked selection, especially when considering that structural variations are not included in the GWAS analyses. 

We agree with the referee in that some of the TIP eQTLs here described might be not the actual cause of expression variability (ej, TIP linked with the causal mutation), although we cannot know the exact fraction. This is stated in several places of the results and discussion sections. However, the fact that TIPs tend to explain more variance than SNPs and that TIP eQTL, but not SNP eQTL, tend to concentrate in the upstream proximal region of genes where most transcription regulatory sequences are located (Figure 1), suggest that TIP eQTLs could be more frequently the causal than SNP eQTLs. We revised the text to ensure that we convey this message appropriately.

Minor comments: 

• Lines 80 to 83: the description of the rice phylogeny should be moved to the introduction. 

Done (Lines 68-72)

• Line 177 to 186: It was unclear to me if the authors checked in the ancestral rice population laced the TIPs described in this section as recently inserted in the indica and japonica ssp. It would be nice to add this information to this section. 

Thanks to the referee comment we noted an imprecision in the text. The approximate 1/3 of subspecies specific TIP-eQTLs refers to the TIPs at 3% MAF (ie, some of these insertions could be present at > 3% in indica, but at < 3% MAF in japonica). We now indicate only the TIPs that are truly specific to any of the two subspecies (frequency is zero in one of the two) and looked for their presence in rufipogon:

59 insertions are indica-specific. Of those, 33 are present in rufipogon.

21 insertions are japonica-specific. Of those, 5 are present in rufipogon.

We have incorporated this information in the manuscript (Lines 185-189). The species-specific TIPs are also available in the Supplementary File 3.

• Line 353: "have two of more TIPs" should be "two or more" 

Done (Line 369)

• Figure 1D: Using a square layout instead of a rectangle layout for the plot will make it easier to interpret. 

Done.

podría ayudar a eliminar las jerarquías

Authors' Response (2 June 2023)

GENERAL ASSESSMENT

The objectives of the study: This paper aims to characterize the dynamics that drive allostery of the adenosine A1 receptor (A1R) via computational analysis of its activation free energy landscape and measurements of the appropriate geometrical parameters. This is done by focusing on the allosteric signaling pathways in different activation states, from inactive to active states via intermediate and pre-active ones, as well as the characterization of putative drug-binding pockets. The long-term objectives are to eventually be able to aid drug discovery efforts for this therapeutically important GPCR.

Key findings and major conclusions: Conventional MD does not enable the sampling of the complete conformational landscape of receptor activation. Instead, enhanced sampling MD simulations are required to achieve this. Using metadynamics, the authors decipher the activation pathway of A1R, decode the allosteric networks and identify transient pockets. The protein energy networks computed throughout the inactive, intermediate active, pre-active and active conformational states unravel the extra and intracellular allosteric centers and the communication pathways that couple them, whereby the pathways are reinforced in the activated state. These conformations primarily differ in the dynamics of the ionic lock motif that couples TM3 to TM6 in the inactive conformation and reveal that G-proteins are required to fully stabilize the active conformation. Support for these findings comes from prior mutagenesis work on the A1R that identified key allosteric residues that in many cases map to identified communication nodes. Finally, the authors identified allosteric pockets throughout the A1R in four different conformational states that support prior experimental and MD studies on the mechanism of the positive allosteric modulator MIPS521 and which could be targeted for the design of modulators. Overall, these findings provide complementary support to a structure-based mechanism of activation and allosteric modulation of A1R, and extend the findings to incorporate dynamics across the full activation pathway.

The perceived strengths and weaknesses: This preprint employs a combination of computational techniques to successfully reconstruct and analyze the conformational ensemble of the A1R activation. The metadynamics simulations supported the aim of the study, the results are clearly presented and the work is very well written. The authors could improve the discussion of how the protein energy network analysis could further advance rational design of specific modulators with a desired mode of action. The computational approach needs to be refined to be robust, with a focus on characterizing the convergence of the free energy landscapes. Overall, A1R is a good choice as the target for this study as there is existing structural and pharmacological data to support preliminary findings. Moreover, the framework presented herein could be adapted and scaled to other GPCRs with structural templates, which might enable comparison of allosteric pathways across families and classes.

We thank the reviewers for contextualizing the findings of this study and for highlighting that our work provides complementary support to the mechanism of activation and allosteric modulation of A1R. We also thank the reviewers for their comments and suggestions, which had a great contribution to improve the quality and significance of the revised version of the preprint.

RECOMMENDATIONS

Revisions essential for endorsement:

1. The paper could better demonstrate how the insights gained herein will or could lead to progress in the rational design of specific modulators with a desired effect. The authors should outline and discuss how they envision the modeling pipeline they have designed will be used towards this goal and tone-down or explain why "this information is essential to ease the design of allosteric modulators for A1R.". A recent study on FFAR1, where the authors targeted a specific dynamic pocket could be helpful in this respect (https://www.pnas.org/doi/full/10.1073/pnas.1811066116). Specifically, this might entail: How does specificity for a receptor correlate with pockets forming in a specific state? From this, how does one design an agonist vs. an antagonist vs. an inverse agonist? Does breaking a specific network select a function of the drug? How would another group follow up on this work, for example in a virtual screening campaign?

We agree with the reviewers that a more comprehensive discussion of the points they mention is highly relevant to the study. Firstly, we have rephrased the last sentence of the abstract as "This information can be useful to ease the design of allosteric modulators for A1R" to ensure the significance of our results is not overstated. However, to address the reviewer's feedback more thoroughly, we conducted additional simulations with a positive allosteric modulator (MIPS51) and added an additional sub-section in the results, which includes three new figures (Figure 7-8 and S12):

"ADO and MIPS51 PAM have a significant impact on the energy networks. In order to establish a connection between the energy networks and the mode of action of allosteric modulators, we focus on exploring the effect of MIPS521 positive allosteric modulator (PAM) and ADO agonist as a proof of concept. Experimental assays and Gaussian accelerated MD determined that MIPS521 PAM increases the binding affinity of ADO in the orthosteric site.[19] Thus, PB and PD must be allosterically coupled. Among MIPS521 PAM pocket residues, only L2426.43, L2456.46, S2466.47 and G2797.44 were experimentally found to affect the PAM cooperativity (Figure S11). Interestingly, the PEN obtained in presence of ADO captures these key residues along activation, including TM6 (L2426.43 and L2456.46) in the intermediate, L2426.43 and S2466.47 in the pre-active and L2426.43 and TM7(G2797.44) allosteric residues in the fully-active ensemble (Figure 7). Indeed, G2797.44 becomes a key node in the PEN of the fully-active ensemble. This evidence suggests that although both PD and PB are open in all conformational states, their energy coupling is particularly stronger during the receptor activation.

This prompted us to investigate whether the binding of ADO and the MIPS521 PAM can affect the allosteric communication between PB and PD sites. To that end, we performed cMD of the heterotrimeric Gi2 protein ADO-A1R-Gi2 complex in presence of the PAM (PAM-ADO-A1R-Gi2 complex) and in absence of adenosine (A1R-Gi2 complex) in order to compute their conformational landscape and energy networks following the same protocol for the ADO-A1R-Gi2 complex (Figure 8 and S12). The analysis of the PEN of A1R-Gi2 complex reveals that in the absence ADO, the receptor displays a reduced allosteric communication between PB and functional regions of the receptor, such as the extracellular allosteric center, TM6 and PD allosteric site. As expected, the presence of ADO restores the allosteric coupling between PB and TM6, which could explain the increase of receptor activity associated with agonist binding. Additionally, our analysis of the PAM-ADO-A1R-Gi2 complex shows that the PAM reinforces the TM7-ECL3-ECL2 allosteric pathway that couple PD with PB, and ECL2 now communicates to the intracellular region through TM5 (Figure 8). Notably, a recently published study reported that the orhosteric pocket contracts after ADO binding, as demonstrated by shortened distances of the so-called vestibular lid (defined as the sum of length of the triangle perimeters formed by E17045.51-Y2717.36-E17245.53 interacting residues) and the E17245.53-K26567 salt bridge.[48] Remarkably, the TM7-ECL3-ECL2 enhanced pathway by PAM effect contains the vestibular lid and the E17245.53-K26567 salt bridge residues (Figure 8). This suggest that PAM promotes the contraction of PB, leading to the stabilization of the ADO-bound state. Thus, the enhanced energy coupling between PB and PD may be responsible for the increase in the binding affinity of ADO in presence of the PAM, as observed experimentally.[19] This data indicates that allosteric modulators are able to enhance and redistribute the energy networks, which is likely attributed for their effects on the receptor activity."

The new insights gained from our additional simulations have significantly enriched the discussion on how the protein energy network analysis can contribute to the rational design of specific modulators with desired modes of action. In light of these finding, the last paragraph of the discussion has been rephrased as:

"As a proof of concept, we focus on the PD, which corresponds to the binding site of MIPS52, a positive allosteric modulator (PAM) that increases Adenosine binding affinity in the orthosteric site (PB). Although PD is open in all conformational states the communication between PB and PD is enhanced along activation capturing the allosteric residues that were found to affect its PAM from the intermediate to the fully active. Based on this observation, we hypothesize that drugs that bind pockets and interact with PEN residues, which progresses towards regions of the receptor where function can be altered, may potentially affect the receptor activity through allosteric effects. Additionally, the pocket where the drug binds must be open at least in the conformation state that is targeted. As a practical aspect, virtual screening campaigns could use this information during the design procedure by selecting drug candidates that perform stronger interactions with the PEN contained in the pockets.

To further support this hypothesis, we explored the allosteric effects of ADO and MIPS52 PAM on the PEN. Interestingly, we observed that ADO is crucial for the formation of the extracellular center and its connection with TM6 pathway. Furthermore, MIPS52 PAM reinforces the pathway that connects PB and PD pockets and redistribute other connections. These alterations in the PEN can be related with their mode of action. ADO may increase the activity of the receptor through its communication with TM6 and the PAM may increase ADO binding affinity though stronger energy coupling between PD and PB pockets. These findings imply that the mode of action of allosteric drugs could be predicted depending on how they redistribute the PEN."

Accordingly, the last paragraph of the conclusions has been rephrased as:

"As a proof of concept, Adenosine and a previously experimentally determined positive allosteric modulator were found to enhance and redistribute the energy networks of the receptor in a manner that is consistent with their respective functions. The prediction of drug effects depending on how they redistribute the protein energy networks presents a promising avenue for drug discovery. All these system-specific structural dynamics understanding provides useful information to advance the design of A1R allosteric modulators on the basis of structure-based drug design. This computational approach can be also transferable to other GPCRs and related receptors, which is of interest for the design of novel allosteric drugs."

1. Free energy calculations:

a. A proof of convergence of the free energy calculations is missing. The authors argue that obtaining landscapes that do not change over time is proof of convergence, but this is incorrect in well-tempered metadynamics. The fact that the heights of the Gaussians decrease over time guarantees that the landscape will be stable over time, and the way to check convergence is to show that the collective variables become diffusive after convergence. In addition, to validate that the choice of collective variables (CV) is actually appropriate, they should check that CVs that were not biased are also diffusive. This would be best studied by looking at the behavior of microswitches that were not considered, such as ones describing the PIF motif, the NPxxY motif, the ligand binding pose, etc.

The goal of the well-tempered approach [Phys. Rev. Lett. 2008, 100, 020603] is to improve the convergence of the energy landscapes. This is achieved by gradually decreasing the height of gaussians over simulation. In this fashion, the height of the Gaussians is proportional to a decaying exponential function of the potential deposited in the currently visited point of the CV space. This technique has the added benefit of constraining reconstruction to the region of interest, reducing the risk of irreversible movement towards physically irrelevant regions of the CV space. As noted in the plumed tutorial (https://www.plumed.org/doc-v2.7/user-doc/html/master-_i_s_d_d-2.html), the fact that the Gaussian height is gradually decreasing should not be used as a measure of convergence. Rather, convergence can be assessed by monitoring the energy differences between chosen regions of the energy landscape over time (e.g. the inactive, intermediate and pre-active local energy minima used in our work). If this energy differences do not change significantly as a function of time, this can be taken as an indicator of convergence. We also would like to emphasize that our aim is to recover the major conformational states involved in the pathway of receptor activation rather than the study of subtle energy barriers and relative stability differences of the energy minima upon system perturbations. This objective has been made clear in the text.

We agree with the reviewers' comment that our measure of the convergence could be strengthened by additional analysis that verify the computed conformational pathway of receptor activation.

As suggested by the reviewers, we have plotted the CV1 values over time to asses convergence of our simulations (Figure S4). However, it should be noted that in this study, we have employed the walkers approach [J. Phys. Chem. B 2006, 110, 3533], which utilizes 10 replicas (walkers) to parallelize free energy reconstruction. Each walker simultaneously reconstructs the energy landscape by reading the Gaussian potentials deposited by the other walkers. Consequently, the correct time to stop the metadynamics simulation using the walkers approach becomes more problematic. To facilitate efficient sampling of the CV space and achieve convergence more rapidly, we utilized a sampling strategy that involved starting the simulation with walkers that spanned the entire CV space of interest. In this context, the fact that the walkers do not become trapped in the initial CV space and are able explore and cross into regions occupied by other walkers may serve as a useful indicator of convergence. This assessment of the convergence has been implemented before for Tryptophan Synthase, in which the resulting energy landscapes were consistent with experimental data. [J. Am. Chem. Soc. 2019, 141, 13049] and [ACS Catal. 2021, 11, 13733]

We have added the following paragraph in the convergence section including Figure S4:

"We have also assessed convergence by analyzing the CV1 values over simulation time. Figure S4A shows that during the first 100ns, walkers primary oscillates around their initial CV1 values. Subsequently, at around 200 ns walkers exhibit a higher frequency of crossing into regions occupied by other walkers. This is further supported by the exploration of W1 and W10, as shown in Figure S4B. These two walkers initially start the landscape reconstruction at the opposite extremes of the CV space. At 120 ns, they are able to escape from their respective basins and approach each other, sampling similar CV values (at approximately 240 ns). At this point of the simulation, only these two walkers have covered the entire conformational space of activation. Subsequently, they tend to return to previously sampled CV space. The observation that walkers do not become trapped in their initial CVs region, but instead explore and cross into other regions suggests that our sampling strategy, which involved starting the simulations with walkers that spanned the entire CV space of interest, has facilitated the exploration of the relevant conformational space. Although we cannot guarantee full convergence of the free energy landscape under these conditions, we successfully reconstructed the major conformational states of the receptor activation at 250 ns."

Accordingly, in the results section we have replaced "After 250 ns of accumulated time the FEL was considered to be converged (see Figure S3 and S4)" by "After 250 ns of accumulated time, we successfully reconstructed the major conformational states of the FEL (see convergence assessment in Figure S3 and S4)."

To further verify the accuracy of the collected conformational landscape, we have conducted additional analysis, which include the following:

"As a complementary analysis, we conducted the reweighting of the metadynamics simulations[28] to determine the free energy as a function of previously identified A1R micro-switches (ionic-lock, PIF motif, water-lock and toggle switch). The fact that we capture the distinct energy barriers associated with unbiased micro-switches highlights the accuracy of the metadynamics simulations in reproducing the pathway of activation and provides useful information to guide the selection of collective variables for future GPCR landscape calculations (Figure S5, S6 and S7)."

b. The authors should characterize the uncertainties/statistical errors on the measured free energy profiles to better evaluate the significance of change (e.g. for inspiration: https://www.plumed.org/doc-v2.7/user-doc/html/masterclass-21-2.html).

In response to the reviewers' comment, we have included a new sub-section in materials and methods together with an additional figure in the Supplementary Information (Figure S13), as follows:

"Error: We estimated the error on the 2D free energy landscape of the first collective variable (CV1), which is the TM3-TM6 intracellular ends distance (Figure S13) using the block averaging technique, as described in the PLUMED tutorial on calculating error bars (https://www.plumed.org/doc-v2.8/user-doc/html/lugano-4.html). We calculated the weights using the metadynamics bias potential obtained at the end of the simulation, and assuming a constant bias during the entire course of the simulation.[28] Specifically, we calculate the error using blocks of histograms of 25 ns each, covering the entire 250 ns simulation time."

c. In the cMD trajectories, a large part of phase space is sampled, which does not appear consistent with what one would expect based on the free energy landscapes. For instance, it does not seem reasonable to cover an almost complete conformational transition in 500ns when the barrier of the system is on the order of 5-8kcal/mol. The definition of CVs may have led to an overestimation of the free energy barrier. Hence an independent validation of the free energy barrier height is needed, by e.g. changing the CV definition.

We agree with the reviewers' that the cMD simulations cover a large part of the phase space. This is in part due to running simulations staring from both the inactive and active structures. For the latest, we removed the G-proteins from the receptor. This situation increases the flexibility of the receptor and induces a population shift respect to the starting point. However, as expected, we did not observe any complete transitions in our cMD simulations either staring from the inactive or active structures (see Figure 1C and S2).

Regarding the activation energy barrier, we would like to clarify that the aim of our study is not to compare subtle differences in energy barriers after system perturbations or compare them with experimental data. As far as we know, there is not NMR data available that confirms the exact time-scale of activation for A1R receptor, suggesting that A1R could be highly flexible. Notably, we report a rather low activation energy barrier of approximately 4 kcal/mol derived from the metadynamics simulations of A1R in the presence of adenosine. This is consistent with other computational studies of A1R where a complete transition from active to inactive [PNAS 2022, 119, E2203702119] and from inactive to pre-active [Nature 2021 597, 571] states is sampled in the course of nanosecond-scale Gaussian accelerated MD simulations. In addition, similar activation barrier values have been computed for the b2 adrenergic GPCR in the presence of adrenaline using different collective variables, as reported in [PLoS Comput. Biol. 2011, 7, e1002193] and [eLife 2021, 10, e60715]."

After careful consideration, we found relevant to validate our path of conformations by reweighing of our free energy into other collective variables. As previously stated, we have reweighted the original free energy into the ionic-lock, PIF motif, water-lock and toggle micro-switches (refer to the last paragraph of Essential revision 2.1 and Figure S5 and S6). Our analysis reveals that our original CV2 (TM6 torsion), the PIF motif, and the toggle display modest energy barriers, while our original CV1 (TM3-TM6 distance) presents a rather high energy barrier. Moreover, the ionic-lock and the water-lock exhibit the highest energy barriers. Thus, if we had chosen to use our initial CV1 and the PIF motif, we would have obtained a similar energy barrier, whereas choosing our initial CV1 and the water-lock would have resulted in a higher energy barrier, as predicted by our reweighting calculations (see Figure S7).

As mentioned in the manuscript, this data highlights the ability of our simulations to reproduce the activation pathway and provides interesting insights that can guide the selection of collective variables for future GPCR landscape calculations.

1. Configurations extracted from both conventional MD and wt-metadynamics are mixed in the analyses of the allosteric networks and the pockets. A more accurate way to integrate these datasets would be to modulate the weights of the configurations by their statistical weights, which can be retrieved from the metadynamics simulations.

We thank the reviewers for this suggestion and we will consider to use configurations by their statistical weights in future work. For this case, we aimed to include as much as configurations as possible from each conformational state. We then included all configurations from the inactive, intermediate and pre-active derived from the metadynamics and for the fully-active we applied a stride value in order to collect a similar number of structures.

1. Related to Figure S6, it is essential to compare the dynamics for all of the key class A activation motifs including the Na binding site, PIF motif, and NPxxY.

Based on the reviewers' comments, we have generated histograms for the relevant micro-switches corresponding to the inactive, intermediate, and pre-active states (see Figure S10). This analysis provides further support to the activation pathway derived from the metadynamics simulations. Notably, the population distributions of these micro-switches in the inactive, intermediate, and pre-active states exhibit a correlated progression that mirrors the receptor's activation pathway.

1. Please provide clarification on why 500 ns was chosen as the time-scale of the MD simulations and inclusion of the time course for the three independent MD simulations for each of the key structural features (e.g. TM6 torsion angles and TM3-TM6 distances).

To ensure adequate initial sampling of receptor activation, we performed three independent MD replicas of 500 ns each, starting from both the inactive and active structures. The purpose of these MD simulations is to provide an initial sampling of the receptor instead of describing the complete activation pathway. Based on this initial sampling, we selected a path of 10 conformations as starting points for the walker metadynamics simulations. We have added this information to the text for clarification.

"For each starting point we computed 3 replicas of 500ns, which is a reasonable simulation time to provide an initial sampling of the receptor activation."

1. The validation of the results in the form of previously published mutagenesis results does not appear completely convincing. Large parts of the protein are included in the allosteric network, making it likely that mutations in some of these residues will have an effect if mutated. In addition, the fact that mutations in ECL2 and ECL3 affect allostery is expected and does not constitute a good validation of the results. If no other results are included, we recommend that the language be toned down so as not to overstate the significance of the results.

Following the reviewers' comment, we have removed the following sentences from the results:"Among the PEN residues, we successfully captured most of the allosteric residues previously identified by mutagenesis studies, which highlights the reliability of the allosteric networks computed" and "The high predictive power of the PEN to identify allosteric residues highlights the reliability of the characterized allosteric pathways"

1. What is the justification for using an energy-based scoring for network analysis, given that a conventionally correlation-based approach has been used successfully in the field? The concern with an energy-based approach is that the interaction energy calculations do not consider the dielectric effect, i.e., if water molecules interfere with two interacting residues. Since the dynamic network is one of the critical aspects of this study, we believe the authors need to explore other tools such as the one implemented in VMD (https://www.ks.uiuc.edu/Research/vmd/plugins/networkview/) and compare the results.

We decided to perform protein energy networks analysis because our aim was to investigate how the networks change in different conformational states along activation. We selected this approach because energy networks can provide a more detailed insight into how communication within the protein changes during activation, as compared to cross-correlation networks, which are more suited to characterizing communication through correlated motions in the global ensemble. In order to compare both protein energy networks and correlation networks we performed the cross-correlation analysis in the global ensemble. Note that both approaches yield some similarities and provides complementary information.

We also would like to thank the reviewers for raising concerns about the methodology employed in our work.We acknowledge that gRINN, which we used to generate the pairwise residue mean interaction energy matrix, does not include water molecules and ligands during the matrix generation process. As a result, we are unable to capture communication pathways that involve water-mediated connections or interactions between ligands and residues. For example, in the pre-active ensemble, Y2005.58 and Y2887.53 from the NPxxY motif are connected by a hydrogen bond facilitated by a bridging water molecule (the son-called water-lock). However, such communication is not captured in our analysis due to the absence of water molecules (Figure 3). We highlight this major limitation in the text. Another example can be observed in the simulation of the PAM-ADO-A1R-Gi2 system, where communication between L242 and S246, two residues involved in the Positive Allosteric Modulator (PAM) binding site, is missing in the PEN (Figure 8). Since these residues must be connected through the PAM, our methodology cannot detect their communication. A promising tool to considered in future studies is webPSN v2.0 [Nucleic Acids Res. 2020, 48, W95], a protein structure network analysis that includes nucleic acids and more than 30,000 biologically relevant molecules and ions, which is highly advantageous to study the effect of drugs on the protein communication.

1. Provide generic residue numbers such as GPCRdb or Ballesteros Weinstein numbering for all mentioned residues in text and figures, as is standard for structural papers.

As the reviewers suggested, we have renumbered all residues mentioned in the text and figures according to the Ballesteros Weinstein numbering scheme.

Additional suggestions for the authors to consider:

1. For the PEN analysis it would be useful to digest these communication networks with respect to the established structural activation motifs of class A GPCRs (Na binding site, PIF, and NPxxY) that are present at the A1R.

In order to make the PEN analysis more digestive, we have revised the second paragraph of the "Energy Networks captures the dynamic allosteric pathways along A1R activation" results section. Specifically, we have highlighted the most relevant micro-switches captured in the PEN, with a particular focus on the ionic and water-lock switches, which are the most prominent for the protein communication.

1. It is unclear why the authors chose two largely correlated CVs (See comment 2c). In addition, the choice of CV is likely contributing to the distortion of S6, as displayed in Figure 1E. It has been shown that choosing a different CV set that describes the motion between states in a more distributed way is more likely to lead to a converged conformational ensemble. We suggest repeating the metadynamics simulations with a more distributed CV set that encompasses all of the microswitches in the receptor.

Regarding the concern raised by the reviewers about the distortion observed in the TM6 end, we want to clarify that it is not attributed to the selection of collective variables (CVs) since it is already explored in the initial conventional MD simulations (see Figure S2B, W5 structure). We selected two CVs that may seem largely correlated, but they actually describe different aspects of the TM6 inward-to-outward transition. The first CV, which measures the distance between the center of mass of TM3 and TM6, is more related to the dynamics of the ionic lock in the intracellular region. On the other hand, the second CV (TM6 torsion) is related to the forces sensed by the upper region of TM6, including the dynamics of the W2476.48 toggle switch. Therefore, we believe that the combination of these two CVs provides a comprehensive description of the TM6 transition.

It is also worth mentioning that a more distributed set of CVs may be beneficial to better reproduce the activation energy barriers of the receptor. In fact, as shown in the reweighting calculations of the metadynamic bias potential (see Figures S6 and S7), using the TM3-TM6 COM end distances and the Y2005.58-Y2887.53 distance (water-lock) as CVs appears to be a good choice for this purpose.

1. To support the vision on how the analysis of activation pathway, energy networks and transient pockets could be used "to ease the design of allosteric modulators for A1R" (last sentence of the abstract), it might be necessary to show that the combination of these methods can indeed be predictive for the binding and effect of known ligands. This might provide a first step towards establishing that molecules that bind to pockets "near allosteric networks" is a promising avenue for drug discovery.

This highly relevant point has been addressed in Essential revisions 1.

1. The specific TM3-TM6 residues should be specified in figures and text. Commonly used TM3-TM6 comparisons include the measured maximum distance between 2x46 to 6x37, which could be used here also (e.g. see https://docs.gpcrdb.org/structures.html#structure-descriptors).

The specific residues of TM3 and TM6 that were used in the analysis have been clearly specified in both the Materials and Methods section and the figure captions of Figure 1 and 4.

1. Even though the "A1R in complex with PSB36 (PDB 5N2S)" is an inactive structure, PSB36 is an agonist. Hence, the authors should consider using the DU172 antagonist-bound structure for comparison (PPDB 5UEN)

According to literature, PSB36 is selective antagonist for A1R. In fact, experimental data showed that PSB36 exhibit low inverse agonist activity [Chem. Med. Chem. 2006, 1, 891]. Although PDB 5N2S and PPDB 5UEN structures are almost identical, the bulkier DU172 ligand causes a displacement of TM2 in the extracellular region. Therefore, we chose to use the 5N2S structure in our study. However, we will consider using the 5UEN structure in future studies.

1. How does adenosine and MIPS521 binding impact the different conformational states and PEN.

This highly relevant point has been addressed in Essential Revisions 1. For a more detailed analysis, refer to Figure 8 and S12, which shows the impact of MIPS521 on the PEN and the conformational landscape, respectively.

1. It would be interesting to note how the findings from this study compare/contrast to a very recently published report by Li et al, PNAS, 2022 "The full activation mechanism of the adenosine A1 receptor revealed by GaMD and Su-GaMD simulations". Similarly with regards to the determination of allosteric binding pockets in this recent publication: "The pocketome of G-protein-coupled receptors reveals previously untargeted allosteric sites" (https://doi.org/10.1038/s41467-022-29609-6)

According to the reviewers' suggestion, we have compared our findings to those of a recently published study [PNAS 2022, 119, E2203702119], which explored the full activation mechanism of A1R using both su-GaMD and GaMD.

This published work serves to further confirm the combined activation mechanism that we observed for A1R in our study, which entails the formation of a pre-active state in the presence of Adenosine and the stabilization of a fully-active state in the presence of both Adenosine and G-proteins. Moreover, their study reports the pre-activation of the receptor from the inactive state within 150 ns of GaMD, indicating a rather low activation energy barrier of the receptor in presence of Adenosine. This is consistent with the approximately 4 kcal/mol activation energy barrier we calculated for A1R-ADO in our 250 ns metadynamic simulations.

We would also like to highlight an additional noteworthy point we have included in the results as: "Notably, a recently published study reported that the orthosteric pocket contracts after ADO binding, as demonstrated by shortened distances of the so-called vestibular lid (defined as the sum of length of the triangle perimeters formed by E17045.51-Y2717.36-E17245.53 interacting residues) and the E17245.53-K26567 salt bridge.[48] Remarkably, the TM7-ECL3-ECL2 enhanced pathway by PAM effect contains the vestibular lid and the E17245.53-K26567 salt bridge residues (Figure 8). This suggest that PAM promotes the contraction of PB, leading to the stabilization of the ADO-bound state. Thus, the enhanced energy coupling between PB and PD may be responsible for the increase in the binding affinity of ADO in presence of the PAM, as observed experimentally.[19]"

1. A major advantage of allosteric drugs is the potential to achieve higher selectivity. Expansion of this study to include other adenosine receptor subtypes or linking to other types of molecular pharmacology (e.g. biased signalling, subtype selectivity, etc.) would be a major benefit to the field.

We are grateful to the reviewers for recognizing the potential impact of our work in various applications beyond our initial scope. We will consider to incorporate their valuable suggestions in our future research endeavors.

1. Consider including an explanation of the physiological and pharmacological relevance of A1AR in the introduction.

According to this suggestion, we have incorporated a new sentence in the introduction section as follows:

"The adenosine A1 receptor (A1R) is a member of the class A G protein-coupled receptor (GPCR) family that preferentially couples with Gi/o proteins. It is widely distributed in multiple organs mediating a variety of physiological processes, including those in the brain and the heart. Thus, A1R has significant therapeutic potential in the treatment of numerous diseases and disorders.[18]"

1. Even if not entirely necessary for the results, it would be more consistent if the study would include metadynamics of the G-protein bound state.

Performing a metadynamics calculation of the G-protein bound state is challenging as it requires careful consideration of the G-protein binding process. As a reference work, Giulio Mattedi et al. successfully implemented this calculation for the glucagon receptor [Proc. Natl. Acad. Sci USA, 2020, 117, 15414]. However, in our study the effect of the G-proteins in the activation landscape is a minor remark. Our study places a greater emphasis on sampling the most stable conformations associated with the fully-active conformational state to compute the protein energy networks.

1. Methods: "In other words, once the free energy surface does not change significantly during a relatively long period of time in the last part of the simulation". What is "relatively long period of time" and "change significantly". The convergence, should be stated as a quantitative description of the observed energy differences.

We have addressed this technical issue in Essential revisions 2. It is worth noting that "the relatively long period of time" required for convergence may vary depending on the specific system under study. Nonetheless, we believe that observing a stable energy surface over the course of 50-100 ns, while the system explores different relevant regions of the CV space, provides a good criterion for convergence."

1. The authors should strongly consider making their analysis code and simulation data publicly available (e.g. on GitHub or Zenodo) so that others can replicate and build upon this work.

We thank the reviewers for this suggestion. We will make all the output files generated from the get Residue Interaction eNergies and Networks (gRINN) calculation available to the public.By doing so, users will be able to visualize the results in the gRINN visual interface and perform customized network analysis using the pairwise residue mean interaction energy matrices. Additionally, we will provide all Pymol sessions that include the protein energy networks as well as the Isosurface representation of the frequency maps of the transient pockets. We believe that these materials will provide better visualization compared to the current figures presented in the manuscript and supporting information, which will be helpful in guiding future structure-based drug design campaigns.

(This is a response to peer review conducted by Biophysics Colab on version 3 of this preprint.)

The desire to be seen as "feminine" by men highlights how society values external validation, linking femininity to approval from men. This belief reinforces the idea that a woman's worth is often based on her ability to conform to societal expectations.

Asociación de los países y territorios de Ultramar: La Asociación de los países y territorios de Ultramar (PTOM) se refiere a una agrupación de territorios y países que tienen una relación especial con un Estado miembro de la Unión Europea. Estos territorios pueden ser islas o regiones situadas fuera del continente europeo. La asociación busca promover el desarrollo económico, social y político de los PTOM y establecer una cooperación estrecha con la Unión Europea.

Los créditos a la exportación son préstamos o financiamiento proporcionados a empresas para apoyar sus actividades de exportación, facilitando la venta de productos y servicios en mercados internacionales.

Cláusulas de salvaguardia: Estas cláusulas permiten a un país tomar medidas temporales para restringir las importaciones de determinados productos cuando se produce un aumento repentino y significativo de las importaciones que amenaza con dañar a la industria nacional. Las cláusulas de salvaguardia pueden incluir el aumento de aranceles aduaneros o la imposición de cuotas de importación durante un período determinado. Estas medidas tienen como objetivo brindar un "alivio" temporal a las industrias afectadas para que puedan ajustarse y competir en mejores condiciones.

Cláusulas de vigilancia: Estas cláusulas permiten a un país monitorear las importaciones de ciertos productos para identificar tendencias o patrones que puedan indicar una amenaza para su industria nacional. A través de la vigilancia, los países pueden recopilar información sobre las importaciones y evaluar si se están produciendo prácticas comerciales desleales, como dumping (venta de productos por debajo de su valor real) o subsidios injustos a las exportaciones. Si se detectan tales prácticas, el país puede considerar la aplicación de medidas correctivas, como investigaciones antidumping o compensatorias, para proteger a su industria y mantener un comercio justo

I was wondering if we can use this tool to create our own website unrelated to our classes or teaching

finally got it :)

ECO- REGIMENES.

Los eco-esquemas son reglas y acciones que los agricultores pueden seguir para cuidar mejor del medio ambiente mientras trabajan en sus campos.

Los eco-esquemas están diseñados para ayudar a proteger la naturaleza y la biodiversidad, reducir el uso de productos químicos dañinos y promover prácticas más sostenibles en la agricultura. Por ejemplo, podrían incluir cosas como plantar setos o árboles para crear hábitats para los animales, usar menos pesticidas y fertilizantes, o rotar los cultivos para mantener el suelo saludable, fruta ecologica,

Los agricultores que participan en los eco-esquemas reciben un apoyo económico adicional por adoptar estas prácticas sostenibles. Un pago anual por todas las hectareas cualificables, solo una practica por hectarea

dividida en muchas partes pequeñas, en lugar de ser concentrada o unificada

This is not the only porcupine to appear in Levi’s writing. In The Truce, we learn that Levi’s companion Cesare, disappointed after a misadventure on the black market, spent two days ‘huddled on his bed, bristling like a porcupine’ (CW I, 338). In The Wrench, Faussone identifies a clearing in which ‘a porcupine was advancing cautiously, with brief stops and starts’ (CW II, 1025). These English translations suggest a possible connection to SQ that is less obvious in the original Italian, where the text refers not to an ‘istrice’ but rather to a ‘porcospino’. In La tregua, Cesare is described as ‘ispido come un porcospino’ (OC I, 417), and in La chiave a stella, Faussone points out that ‘un porcospino avanzava cauto, con brevi arresti e riprese’ (OC I, 1099).

The terms ‘istrice’ and ‘porcospino’ refer to animals of the same family, Hystricidae, and identify the same species, Hystrix cristata, the crested porcupine, which is native to Italy. In the Grande dizionario italiano dell’uso, ‘porcospino’ is listed as a synonym of ‘istrice’, which is defined scientifically as a ‘piccolo mammifero con il corpo coperto di aculei appuntiti ed erettili’, with a second figurative meaning as a ‘persona intrattabile, scontrosa’ (805).

Despite their similarity, there is a notable difference between the two synonyms with regard to their literary resonances. As the Tesoro della lingua Italiana delle Origini demonstrates, ‘istrice’ was the preferred term for medieval philosophers, historians, and poets, including Boccaccio, who used it in his Caccia di Diana and Ameto, where the husband’s beard is described as being ‘né più né meno pugnente che le penne d’uno istrice’ (Tutte le opera di Giovanni Boccaccio, 774). The Grande dizionario della lingua italiana attests subsequent citations from Parini, D’Annunzio, De Amicis, and Foscolo, with the latter two adopting the term metaphorically to refer to a person who is taciturn and cagey (615).

I suspect that Levi had another literary reference in mind when he opted for ‘istrice’ rather than ‘porcospino’ in SQ. Here are the words with which the Ghost in Shakespeare’s Hamlet reveals both his identity and the infernal torments he suffers in the afterlife:

I am thy father’s spirit,

Doom’d for a certain term to walk the night,

And for the day confined to fast in fires,

Till the foul crimes done in my days of nature

Are burnt and purged away. But that I am forbid

To tell the secrets of my prison-house,

I could a tale unfold whose lightest word

Would harrow up thy soul, freeze thy young blood,

Make thy two eyes, like stars, start from their spheres,

Thy knotted and combined locks to part

And each particular hair to stand on end,

Like quills upon the fretful porpentine:

But this eternal blazon must not be

To ears of flesh and blood. List, list, O, list! (Hamlet, Act I, Scene 5, vv. 14-28)

In standard Italian translations dating back at least to the early nineteenth century, Shakepeare’s ‘fretful porpentine’ is rendered as a ‘pauroso istrice’ (Amleto, 59). This word, and these lines, would seem to resonate remarkably well with Levi’s description of the hell of Auschwitz, which is the context for his invocation of ‘la difesa dell’istrice’.

After all, Hamlet’s Ghost is compelled to speak quickly, in the brief interval he has been granted in his eternal suffering:

My hour is almost come,

When I to sulphurous and tormenting flames

Must render up myself (Hamlet, Act I, Scene 5, vv. 5-7)

Cannot Levi and Jean say the same thing? The ‘lungo giro’ that Jean has arranged buys them a brief respite, but this precious time has begun to disappear as soon as it arrives: ‘quest’ora già non è più un’ora’. Cannot Hamlet’s Ghost say the same thing?

The moment of connection and communication at the heart of ‘Il canto di Ulisse’ can be said to have begun with Jean’s clever strategy to curry favour with cruel Alex, the Kapo Levi describes as ‘un bestione violento e infido’, who is won over by Jean’s ‘opera lenta cauta e sottile’, finally ceding to him the coveted role of Pikolo. It is this victory that Levi describes as penetrating ‘the porcupine’s defence’. And it is this victory that frees Jean to choose Levi for the task of fetching the daily soup ration, enabling the disquisition on Dante that gives the chapter its title.

If I am correct that the reference to ‘la difesa dell’istrice’ is thus evidence that Levi and Jean’s Dantean voyage begins under the sign of Hamlet, this would be a particularly elegant literary manoeuvre, since the voyage concludes under the very same sign. As their hour runs out once they have reached the kitchen, Levi finds himself unable to say all that needs to be said and is forced to concede that ‘il resto è silenzio’, an unmistakable echo of Hamlet’s final words: ‘the rest is silence’ (Hamlet, Act V, Scene 2, v. 395).

If further evidence for a Shakespearean source text is warranted, I would note that Levi included in Ad ora incerta a poem that explicitly references Hamlet’s Ghost, who is referred to as an ‘old mole’ because he continues to speak from beneath the floorboards (Hamlet, Act I, Scene 5, v. 183). Italian translations render this line as ‘vecchia talpa’, words that Levi borrowed for the title of a 1982 poem, which literalises the reference - the poem is in fact written from the perspective of an old mole - while nevertheless conveying the sense of the original, with its profound intimations of the latent power of buried knowledge.

In altri tempi seguivo le femmine,

E quando ne sentivo una grattare

Mi scavavo la via verso di lei:

Ora non più; se capita, cambio strada.

Ma a luna nuova mi prende il morbino,

E allora qualche volta mi diverto

A sbucare improvviso per spaventare i cani. (OC II, 727)

The reference to an ‘istrice’ in ‘Il canto di Ulisse’ similarly suggests hidden depths.

CLL

The late Stuart Woolf (1936-2021) must have smiled to himself when he first translated these lines, as a young historian working on his PhD in 1950s Turin. Woolf is the only published English translator of SQ; his fluid and immediate rendering of Levi’s words remains the version known to millions of anglophone readers. While the task of a translator is never easy, it may be that the clarity and simplicity of Levi’s style lends itself to translation and grants his writing a certain universality - almost like a chemical formula.

‘The Canto of Ulysses’ can be read as an ode to translation, not just from one language to another, but in a metaphorical sense, in the repositioning of meaning between people and time. This goes back to the idea implied in the etymology of the word ‘translation’, which comes from the Latin translatio, to ‘carry over’, to ‘bring across’. In this chapter, instances of translation form a mise en abyme that ‘carries over’ from Homer to Virgil, Virgil to Dante, Dante to Levi, Levi to Pikolo, Italian to French, Italian to English, and text to reader.

This more conceptual idea of ‘translation’ has become a way of understanding the testimonial act, central to Holocaust studies (Insana 2009; Felman and Laub 1992). Witnesses ‘translate’ into words their experience and their trauma. This process is often thought of as entailing a loss: an ineffable residue that cannot be communicated through language. However, the exchange that takes place between Levi and Jean in ‘The Canto of Ulysses’ invites us to rethink translation in terms of expansion, with each new version becoming part of the original’s harvest. The non-Italian reader’s lack of familiarity with ‘Who Dante is’, ‘What the Comedy is’, may at first seem a disadvantage. And yet, this has the enriching effect of aligning us with Jean: the reader/Pikolo attempts to overcome a linguistic and cultural barrier, to be in communion with the narrator/Levi. Conversely, Italian readers are likely to identify more closely with Levi, as they try, with him, to remember lines learned in their schooldays.

The new interpretative perspectives created by the translated text respond to the original and form a polyphony. This polyphonic effect works on two different levels: first, just as a piece of music sounds different when sung by a different voice, a translation performs a text in another language, with another instrument. Second, by co-existing in the literary universe of the original text, the many translations of this chapter embody the multiple voices that have resonated from Levi’s writing. As Levi and Jean walk, we see the process of translation unfold. As they come to understand each other, communication through words falters, and another kind of translation begins to happen:

O forse è qualcosa di piú: forse, nonostante la traduzione scialba e il commento pedestre e frettoloso, ha ricevuto il messaggio, ha sentito che lo riguarda, che riguarda tutti gli uomini in travaglio

The ‘something more’ is in the polyphony of their exchange, where the ensemble is greater than any individual line. It takes on a special significance in Woolf’s translation - or in any translation of these lines, for it becomes another performance, or layer, of the initial translational act. The message of the original seems to swell, rather than subside. And, just as a melody transcends individual notes, the concern for individual words is eventually superseded by the harmony between Levi and Jean. Describing Dante’s approach to divine grace in Paradiso, George Steiner writes:

But as the poet draws near the Divine presence, the heart of the rose of fire, the labour of translation into speech grows ever more exacting. Words grow less and less adequate to the task of translating immediate revelation (Steiner 1967).

Levi draws us to a similar source, one that sounds ‘like the blast of a trumpet, like the voice of God’. This ‘something more’ that is not bound to language has the universality of music. It reaches towards an inexpressible goodness or enlightenment. This is in direct contrast to the negative ‘ineffability’ that is so often used to describe elements of testimony in Levi and others, in the challenge the Holocaust posed to language, in the impossibility of its translation. Here, language does not drift towards a void of suffering, but towards a chorus of joyful expression, a blast of trumpets. Unlike elsewhere in SQ, the ambiguity present in the meeting of languages is not represented as a chaotic and hellish Tower of Babel, but as a fecund, creative space. Translation is momentarily reclaimed, and acts as an implicit resistance to the obsessive uniformity of Nazi ideology. But their ‘canto’ is interrupted by the cacophony of Auschwitz, and this revelatory chink is closed with a tragic, symphonic surge:

Infin che ’l mar fu sopra noi rinchiuso.

RMur

Very often, when we think about ‘Il canto di Ulisse’, we tend to recall only the most famous pages in which Levi tries to remember Dante’s canto. The depth and sense of urgency of the Ulyssean passages are so overwhelming and passionate that they may distract us from other elements in the chapter. However, if we go back to the text and read it closely, we cannot avoid noticing that, after a brief opening in which Levi introduces Pikolo and narrates how he came to be Pikolo’s ‘fortunate’ chaperone to collect the soup for the day, ‘Il canto di Ulisse’ also dwells quite significantly on a moment of domestic memories. While going to the kitchens, Levi writes: ‘Si vedevano i Carpazi coperti di neve. Respirai l’aria fresca, mi sentivo insolitamente leggero’. This is the first moment in the chapter in which Levi refers to the mountains as something that revitalises him and makes him feel fresh and light, both physically and mentally.

This moment foreshadows another, also in this chapter, when Levi goes back to his mountains, those close to Turin, and compares them to the mountain that the protagonist of Dante’s canto, Ulysses, encounters just before his shipwreck with his companions:

... Quando mi apparve una montagna, bruna

Per la distanza, e parvemi alta tanto

Che mai veduta non ne avevo alcuna.

Sì, sì, ‘alta tanto’, non ‘molto alta’, proposizione consecutiva. E le montagne, quando si vedono di lontano... le montagne... oh Pikolo, Pikolo, di’ qualcosa, parla, non lasciarmi pensare alle mie montagne, che comparivano nel bruno della sera quando tornavo in treno da Milano a Torino! Basta, bisogna proseguire, queste sono cose che si pensano ma non si dicono. Pikolo attende e mi guarda. Darei la zuppa di oggi per saper saldare ‘non ne avevo alcuna’ col finale.

The significance of the mountains in Levi’s narration is confirmed in this passage. For him, the mountains represent his experience of belonging, his youthful years, and his work as a chemist – the job he was doing when he commuted by train from Turin to Milan. At the same time, Levi’s own memories of the mountains intertwine and overlap with another mountain, Dante’s Mount Purgatory. Here, a deep and perhaps not fully conscious intertextual game starts to emerge and to characterise Levi’s writing. The lines that Levi does not remember are these (compare, on the Dante page):

Noi ci allegrammo, e tosto tornò in pianto,

ché de la nova terra un turbo nacque,

e percosse del legno il primo canto.

For Dante’s Ulysses, Mount Purgatory signifies the final moment of his adventure and his desire for knowledge. The marvel and enthusiasm that Ulysses and his company feel when they see the mountain is suddenly transformed into its contrary. From the mountain, a storm originates that will destroy the ship and swallow its crew: ‘Tre volte il fe’ girar con tutte l’acque, | Alla quarta levar la poppa in suso | E la prora ire in giù, come altrui piacque’. Dante’s Mount Purgatory, so majestic and spectacular, represents the end of any desire for knowledge that aims to find new answers to and interpretations of human existence in the world without God’s word.

Going back to Levi’s text, we find that, instead, in a kind of reverse overlapping between his image and that of Ulysses, the image of the mountain of Purgatory suggests to Levi a very different set of thoughts that, although seemingly and similarly overwhelming, opens up new interpretations: ‘altro ancora, qualcosa di gigantesco che io stesso ho visto ora soltanto, nell’intuizione di un attimo, forse il perché del nostro destino, del nostro essere oggi qui’. For a moment, it is almost as if Levi, a new Dantean Ulysses in a new Inferno, stands in front of Mount Purgatory and forgets the terzine and the shipwreck. Maybe Levi cannot or does not want to remember those terzine because the mountain in Purgatory represents something very different for him than for Dante’s Ulysses. Levi’s view of the mountain does not lead to a moment of recognition of sin, as it does in Dante’s Ulysses. For him, the mountain, like his mountain range, is the gateway to knowledge, enrichment, and illumination and to a world that lies beyond the imposed limits of traditional, constricting, and distorted views and that awaits discovery (‘qualcosa di gigantesco che io stesso ho visto ora soltanto’). Something about and beyond the Lager.

To better understand how the mountains are central in ‘Il canto di Ulisse’, we have to remember that Levi’s view of the mountains strongly depends on his anti-Fascism, which he expressed particularly vigorously in two moments of his life: during his months in the Resistance, just before he was captured and sent to Fossoli, and, even more intensely, during the adventures of his youth, when he was a free young man who enjoyed climbing the mountains surrounding Turin. As Alberto Papuzzi has suggested, ‘le radici del suo rapporto con la montagna sono ben piantate in quella stagione più lontana: radici intellettuali di cittadino che cercava sulla montagna, nella montagna, suggestioni e risposte che non trovava nella vita, o meglio nell’atmosfera ispessita di quella vita torinese, senza passato e senza futuro’ (OC III, 426-27). Indeed, reports Papuzzi, Levi confirms that:

Avevo anche provato a quel tempo a scrivere un racconto di montagna […]. C’era tutta l’epica della montagna, e la metafisica dell’alpinismo. La montagna come chiave di tutto. Volevo rappresentare la sensazione che si prova quando si sale avendo di fronte la linea della montagna che chiude l’orizzonte: tu sali, non vedi che questa linea, non vedi altro, poi improvvisamente la valichi, ti trovi dall’altra parte, e in pochi secondi vedi un mondo nuovo, sei in un mondo nuovo. Ecco, avevo cercato di esprimere questo: il valico.

The heart of that epic story made its way into the chapter ‘Ferro’ in Il sistema periodico. The discovery of this (brave) new world, ‘mondo nuovo’, is an integral part and a direct achievement of Levi’s experience in the mountains. The mountains open a new understanding and a new perspective on the world.

Something that escapes common understanding is revealed through the experience of the mountains, both in Levi’s memories of his youth and in his literary recounting of Auschwitz. Reciting Dante in ‘Il canto di Ulisse’ is therefore not only an intertextual exercise for Levi. Only by inserting Levi’s literary references in the complexity of his own experience – before, during, and after Auschwitz – can we fully capture the depth of his reflections. Levi mentally and metaphorically brought to Auschwitz not only Dante but also his ‘metafisica dell’alpinismo’. Together, they contributed to his attempt to come to terms with that reality.

‘The Canto of Ulysses’ records a truly remarkable feat of cultural memory, as Levi recalls and recites Dante amidst the ruin of Auschwitz. He does so to teach Italian to his campmate Jean. But as he recites the words of Dante, Levi also feels his past come back to him. He begins to recover something of his identity and his humanity in the degraded world of the Lager, to feel again that he is ‘a man’.

Dante is not, however, the only canonical writer to appear in ‘The Canto of Ulysses’. So too does Shakespeare. <br /> Levi admits to ‘gaps’, ‘holes’, and ‘lacuna[e]’ in his memory as he recites Dante, some of which would seem to be ‘irreparable’. Unable to recall his Dante, Levi is forced to confront ‘silence’:

I would give today’s soup to know how to connect ‘the like on any day’ to the last lines. I try to reconstruct it through rhymes, I close my eyes, I bite my fingers – but it is no use, the rest is silence [il resto è silenzio]. Other verses dance in my head: ‘…The sodden ground belched wind …’, no, it is something else. [Woolf’s translation.]

‘[T]he rest is silence’: Levi is quoting the last words that are spoken by Hamlet, before he dies:

O, I die, Horatio!

The potent poison quite o’ercrows my spirit.

I cannot live to hear the news from England.

But I do prophesy th’election lights

On Fortinbras; he has my dying voice.

So tell him, with th’occurrents more and less

Which have solicited – the rest is silence.

With his ‘spirit’ succumbing to the ‘potent poison’ Claudius and Laertes have used to kill him, Hamlet undertakes the impossible: to testify to his own death, to ‘tell’ in words his fall into deathly silence. This is his ‘dying voice’.

What is Hamlet doing in ‘The Canto of Ulysses’? Why does Levi place it where he does, in ‘the caesura’, the abyssal gap between Auschwitz and his cultural identity and memory, embodied by Dante? It is certainly ironic that Levi draws once again on the Western canonical literary tradition to record the moment of its ostensible breakdown. What emerges from the lapse, the silence that Levi testifies to, is another tie to his compromised past, and the literary culture that would seem to have been obliterated by the Holocaust – even if Levi does not choose to bring obvious notice to his allusion by using quotation marks or by writing, ‘as Hamlet says’. The poetry of Hamlet appears to be among the ‘other verses’ Levi has confusedly ‘dancing in his head’ while he tries to fill the gaps in his memory.

By quoting Hamlet, Levi would appear to again testify to the power of literature as a mainstay of culture and humanity, evincing his commitment to humanist ideals. This is certainly the positive interpretation of Hamlet in ‘The Canto of Ulysses’. I would make the case, however, that Levi is using Shakespeare for an altogether more challenging purpose. By appropriating the ‘dying voice’ of Hamlet, Levi records the place where his memory and his identity collapse, testifying to the reduction of the camp inmate to silence and oblivion. Bryan Cheyette writes that ‘even when his memory self-consciously fails him’, Levi is always ‘at pains to bear witness to those moments of failure’ (Cheyette 1999, 64). Levi seeks to testify to the silence, to show that ‘something has occurred even if it cannot be understood’ (Druker 2009, 64). This is the role played by Hamlet in ‘The Canto of Ulysses’. Levi does not use the play to testify to the endurance of the human spirit in the camps, but to silence, to a ‘world of negation’ (OC I, 235). Hamlet, perhaps ironically given its near-unrivalled canonical and cultural status, marks the space beyond language and culture, into which Levi is at risk of falling. This, as Levi called it, is the ‘black hole’ of Auschwitz (OC II, 1663).

Through his allusion to Hamlet, Levi rewrites Shakespeare from the perspective of Auschwitz, endowing the play with new meanings as he confronts the lacunae and voids produced by the world of the concentration camp. Levi uses the play to record the disintegration of humane values in Auschwitz, as memory and language are brought to the point of collapse. Jacques Derrida does much the same in his own work on the play. He draws Hamlet into conversation with Holocaust testimony when he compares the play to the poetry of survivor Paul Celan in his 1995 piece ‘The Time is Out of Joint’. Derrida contends that the paradox of testimony is that the witness must uncannily ‘outlive his life’ (3.2.117) – or ‘survive’ that which is not ‘survivable’: the collapse of all meaning and death (Derrida 1995). This is the sense in which Hamlet is a play about the ‘impossible possibility of testimony’, as Derrida calls it. Hamlet has ‘seen the worst’ and is ‘the witness of the worst disorder, of absolute injustice’, writes Derrida. Hamlet has witnessed too much for words – but testimony, ‘though it hath no tongue, will speak’ (2.2.546).

Levi must also confront and testify to the painful death of memory, the destruction of human identity and culture, before the event of physical death itself – or as Jacques Lacan would call it, ‘symbolic’ before ‘actual’ death, the death of the self before physical death. Lawrence Langer uses the phrase ‘deathlife’ to name the same phenomenon, of ‘dying while one is living’ (Langer 2021, 13). Not unlike the melancholic prince, Levi attempts the impossible of testifying to his own demise. He deploys Hamlet to record his fall into a place beyond humanity and beyond culture – even beyond language. It is, to adopt the words of Jean Améry, an act of both resignation and revolt: resignation to silence, and a determined revolt against oblivion, by testifying to it. It is an astonishing moment.

RA

Among many other layers to this chapter and its revelations, the ‘qualcosa di piú’ is also a reference to poetry as a means to escape Auschwitz. Poetry itself becomes the means to resist the process of bestialisation and reification.

MJ

Scholars tend consistently if not quite unanimously to emphasise the ambiguity of Levi’s term ‘gigantesco’. The discussion of Dante’s Ulysses is ‘broken off’, as Hayden White puts it, before Levi can tell us ‘what we are supposed to conclude’ (2015, 12). As a result, ‘there is no final manifestation of the message, of the meaning that Levi is so desperately trying to grasp and communicate’, argues Giuseppe Stellardi (2019, 715). ‘Nessuno potrà mai affermare nulla con sicurezza’, assert Alberto Cavaglion and Paolo Valabrega (515). The lack of certainty regarding the term’s meaning is perhaps responsible for the substantial divergence between the critical interpretations that this passage has inspired.

There are those who argue that Levi’s ‘gigantic’ discovery when reciting Dante in Auschwitz is an unconquerable ‘faith in his culture’ (Hartman 1996, 52), and those who claim, conversely, that Levi instead recognises how ‘behind the gas chambers, the ovens, the starvation rations, and the astonishing otherworldly everyday viciousness and cruelty can be found at the heart of Western culture’ (Feinstein 2003, 365). In other words, while some hold that Levi finds in Dante the antidote to Auschwitz, others argue that he finds the cause.

Levi’s subsequent glosses on this passage have done little to alleviate the uncertainty. In the version of SQ that he prepared for a 1964 radio broadcast, he clarified the meaning of the quoted phrase ‘come altrui piacque’ (OC I, 1237); for the 1973 Schools edition of SQ, intended for an audience of Italian students, he provided footnotes explaining the term ‘anacronismo’ and the phrase ‘il perché del nostro destino’ (OC I, 1417-18). In both instances, Levi left ‘gigantesco’ undefined. This apparent authorial reticence should inspire some restraint in our critical exegesis. There is no need to pursue false certainty where the text offers legitimate ambiguity.

What we may note, however, is that elsewhere in SQ, and with significant frequency in Levi’s subsequent writing, the term ‘gigantesco’ serves to identify the monstrosity of the Lager. In the chapter ‘I sommersi e i salvati’, he describes Auschwitz as ‘una gigantesca esperienza biologica e sociale’ (OC I, 217). In the aforementioned school edition of SQ, he explains the historical shift in Nazi policies that transformed concentration camps into ‘gigantesche macchine di morte’ (OC I, 292). In a 1955 article celebrating the tenth anniversary of Italy’s liberation from Fascism, he describes how the Nazis ‘[h]anno lavorato con tenacia a creare la loro gigantesca macchina generatrice di morte e di corruzione’ (OC II, 1293). In a 1968 preface to a book on Auschwitz, he argues that the vital question remains ‘per quali ragioni e cause, prossime o lontane, abbia potuto nascere in questo civile continente una gigantesca fabbrica di morte’ (OC II, 1357). In the 1975 article ‘Così fu Auschwitz’, he describes what he calls the Nazis’ ‘costituzione di un gigantesco esercito di schiavi, non retribuiti e costretti a lavorare fino alla morte’ (OC II, 1374). In a 1979 response to the broadcast of the TV mini series Holocaust, he notes how the public seemed to focus on the question of why the genocide of the European Jews had occurred, ‘e questo è un perché gigantesco, ed antico quanto il genere umano: è il perché del male nel mondo’ (OC II, 1456). The accretion of these examples is by no means definitive; the ‘qualcosa di gigantesco’ that Levi discovered in discussing Dante with Jean may well differ from the ‘perché gigantesco’, the ‘perché del male nel mondo’, on which he deliberated decades later. Nevertheless, there would appear to be a pattern.

It is a pattern, moreover, that obtains well beyond Levi’s own work. To cite just one relevant example, the Italian anti-Fascist expatriate Giuseppe Antonio Borgese entitled his 1937 study of the totalitarian movement in Italy Goliath: The March of Fascism. And in that text Borgese lay the ultimate blame for the enormity of Fascism not on the Duce—‘it is futile […] to explain Fascism as if it were the creation of a single man, Mussolini’—but rather on another ‘gigantic individuality’ in the Italian national pantheon: Dante, who ‘distorted the soul of his people’, giving rise to ‘Nationalism and Racialism’ (46). Many Fascists, too, claimed Dante as the founder of their movement (Albertini 2013). Not for nothing did ‘Giovinezza’, the Fascist anthem, boast that ‘la vision dell’Alighieri, | oggi brilla in tutti i cuor’ (Pugliese 2001, 55). A 1927 study of Dante Alighieri e Benito Mussolini argued that the Duce’s Italy was closer than ever to Dante’s ideal (7-8). The Fascist Party’s own 1940 Dizionario di Politica made the same claim (735). ‘Che Dante sia Fascista lo dimostrano tutte le sue opere’, insisted one of the regime’s intellectuals; ‘solo oggi possiamo riconoscere in Dante il profeta del nostro destino’, maintained another (Scorrano 2001, 92-93, 198).

It is perhaps tempting to hear the echo of such sentiments in Levi’s epiphany that in Dante’s ‘Canto di Ulisse’ is to be found ‘il perché del nostro destino’. After all, Levi had learned his Dante in an Italian school system that had been turned to Fascism’s totalitarian aims. If he is indeed suggesting to Jean that abuses of Dante - the appropriation of tradition to support the arrogation of power in the present; the fraudulent claims to a divine warrant for violence - bear responsibility for the Häftlinge’s tragic fate, he has good reason.

Yet those who interpret Levi’s Dante lesson in a more liberatory manner have good reason as well. If the inhuman contrapasso of eternal damnation ‘come altrui piacque’ is to be found in Dante, so, too, is Ulysses’ call for an innate and inviolable human dignity: ‘considerate la vostra semenza’. Borgese in Goliath recognised this duality (12). So, too, I suspect, does Primo Levi in ‘Il canto di Ulisse’. Perhaps this is the ‘gigantic’ discovery that Levi has made in his meditation on Dante’s Inferno: that in our cultural inheritance are to be found both the roots of Fascism and the seeds of resistance.

CLL

los ingresos proyectados deben ser iguales o superiores a los gastos previstos

Diferencias entre presupuesto y perspectivas financieras:

Enfoque temporal: El presupuesto se centra en un período específico, generalmente anual, mientras que las perspectivas financieras abarcan un horizonte temporal más amplio, que puede ser de varios años.

Detalle y especificidad: El presupuesto es más detallado y específico, ya que se basa en cifras concretas y desglosadas por categorías presupuestarias, como ventas, gastos operativos, salarios, etc. En cambio, las perspectivas financieras suelen ser más generales y se basan en supuestos y estimaciones.

Propósito: El presupuesto se utiliza principalmente para la planificación y el control financiero a corto plazo, mientras que las perspectivas financieras se utilizan para analizar el rendimiento financiero futuro y evaluar la viabilidad de las estrategias a largo plazo.

Naturaleza flexible: El presupuesto es más rígido y se actualiza anualmente, mientras que las perspectivas financieras son más flexibles y se pueden ajustar y modificar a medida que cambian las circunstancias o se obtiene nueva información.

Audiencia y uso: El presupuesto es una herramienta interna utilizada por la gerencia y los responsables de la toma de decisiones dentro de la organización. Las perspectivas financieras, por otro lado, se utilizan tanto interna como externamente, ya que pueden ser presentadas a inversionistas, accionistas, instituciones financieras y otros interesados en la empresa.

Facilidad de depósito: La facilidad de depósito permite a los bancos comerciales depositar fondos en el banco central y recibir a cambio un rendimiento de interés. Esta facilidad se utiliza cuando los bancos comerciales tienen exceso de liquidez y desean guardar sus fondos de forma segura y obtener un rendimiento sobre ellos. El tipo de interés ofrecido en la facilidad de depósito suele ser menor que la tasa de referencia establecida por el banco central.

Facilidad de préstamo (también conocida como facilidad de crédito o facilidad marginal de préstamo): La facilidad de préstamo permite a los bancos comerciales obtener préstamos a corto plazo del banco central en caso de que necesiten liquidez adicional. Los bancos comerciales pueden acudir a esta facilidad cuando experimentan una escasez temporal de fondos para cubrir sus obligaciones. El tipo de interés aplicado en la facilidad de préstamo suele ser mayor que la tasa de referencia del banco central.

Las standing facilities ofrecen una forma conveniente para que los bancos comerciales gestionen sus necesidades de liquidez de manera rápida y flexible. Al utilizar estas facilidades, los bancos pueden ajustar su posición de liquidez y cumplir con los requisitos regulatorios y las necesidades de sus clientes.

El mecanismo de crédito en torno al FECOM se refiere al Fondo Europeo de Cooperación Monetaria (FECOM), que fue establecido como parte del Sistema Monetario Europeo (SME) en la década de 1970. El FECOM fue creado para brindar asistencia financiera a los países miembros de la Comunidad Económica Europea (CEE) que enfrentaban dificultades en sus balanzas de pagos o en la estabilidad de sus monedas.

El DUA es como una especie de pasaporte para los productos que entran o salen ENTRE UN PAIS MIEMBRO Y TERCEROS. Cuando importas o exportas algo, necesitas llenar este documento para informar a las autoridades aduaneras sobre los detalles del producto, como su origen, su valor y su cantidad. Es como un formulario que debes completar para que todo esté legal y en orden.

es como un gran diccionario que se utiliza en la Unión Aduanera Común de Europa para clasificar los diferentes productos que se importan y exportan. La NC es muy importante porque ayuda a tener un sistema de comercio justo y claro. Cuando importas o exportas un producto, necesitas saber en qué categoría de la NC se encuentra para cumplir con todas las reglas y requisitos. También es útil para las estadísticas comerciales, ya que se puede seguir la cantidad y el valor de los diferentes productos que se comercian entre los países.

Los prélèvements son impuestos o tasas adicionales que se aplican a ciertos productos importados en algunos países miembros de la UE. Estas tasas se utilizan para financiar programas específicos o para abordar necesidades particulares en esos países

Estándares de calidad: Cada país puede tener reglas diferentes sobre la calidad y seguridad de los productos. Si un juguete no cumple con los estándares de calidad del país al que quieres importarlo, no podrás venderlo allí. Esto asegura que los productos sean seguros para los consumidores.

Licencias y permisos: Algunos productos pueden requerir licencias especiales o permisos del gobierno para ser importados o exportados. Por ejemplo, si quieres exportar alimentos, es posible que necesites obtener un permiso sanitario.

Cuotas de importación: Algunos países limitan la cantidad de productos que se pueden importar de ciertos países o en general. Esto puede hacer que sea más difícil y costoso importar grandes cantidades de un producto específico.

Embalaje y etiquetado: Cada país puede tener requisitos específicos sobre cómo deben ser empaquetados y etiquetados los productos. Si no sigues estas reglas, es posible que tus productos no puedan entrar al país de destino.

Barreras técnicas: Algunos productos requieren pruebas y certificaciones especiales para demostrar que cumplen con ciertos estándares técnicos. Por ejemplo, los productos electrónicos pueden necesitar cumplir con ciertos requisitos de seguridad eléctrica antes de poder ser vendidos en otro país.

Un acuerdo restrictivo de la competencia es un acuerdo o práctica entre empresas que tiene como objetivo restringir, distorsionar o limitar la competencia en un mercado determinado.

Acuerdos de exclusividad por ejemplo: Cuando una empresa acuerda vender o distribuir exclusivamente los productos de otra empresa, limitando así la disponibilidad de productos de la competencia en el mercado.

Cuando las empresas se ponen de acuerdo para dividir un mercado o territorio entre sí, evitando la competencia directa

Cuando dos o más empresas acuerdan establecer precios mínimos o máximos para sus productos o servicios, lo cual limita la competencia en términos de precios y puede resultar en precios más altos para los consumidores.

En la práctica, el Principio de Reconocimiento Mutuo implica que un producto o servicio legalmente comercializado en un Estado miembro pueda ser comercializado en los demás Estados miembros sin tener que cumplir con nuevos procedimientos de autorización o requisitos adicionales

Los actos delegados permiten a la Comisión Europea adaptar la legislación a los desarrollos técnicos o científicos, asegurando así su actualización y eficacia continua, determinan los pequeños detalles. Sin embargo, estos actos están sujetos a un control de supervisión por parte del Parlamento Europeo y el Consejo.

para garantizar la correcta aplicación y ejecución de la legislación de la UE. Son como los detalles de como tiene que aplicarse en la pratica una directiva o regla, como el tamaño de una etiqueta, el tipo de letra...

La variación del tipo de cambio puede ser necesaria para hacer frente a ajustes en la economía debido a choques simétricos o asimétricos.

Se plantean dos posibilidades de ajuste en una unión monetaria sin la opción de devaluar el tipo de cambio. La primera es a través de la flexibilidad de salarios y precios, donde una disminución del desempleo en un país mejora la competitividad de sus productos. La segunda es a través de la movilidad del trabajo, donde la migración de mano de obra desempleada hacia otro país con exceso de demanda de trabajo puede solucionar el desequilibrio.

Se discute la posibilidad de choques asimétricos en una unión monetaria y se menciona que pueden ocurrir debido a factores específicos de un país o a diferencias en las economías de los diferentes países. La integración en el mercado de productos, ya sea interindustrial o intraindustrial, también puede influir en la probabilidad de que los choques sean asimétricos.

Se menciona que la eliminación de barreras comerciales en un mercado único puede aumentar la integración intraindustrial y reducir la probabilidad de choques externos asimétricos en las economías de la unión monetaria. Sin embargo, existen críticas basadas en los efectos de aglomeración, que podrían llevar a una mayor especialización regional y aumentar la probabilidad de choques asimétricos.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary

Ge et al. defined the role of Gli1 in M1 macrophage activation and osteoclast differentiation in physiological conditions and inflammatory arthritis. The authors found that Gli1 expression is elevated in human RA synovial tissue relative to that in healthy donor controls. Moreover, the authors showed that the administration of GANT58, a Gli1 inhibitor, ameliorates inflammation and bone erosion in CIA mice. Gli1 expression is suppressed by LPS/IFN-____γ stimulation in Raw264.7 cells while being induced by RANKL stimulation in Raw264.7 cells. However, GANT58 suppressed LPS/IFN-____ɣ -induced expression of inflammatory cytokines and iNOS and osteoclastogenesis. The authors also identified DNMT1 and DNMT3a as downstream effectors of Gli1. Transcriptomic analysis of GANT58 treated Raw264.7 cells identified diminished protein expression of DNMT1 and DNMT3a by GANT58. Gli1 also directly interacts with DNMT1. Intriguingly, DNMT1 overexpression restores the effect of GANT58 on LPS/IFN-____ɣ-mediated activation, while DNMT3a overexpression reverses the effect of GANT58 on RANKL-induced osteoclastogenesis. Since this study defines the role of Gli1 in the function and differentiation of myeloid cells, this is interesting. In addition, GANT58 nearly completely protects mice from arthritis, suggesting a therapeutic potential of Gli1 targeting in RA. However, the details of experiments are not clearly described, and the authors present the mixed data from Raw264.7 cells and BMMs without any explanations.

Reply: Many thanks for your recognition and constructive comments on our research. In this study, used mouse macrophage-like cell line RAW264.7 and primary bone marrow-derived macrophages (BMMs). The RAW264.7 is the most commonly used mouse macrophage cell line in medical research, and it is one of the most commonly used in vitro models for osteoclasts and inflammation research. In addition, compared with cell lines, primary cells have the characteristics of unchanged genetic material and biological characteristics closer to cell physiology in vivo. Therefore, in addition to cell lines, we also extracted primary macrophages from bone marrow for experiments to improve the reliability of this study. According to your comments, we have revised the manuscript, and our point-by-point responses are shown as follows.

Major comments

Comment 1. Figs 1h and i. The author should show the histological score.

Reply: Thanks for the constructive comment. According to your suggestion, we have scored the results of H&E staining histologically and added quantitative results.

Comment 2. Pharmacological inhibitors often show non-specific effects. To complement their findings showing the effect of GANT58 on M1 macrophage activation and osteoclastogenesis, the authors should utilize Gli1-deficient cells that can be obtained by siRNAs-mediated knock down or Gli1 deletion.

Reply: Thanks for the professional and constructive comment. To make the results more reliable, we have synthesized siRNA and supplemented the related experiments to verify the role of GLI1 in M1 macrophage activation and osteoclastogenesis, which showed the same trend as GANT58 intervention. In the revised manuscript, the relevant results were shown in the Response to Reviewer File.

Comment 3. Figure 4d: The authors should measure DNMT1 and DAMT3a RNA expression in LPS/IFN-____ɣ- treated (Fig 2c and d) or RANKL treated Raw264.7 cells.

Reply: Thanks for your constructive comment. According to the suggestion, we have added the RNA expression of DNMT1 and DAMT3a to the revised Figure 4. At the same time, the corresponding contents are also described in the Results part.

__ detailed information of RNA-seq including how many genes are regulated by GANT58 and what is their cutoff (fold induction and FDR). The authors should deposit their RNA seq data in the public databases repository such as GEO.__

Reply: Thanks for the professional and constructive comment. In the revised manuscript, we have made a more detailed analysis of the sequencing results and the detail information of RNA-seq have been added in the supplementary information.

Revised in the manuscript:

2.4. GLI1 regulates the expression of DNMTs in distinct ways during the different fates of macrophages

As a nuclear transcription factor, GLI1 exerts an active effect through nuclear entry. In order to explore the potential downstream regulation mechanism of GLI1, RNA sequencing (RNA-seq) on the macrophages before and after GLI1 intervention was performed then to observe gene expression changes. The seq data showed that more genes were down-regulated (143) than up-regulated (74) in GANT58 treated cells (Fig. S7a, b). Among these differentially altered genes, we revealed through Gene Ontology (GO) analysis that GANT58's intervention in GLI1 affected multiple biological processes including macrophage chemotaxis and macrophage cytokine production (Fig. 4a). What’s more, the results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the pathway team enrichment was then performed and we showed the TOP30 enriched pathway. In these pathways, we classified them into cellular processes (red), human diseases (blue) and organismal systems (green) respectively. It showed that these down-regulated genes were involved in the development of human diseases such as rheumatoid arthritis, as well as organismal systems such as osteoclast differentiation (Fig. S8c; ____Fig. 4b). These evidences confirmed our previous results. Specifically, GANT58 reduced some of the osteoclast and inflammation-related genes in the cell resting state.

Comment 5. Figure 5c. The authors should add non-stimulating condition as a control.

__Reply: __Thanks for your constructive comment. We have re-conducted the experiment and added the control group.

Comment 6. Figure 6C: DNMT3a deficiency regulates limited number of genes such as IRF8. The authors should measure IRF8 RNA or protein expression in RANKL-treated cells.

Reply: Thanks for your constructive comment. It is reported that DNMT3a can affect the activity of IRF8 and regulate the formation of osteoclasts. Thus, according to your suggestion, we have added IRF8 gene expression detection in the revised manuscript. As shown below, the gene expression of Irf8 was decreased after being treated by RANKL. However, the expression of Irf8 was reversed by Dnmt3a knock down.

Comment 7. Although the effects of Gli1 on bone metabolism in the literature are inconclusive, Gli1 is expressed on other cell types in bone. Gli1 haplodeficiency in mice decreased bone mass with reduced bone formation and enhanced bone resorption compared to control mice (PMID:25313900). Gli1 is also used as a marker for osteogenic progenitors which are precursors of chondrocytes and osteoblasts (PMID: 29230039). Thus, the beneficial effect of GANT58 on inflammation and bone erosion in CIA mice may result from the effects of GANT58 on multiple cell types other than F4/80+ cells. The authors should include these references in the discussion on pg.9 and expand their discussion.

__Reply: __Thank you for your constructive comments. Indeed. there have been some divergent conclusions about the function of hedgehog and GLI1 in bone metabolism, which suggests that GLI1 may have multiple roles. According to your suggestion, we have expanded the relevant discussion and added related references in the Discussion part.

Discussion:

… … Although we have demonstrated that the inhibition of GLI1 by GANT58 can reduce the inflammatory response and inhibit osteoclast formation and that this mechanism is achieved through the downregulation of DNMTs, these findings also raise new questions. In the previous research report, Gli1 haplodeficiency in mice decreased bone mass with reduced bone formation compared to control mice, which was due to the osteoblasts with weakened function [44]. In this process, the osteogenic differentiation of mesenchymal stem cells also affected the function of osteoclasts. In addition, GLI1 is also used as a marker for osteogenic progenitors which are precursors of chondrocytes and osteoblasts [45]. These studies suggest that the regulation of GLI1 on bone metabolism is complex, and the therapeutic effect of GANT58 on RA may be more than just affecting the inflammatory reaction mediated by macrophages and the bone destruction mediated by osteoclasts. In addition to macrophages and osteoclasts, the functions of synovial fibroblasts and osteoblasts play essential roles in the RA microenvironment. These cells are also closely linked to each other. Synovial fibroblasts OPG and RANKL secreted by osteoblasts are important factors that regulate osteoclasts. Therefore, in a follow-up study, we will extend the study of GLI1 to its regulatory mechanism in osteoblasts.

Reference:

[44] Y. Kitaura, H. Hojo, Y. Komiyama, T. Takato, U.I. Chung, S. Ohba, Gli1 haploinsufficiency leads to decreased bone mass with an uncoupling of bone metabolism in adult mice, PLoS One 9(10) (2014) e109597.

[45] Y. Shi, G. He, W.C. Lee, J.A. McKenzie, M.J. Silva, F. Long, Gli1 identifies osteogenic progenitors for bone formation and fracture repair, Nat Commun 8(1) (2017) 2043.

Minor comments

Comment 1. CIA model: The experiment design of CIA model is not clearly described. The author should specify the time point of GANT58 injection.

__Reply: __Thank you for your comment and we are sorry for the confusion caused by vague method descriptions about animal experiments. We have added the specific design and method description of related experiments in the revised manuscript.

Revised in the manuscript:

Materials and Methods:

… … An emulsion of bovine type II collagen (Chondrex, Redmond, WA, USA) and an equal amount (1:1, v/v) of complete Freund’s adjuvant (Chondrex) was prepared to establish the CIA mouse model. First, 0.1 ml of the emulsion was injected intradermally into the base of the tail on day 0. On day 21, 0.1 mg of bovine type II collagen mixed with incomplete Freund’s adjuvant (Chondrex) was injected. From the 21st day, mice began to receive injection intervention treatment. For vehicle group, mice were injected with the same volume of placebo daily. For treatment groups, mice were injected with GANT58 or 5-AzaC solution daily. All interventions began the day after the second injection of bovine type II collagen. Arthritis score was given every three days from the second immunization. On day 49, all mice were sacrificed (in accordance with the guidelines of the Animal Welfare and Ethics Committee of the Soochow University) for the collection of specimens. … …

Comment 2. Joint inflammation of RA can be caused by many different cells. Abstract needs to be revised.

Reply: Thanks for your constructive comment. According to the suggestion, we have revised relevant descriptions in the abstract.

Abstract:

Rheumatoid arthritis (RA) is characterized by joint synovitis and bone destruction, the etiology of which remains to be explored. Many types of cells are involved in the progress of RA joint inflammation, among which the overactivation of M1 macrophages and osteoclasts has been thought an essential cause of joint inflammation and bone destruction. Glioma-associated oncogene homolog 1 (GLI1) has been revealed to be closely linked to bone metabolism. In this study, GLI1-expression in synovial tissue of RA patients showed to be positively correlated with RA-related scores and was highly expressed in collagen-induced arthritis (CIA) mouse articular macrophage-like cells. The decreased expression and inhibition of nuclear transfer of GLI1 downregulated macrophage M1 polarization and osteoclast activation, the effect of which was achieved by modulation of DNA methyltransferases (DNMTs) via transcriptional regulation and protein interaction ways. By pharmacological inhibition of GLI1, the proportion of proinflammatory macrophages and the number of osteoclasts were significantly reduced, and the joint inflammatory response and bone destruction in CIA mice were alleviated. This study clarified the mechanism of GLI1 in macrophage phenotypic changes and activation of osteoclasts, suggesting potential applications of GLI1 inhibitor in the clinical treatment of RA.

Comment 3. Figure 4g, h: are these experiments done in the resting states?

Reply: Thank you for your comment. This part of the experiments was carried out during the induction of M1 macrophage or induction of osteoclast. In this work, we found that GANT58 can inhibit GLI1 and at the same time reduce the gene expression of DNMT3a but not DNMT1 in the resting state. However, during M1 macrophage and osteoclast induction, GANT58 seemed to be able to inhibit both DNMT1 and DNMT3a protein expression. In view of the discovery that the expression of DNMT1 increased during the polarization of M1 macrophages, while the expression of DNMT3a increased during the activation of osteoclasts, we performed the binding experiment of GLI1 with DNMT1 in the process of LPS/IFN-γ induction, while the binding experiment with DNMT3a in the process of RANKL induction. We have added a detailed description to the revised manuscript.

Reviewer #1 (Significance (Required)):

Strengths: Hedgehog (hh) signaling has been implicated in the differentiation of osteogenic progenitors. Gli1+ mesenchymal progenitors are responsible for both normal bone formation and fracture repair. This study defines a new role of Gli1 in the function and differentiation of myeloid cells. In addition, GANT58 nearly completely protects mice from arthritis, suggesting a therapeutic potential of Gli1 targeting in RA.

Reply: Thank the reviewer for your recognition of our research work.

Limitations: This study mainly uses a pharmacological inhibitor to study the mechanism underlying Gli1's action. In addition, the details of experiments are not clearly described, and the authors present the mixed data from Raw264.7 cells and BMMs without any explanations. Advance: This study provides conceptual advancement for hh signaling research by demonstrating the function of Gli1 in myeloid cells.

Reply: Thank the reviewer for your constructive comments and help us to further improve the manuscript.

Audience: Basic research

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary:

The paper by Ge et al seeks to identify a role for GLI1 in rheumatoid arthritis, as GLI1 is upregulated in the synovium of patients with rheumatoid arthritis. Inhibition of GLI1 by the GANT58 limited inflammation and destructive bone loss in a murine model of arthritis (Collagen Induced Arthritis). Inhibition of GLI1 increased expression of pro-inflammatory cytokines and M1 macrophage differentiation. Inhibition of GLI1 also blocked osteoclast formation. As has been shown in other settings, the function of GLI1 in M1 and osteoclast differentiation was linked to regulation by DNMTs.

Major comments:

Comment 1. There are several main problems with the text. Overall, the authors show an intriguing set of data implicating the use of GANT58 as a means to limit rheumatoid arthritis inflammation and bone destruction. The authors directly link the functions of GANT58 with loss of GLI1 activity by showing that GLI1 protein is reduced or translation to the nucleus blocked. It would be compelling if the authors would leverage a genetic model (either GLI1 knockout, or a CRISPR/siRNA approach) to see if it recapitulates key findings in vitro and in vivo. These data could further their claims that their findings are in fact directly due to GLI1.

Reply: Thanks for the professional and constructive comment. To make the results more reliable, we have synthesized siRNA and supplemented the related experiments to verify the role of GLI1 in M1 macrophage activation and osteoclastogenesis. Related experiments have been updated in the revised manuscript, which are shown in the Response to Reviewer File.

Comment 2. Overall, the paper lacks methodologic clarity that limits thorough interpretation of the data. Multiple experiments are missing from the Materials and Methods, including descriptions of the definition of trabecular bone and its analysis in micro-CT, the means by which cytoplasmic and nuclear fractions were generated, and the timing and dosing of GANT58 in vitro studies. In addition, key details regarding the reagents include the sources of primary antibodies used in the western blots and immunoprecipitation studies. Important methodologies are not well explained, which include the treatment of the Sham animals (presumably healthy) are not explained, that is, whether they receive injections of vehicle or are truly naïve. Finally, there is no statistical methodology, minimal explanation of the RNA-sequencing analyses, and no statement about how the RNA-sequencing data will be made available. This lack of detail makes a thorough assessment of the quality and interpretations of the data challenging and replication of the results impossible.

Reply: Thanks for your careful reading and constructive comments. We are sorry for the lack of some detailed methodological descriptions in the manuscript. In order to better explain how our experiment is carried out and improve the repeatability of the experiment, we have comprehensively improved the description of the experimental method in the revised manuscript.

Materials and Methods:

4.1. Experimental animals and human synovial tissue. Male DBA mice aged 6-8 weeks and weighing 15-20 g were randomly selected and fed in a specific pathogen-free (SPF) environment at a room temperature of 25℃, a relative humidity of 60%, and 12 hours of alternating light. All animal experiments were approved by the Animal Ethics Committee of the Soochow University (201910A354). The animals were divided randomly into groups (6 per group): sham group (healthy mice not received any treatment), vehicle control group (CIA model mice treated with solvent), and GANT58 (GLI1 specific inhibitor; MedChemExpress, New Jersey, USA) group (mice treated with 20 mg/kg GANT58) or 5-AzaC (DNMTs specific inhibitor; MedChemExpress) group (mice treated with 2 mg/kg 5-AzaC). An emulsion of bovine type II collagen (Chondrex, Redmond, WA, USA) and an equal amount (1:1, v/v) of complete Freund’s adjuvant (Chondrex) was prepared to establish the CIA mouse model. First, 0.1 ml of the emulsion was injected intradermally into the base of the tail on day 0. On day 21, 0.1 mg of bovine type II collagen mixed with incomplete Freund’s adjuvant (Chondrex) was injected. For vehicle group, mice were injected with the same volume of placebo daily. For treatment groups, mice were injected with GANT58 or 5-AzaC solution daily. All interventions began the day after the second injection of bovine type II collagen. Arthritis score was given every three days from the second immunization. On day 49, all mice were sacrificed (in accordance with the guidelines of the Animal Welfare and Ethics Committee of the Soochow University) for the collection of specimens. … …

4.3. Micro-CT analysis. The fixed bone samples of mice were collected. The joint samples were placed in a SkyScan 1174 Micro-CT scanning warehouse (Belgium). The parameters were set as follows: voltage 50 kV, current 800 μA, scanning range 2 cm × 2 cm, and scanning layer thickness 8 μm. The scan data were then entered into computer to conduct three-dimensional reconstruction with NRecon software (Bruker, Germany), and the bone tissue parameters were analysed with CTAn software (Bruker, Germany) after data conversion. During this procedure, we performed an analysis of bone parameters including BMD (Bone Mineral Density), BV/TV (Percentage Trabecular Area), Tb.N (Trabecular Number) and Tb.Sp (Trabecular Separation) by selecting the small joint of paws as the region of interest (ROI) in CTAn software. The three-dimensional reconstruction images were exhibited by Mimics Research software (Version 21.0; Materialise, Belgium).

4.11. Western blotting. Cells were seeded in 6-well plates at a density of 1 × 106/well with stimulation with RANKL (50 ng/ml) or LPS (100 ng/ml) + IFN-γ (20 ng/ml). First, cells were collected to extract total protein, and the BCA (Beyotime) method was used to adjust the protein concentration. Total protein was mixed with 5× loading buffer (Beyotime) and boiled at 95 °C for 10 minutes. For cytoplasmic/nucleus isolation, cells were collected and protein was extracted according to the instructions using the nuclear protein and cytoplasmic protein extraction kit (Beyotime). The proteins were separated by SDS polyacrylamide gel electrophoresis (SDS–PAGE; EpiZyme, Shanghai, China) based on their different molecular weights. Electrophoresis was performed using Bio–Rad (California, USA) equipment at 180 V for 40 minutes. Then, the proteins were transferred to a nitrocellulose membrane at 350 mA for 70 minutes using membrane transfer equipment (Bio–Rad). The membrane was removed and placed into western blot blocking buffer for 1 hour at room temperature. The diluted primary antibodies (GLI1, Abclonal, A14675; β-actin, Beyotime, AF5003; Lamin-B1, Abcam, ab16048; NFATc1, Abclonal, A1539; CTSK, Abclonal, A5871; MMP9, Abclonal, A11147; DNMT1, Abclonal, A16729; DNMT3a, Cell Signaling Technology, D23G1; GAPDH, Abclonal, A19056) were placed on the membrane and incubated at 4 ℃ for 12 hours, and then the corresponding secondary antibody was added and incubated for 1 hour at room temperature. Finally, a chemiluminescence detection system (Bio–Rad) was used to observe the results.

4.12. High-throughput sequencing (RNA-seq). To further screen for differential genes, we first subjected RAW264.7 cells to a 24-hour adaptive culture, followed by the addition of GANT58 at a final concentration of 10 μM to the GANT58 intervention group and cultured for a total of 24 h. After the cell treatment was completed, cells of the control group and GANT58 treated group were collected respectively, and RNA-seq detection and analysis were entrusted to a professional biological company (Azenta Life Sciences, Suzhou, China). Briefly, for differential expression gene analysis, the differential expression conditions were set as fold change (FC) > 1.5 and false discovery rate (FDR) 4.14. Statistical analysis. All data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with an unpaired two-tailed Student’s t test for single comparisons with GraphPad Prism 8 (GraphPad Software, CA, USA). One-way analysis of variance (ANOVA) was used to compare data from more than two groups. p values less than 0.05 were considered statistically significant.

The specific statistical methods are marked in Figure legends as well.

Data Availability: The authors declare that all data supporting the findings of this study are available within this paper and its Supplementary Information and raw data are available on request from the corresponding author.

Comment 3. The authors should expand their introduction and Discussion to include a description of the history of other GLI inhibitors (such as GANT61) in rheumatoid arthritis. Further, the authors failed to cite current studies showing that GLI1 is upregulated in RA patients (DOI: 10.1007/s10753-015-0273-3 amongst others).

Reply: Many thanks to your thoughtful reading and constructive comment. According to your suggestion, we have added some revisions, including the description of GLI1 inhibitors, in the introduction and discussion sections. At the same time, we have also added descriptions and citations of GLI1 and RA-related research in corresponding positions.

Introduction:

… … To date, three mammalian GLI proteins have been identified, among which GLI1 usually acts as a transcriptional activator. On the basis of these studies, small molecular compounds such as GANT58 (selective inhibitor of GLI1) and GANT61 (inhibitor of GLI1 and GLI2) are often used as pharmacological interventions of GLI1, so as to achieve the purpose of inhibiting GLI1 activity and regulating the molecular biological process [13, 14]. Many of the physiopathological processes involved with GLIs are complex and worth discussing. Relevant studies have shown that GLI1-activated transcription promotes the development of inflammatory diseases such as gastritis, and antagonizing GLI1 transcription can alleviate the inflammatory degradation of articular cartilage [15, 16]. … …

Discussion:

… … In previous studies, GLI1 signal transduction and other pathways, including the NF-κB signaling pathway, were usually studied in tumor-associated diseases and are considered a response network that promotes cancer development [21, 22]. Qin. et al. found that the content of SHH in RA patients serum increased significantly by comparing with healthy patients [23]. At the same time, our study also showed that GLI1 was more expressed in the joint tissue of RA patients. These results suggest that HH-GLI signaling pathway may be involved in the regulation of the pathological process of RA. However, the research results of the hedgehog pathway in bone metabolism are complex. … …

Reference:

[13] X. Chen, C. Shi, H. Cao, L. Chen, J. Hou, Z. Xiang, K. Hu, X. Han, The hedgehog and Wnt/beta-catenin system machinery mediate myofibroblast differentiation of LR-MSCs in pulmonary fibrogenesis, Cell Death Dis 9(6) (2018) 639.

[14] R.K. Schneider, A. Mullally, A. Dugourd, F. Peisker, R. Hoogenboezem, P.M.H. Van Strien, E.M. Bindels, D. Heckl, G. Busche, D. Fleck, G. Muller-Newen, J. Wongboonsin, M. Ventura Ferreira, V.G. Puelles, J. Saez-Rodriguez, B.L. Ebert, B.D. Humphreys, R. Kramann, Gli1(+) Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target, Cell Stem Cell 23(2) (2018) 308-309.

[23] S. Qin, D. Sun, H. Li, X. Li, W. Pan, C. Yan, R. Tang, X. Liu, The Effect of SHH-Gli Signaling Pathway on the Synovial Fibroblast Proliferation in Rheumatoid Arthritis, Inflammation 39(2) (2016) 503-12.

Comment 4. The antibody for GLI1 seems poor and inconsistent. Knockdown studies to show its specificity, and an example of the whole membrane stained for GLI1 would provide important validation of the reagent.

Reply: Thanks for your comment and we are sorry for showing the western blot results with poor quality. In the revised manuscript, we used the newly purchased antibody (Abclonal, Catalog: A14675) and rearranged the groupings for better comparison of protein expression and replaced the results with clearer blot images. Original images of all western blot results can be uploaded subsequently.

Comment 5. Regarding Figure S1:

The studies of RA patients are underpowered. With only three RA patients and three healthy synovial the distribution of DAS28 scores is clustered at healthy and active disease, and the correlation study is unconvincing.

Reply: Thanks for your constructive comment. We are sorry that the studies of RA patients might not be convincing enough due to the small sample size. In order to avoid controversial conclusions, we left out the results of correlation analysis between GLI1 expression and DAS28. In the follow-up study, we will collect additional clinical pathology data for statistical analysis and quantified the expression of GLI1 in healthy control patients and RA patients.

Comment 6. Regarding Figure 1 f-g and Figure 4j-k:

However, the information on inflammatory bone loss are incomplete. The methodology for the assessment of BMD and trabecular bone parameters in the hind paw is not explained. The 3D reconstructions are of the whole bone hind paw, but the anatomical region where trabecular bone is assayed not defined. It would be convincing if the authors added erosion scores in the hind paws or knees to show that the erosion in the synovium, which contributes to inflammatory arthritis, mirrors what occurs in the trabeculae.

Reply: Thanks for your constructive comment. We are sorry for incomplete description on in vivo experiments, including the micro-CT analysis and histological analysis. In the revised manuscript, we further supplemented and improved the relevant methods. The Inflammatory cell infiltration score and bone erosion score were also added according to your suggestion.

Materials and Methods:

4.3. Micro-CT analysis. The fixed bone samples of mice were collected. The joint samples were placed in a SkyScan 1174 Micro-CT scanning warehouse (Belgium). The parameters were set as follows: voltage 50 kV, current 800 μA, scanning range 2 cm × 2 cm, and scanning layer thickness 8 μm. The scan data were then entered into computer to conduct three-dimensional reconstruction with NRecon software (Bruker, Germany), and the bone tissue parameters were analysed with CTAn software (Bruker, Germany) after data conversion. During this procedure, we performed an analysis of bone parameters including BMD (Bone Mineral Density), BV/TV (Percentage Trabecular Area), Tb.N (Trabecular Number) and Tb.Sp (Trabecular Separation) by selecting the small joint of paws as the region of interest (ROI, bone tissue from ankle joint to toe) in CTAn software. The three-dimensional reconstruction images were exhibited by Mimics Research software (Version 21.0; Materialise, Belgium).

Comment 7. Regarding Figure 2:

-The methods and text do not state the dose of GANT58 used in these assays. Nor do they specify the timing of the GANT58 application in relationship to LPS and IFNg stimulation.

Reply: Thanks for your thoughtful reading and constructive comment. We apologize for not expressing the detailed dose and intervention time of GANT58 in some experiments in detail. In the revised manuscript, we have added drug dose and intervention time cutoff points in the parts of Methods, Results, and Figure Legends.

-The authors conclude that GLI1 limits the differentiation of M1 macrophages and also directly blocks the production of pro-inflammatory cytokines. The data are difficult to parse in that the directionality is not clear. If GLI1 promotes M1 macrophages, there would be less proinflammatory cytokines due to the reduction of their proliferation. To evaluate the role of GLI1 in regulating the cytokines, additional studies showing a transcriptional regulation of these cytokines is warranted.

Reply: Thank you for your professional and constructive comment. We totally agree with you that the release of inflammatory cytokines is affected not only by gene expression but also by the number of cells that proliferate. Therefore, to exclude this interference, we further examined transcriptional expression of cytokines responsible for cellular inflammation under the same conditions. The results shown in the Response to Reviewer File confirmed the inhibition of GANT58 on the expression of pro-inflammatory cytokine mRNAs, which further supported our conclusion.

-To show that the fractionation of the cytoplasm and nuclear compartments was complete, the westerns for GLI1, lamin-B1 and beta actin should be shown in the same blot.

Reply: Thank you for your professional and constructive comment. According to your suggestion, we have rearranged the groupings to show the westerns for GLI1, lamin-B1 and β-actin in the same blot for better comparison in the revised manuscript.

-In Section 2.3 ("the expression of and intranuclear transport..."), the authors state that their previous studies showed GLI was expressed in macrophages (line 80-81). It is unclear whether the authors are referring to studies in this manuscript or a previously published study and a citation is needed.

Reply: Thank you for your careful reading and helpful comment. We are sorry that the description in this part is confusing. In fact, what we want to refer to is the in vivo results described in the first section of the results part. We have changed this description in the revised manuscript.

2.3. The expression and intranuclear transport of GLI1 is involved in osteoclast activation

The over activation of osteoclast is the direct cause of bone destruction in RA. As described of the in vivo experimental results in the first part, we have found that GLI1 is highly expressed in macrophage-like cells in the subchondral bone of the joints, which raised our concerns about GLI1 and osteoclasts. … …

In response to Figure 3:

-The authors show that GANT58 has a potent impact in limiting osteoclast formation. The text states that GANT58 is a pretreatment, but the timing of this is not stated.

Reply: Thanks for your constructive comment. In order to reach the working concentration of drugs at the beginning of some experiments, we usually pretreated cells for 6-8 hours. We have added the specific time in the parts of Materials and Methods or Figure legends.

-It would be interesting to see whether there is a dose-response effect of GANT58.

Reply: Thanks for your comment. According to your comment, we set the concentration of GANT58 to 0, 1, 5 and 10 μM to intervene the induction of M1 macrophages and osteoclasts respectively. As shown in the Response to Reviewer File, with the increase of GANT58 concentration, the mean fluorescence intensity of iNOS in macrophages seems to decrease gradually, but there is no statistical significance when the concentration is below 5 μM. Similarly, when the concentration reached 10 μM, GANT58 significantly inhibited the formation of osteoclasts.

-It is not stated how long the cells are RANKL treated prior to nuclear/cytoplasmic fractionation? (3a, b, c and i).

Reply: Thanks for your constructive comment. For osteoclast induction and intervention, we treated cells for 48 h as cell transcription regulation usually occurs in the early and middle stages of osteoclast differentiation. According to your comment, we have added the description of specific intervention time information in Figure legends and other parts.

-The "Zoom" images in Figure 3j do not have a box to delineate where the higher magnification images are taken from in the top panes. The images appear to be from serial sections. This should be clarified.

Reply: Thanks for your constructive comment. In the revised Figure, we have boxed the area represented by the Zoom images. We can ensure that these images come from different groups of specimen slices. In order to better observe the number of osteoclasts, we chose a larger shooting multiple, which might make the pictures look similar. The revised images are shown in the Figure 3n, o in the Response to Reviewer File.

In Figure 3 and Figure 6e and 6f:

Although the data in BMM showed that there was no impact on cell survival was limited at low concentrations, showing that the differentiating osteoclasts are not more sensitive to apoptosis by GANT58 would be compelling. The large difference in cellularity in the presence of GANT58 provokes this question.

Reply: Thank you for your careful reading and helpful comment. As shown of the CCK8 result, GANT58 had no significant inhibitory effect neither on BMMs nor RAW264.7 cells until the concentration reached 40 μM. In the process of changing the polarization phenotype of macrophages, the cell morphology will also change to some extent. In our research results, the change of cell morphology after GANT58 intervention might be due to the inhibition of M1 macrophages. In order to observe the effect of GANT58 on BMM cell death and apoptosis, we further performed living/dead staining and apoptosis detection by fluorescence after GANT58 intervention. The results showed that GANT58 did not change the level of apoptosis nor increase the number of dead cells at the concentration of 10 μM. However, when the concentration increased to 30μM, the number of apoptotic cells increased. These results suggest that we should pay strict attention to the control of drug concentration in experimental intervention and transformation application. The supplementary results are shown in the Response to Reviewer File.

In Figure 4:

-The IP studies (4g and 4h) lack showing successful pull-down of GLI1 by western blotting as a critical control for the study.

Reply: Thanks for your constructive comment. During the performance of CO-IP experiment, we simultaneously detected the expression of GLI1 to verify the effectiveness of the antibodies used. In the revised Figure 4g and h, we have updated the corresponding results.

Revised Figure 4:

-Details about the steps involved in RNA-sequencing analyses need to be provided.

__Reply: __Thanks for your constructive comment. According to your suggestion, we have provided the steps involved in RNA-sequencing analyses in the Methods.

4.12. High-throughput sequencing (RNA-seq). To further screen for differential genes, we first subjected RAW264.7 cells to a 24-hour adaptive culture, followed by the addition of GANT58 at a final concentration of 10 μM to the GANT58 intervention group and cultured for a total of 24 h. After the cell treatment was completed, cells of the control group and GANT58 treated group were collected respectively, and RNA-seq detection and analysis were entrusted to a professional biological company (Azenta Life Sciences, Suzhou, China). Briefly, for differential expression gene analysis, the differential expression conditions were set as fold change (FC) > 1.5 and false discovery rate (FDR)

-Studies have previously shown a reduction of inflammatory arthritis by 5'-Azac and should be cited.

__Reply: __Thank you for your careful reading and helpful comment. In the discussion part of the revised manuscript, we have cited the related articles, which is shown as below.

Discussion:

… … In addition to normal physiological development, the abnormal expression of DNMTs causes the development of tumors and other diseases [35]. Through the treatment of DNMTs inhibitors, the inflammatory arthritis in mice was significantly relieved, which was consistent with the previous studies [36]. These results suggested that DNMTs might be involved in the inflammatory reaction and bone destruction of RA. Reports have suggested that the absence of DNMT3a inhibits the formation of osteoclasts, which may be due to the methylation of downstream IRF8 by DNMT3a [37]. In our study, we also verified this finding through pharmacological and genetic intervention. … …

Reference:

[36] D.M. Toth, T. Ocsko, A. Balog, A. Markovics, K. Mikecz, L. Kovacs, M. Jolly, A.A. Bukiej, A.D. Ruthberg, A. Vida, J.A. Block, T.T. Glant, T.A. Rauch, Amelioration of Autoimmune Arthritis in Mice Treated With the DNA Methyltransferase Inhibitor 5'-Azacytidine, Arthritis Rheumatol 71(8) (2019) 1265-1275.

-What is the proposed functional consequence for GLI1 binding to DNMT3a? Does GLI1 inhibition lead to hypomethylation of DNA by DNMT?

Reply: Many thanks for your constructive comment. In this study, it is interesting to find that GLI1 can affect the expression of Dnmt3a at the level of gene transcription, and affect the expression of DNMT3a and DNMT1 both in the process of protein expression. Through the CO-IP experiment, we confirmed that GLI1 protein can bind to DNMT1 instead of DNMT3a protein. These results suggested that GLI1 may regulate the expression of DNMT3a and DNMT1 at genetic level and post-translation proteinic level, respectively. Patricia Gonz á lez Rodr í Guez's latest research showed that during autophagy induction, GLI1 is upregulated, phosphorylated, translocated to the nucleus and recruited to the regions closer to the Transcription Start Site (TSS) of the Dnmt3a gene. This may be the direct mechanism of GLI1 regulating the expression of DNMT3a [1]. Theoretically, the expression of DNMTs affects the degree of methylation of related genes [2]. Thus, in the follow-up study, we will further verify the degree of genomic methylation caused by GLI1's regulation of DNMTs, and further explore more possible ways of GLI1's regulation of DNMTs and its potential role in other cell models.

Reference:

[1] P. Gonzalez-Rodriguez, M. Cheray, L. Keane, P. Engskog-Vlachos, B. Joseph, ULK3-dependent activation of GLI1 promotes DNMT3A expression upon autophagy induction, Autophagy (2022) 1-12.

[2] Dura M, Teissandier A, Armand M, Barau J, Lapoujade C, Fouchet P, Bonneville L, Schulz M, Weber M, Baudrin LG, Lameiras S, Bourc'his D. DNMT3A-dependent DNA methylation is required for spermatogonial stem cells to commit to spermatogenesis, Nat Genet 54(4) (2022) 469-480.

Figure 5:

-The groups in 5g are not well defined.

Reply: Thank you for your careful reading and comment. We're sorry that we didn’t clearly show the grouping information. In the revised Figure 5g, we have added the complete information of the groups.

-DNMT1 and DNMT3a reduction by siRNA, CRISPR or knockout would strengthen the inhibitor studies.

Reply: Thanks for your constructive comment. In the revised manuscript, we knocked down the expression of DNMT1 and DNMT3a by siRNA, and supplemented the related experimental results, which are shown in the Response to Reviewer File.

Regarding Figure 5 and 6:

-What is the impact of DNMT1 and DNMT3a overexpression on their own (not in the presence of GANT58)?

Reply: Thanks for your constructive comment. According to your comment, we observed and compared the differences in the polarization of macrophages M1 and the activation of osteoclasts between the DNMTs overexpression group and the control group. The results showed that overexpression of DNMT1 seemed to have no obvious effect on the formation of M1 macrophages. During the osteoclast activation, at day 4 of RANKL induction, the TRAP positive stained osteoclast number seemed to be no significance between WT group and Dnmt3aOE group. However, at day 3, there was more osteoclast in Dnmt3aOE group, which suggested that overexpression of Dnmt3a might accelerate the activation of osteoclasts to some extent. The results are shown in the Response to Reviewer File.

Minor comments:

Comment 1. The authors do not include a description of DNMTs in the introduction.

Reply: Thanks for your constructive comment. According to your suggestion, we have added a description of DNMTs in the Introduction.

Introduction:

DNA methylation is an important epigenetic marker playing an important role in regulating gene expression, maintaining chromatin structure, gene imprinting, X chromosome inactivation and embryo development an important epigenetic modification way to regulate gene expression, which is activated by DNA methyltransferases (DNMTs) [17]. As reported, DNMT1 and DNMT3a are involved in the progress of many physiological disorders, such as immune response and cell differentiation [18, 19]. In this study, … …

Reference:

[17] E. Li, Y. Zhang, DNA methylation in mammals, Cold Spring Harb Perspect Biol 6(5) (2014) a019133.

[18] Y. Fu, X. Zhang, X. Liu, P. Wang, W. Chu, W. Zhao, Y. Wang, G. Zhou, Y. Yu, H. Zhang, The DNMT1-PAS1-PH20 axis drives breast cancer growth and metastasis, Signal Transduct Target Ther 7(1) (2022) 81.

[19] R. Ramabadran, J.H. Wang, J.M. Reyes, A.G. Guzman, S. Gupta, C. Rosas, L. Brunetti, M.C. Gundry, A. Tovy, H. Long, T. Gu, S.M. Cullen, S. Tyagi, D. Rux, J.J. Kim, S.M. Kornblau, M. Kyba, F. Stossi, R.E. Rau, K. Takahashi, T.F. Westbrook, M.A. Goodell, DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells, Nat Cell Biol 25(4) (2023) 528-539.

Comment 2. The descriptions of the groups are often unclear. In Figure 2, the label "GANT58" (blue bars) is presumably for a group that is treated for LPS+IFNg+GANT58 but this is not clarified.

Reply: Thanks for your careful reading and we are sorry for the ambiguous labeling. We have checked the whole manuscript and changed the related labeling information.

Comment 3. The distinction of Figure 3g as multinuclear giant cells (vs TRAP+ OCs in panel 3d) should be explained.

Reply: Thanks for your comment. Osteoclast is defined as a multinucleated giant cell with bone absorption function, which is composed of multiple monocytes/macrophages [1]. As osteoclasts mature, their cytoskeleton will undergo drastic reorganization. Filamentous actin (F-actin) firstly constitutes a podosomes with a highly dynamic structure, thereby completing the cell adhesion, migration, dissolution of bone minerals and digestion of organic matrix [2]. Therefore, in addition to observing the formation of osteoclasts by TRAP staining, we also carried out immunofluorescence staining to observe the F-actin ring formation to further evaluate the functional maturity of osteoclasts. Osteoclasts usually have 2-50 nuclei, so we mainly regarded multinucleated giant cells with complete F-actin rings as mature osteoclasts during the quantification process.

Reference:

[1] da Costa CE, Annels NE, Faaij CM, Forsyth RG, Hogendoorn PC, Egeler RM, Presence of osteoclast-like multinucleated giant cells in the bone and nonostotic lesions of Langerhans cell histiocytosis. J Exp Med 7;201(5) (2005) 687-93.

[2] Portes M, Mangeat T, Escallier N, Dufrancais O, Raynaud-Messina B, Thibault C, Maridonneau-Parini I, Vérollet C, Poincloux R, Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts, Elife (11) (2022) e75610.

Comment 4. The labels in 4C of "R1, R2, R3" standing for GANT58 is confusing

__Reply: __We are sorry for the confusing labeling. In the revised manuscript, we have added specific grouping information in the Figure legend, as shown below.

*Figure 4. DNA methyltransferases might be a regulatory target downstream of GLI1. a Biological process GO analysis of RNA-seq results for macrophages with or without GANT58 treatment. b KEGG rich analysis of RNA-seq results. c Heat map of parts of the relevant gene transcriptional expressions (C = control group; R = GANT58 treated group; red: increased expression; blue: decreased expression). d Relative mRNA expression of Gli1, Dnmt1 and Dnmt3a in macrophages with or without GANT58 treatment. Statistical analysis was performed using two-way ANOVA test. e RAW264.7 cells were stimulated by LPS and IFN-γ for 24 h, with or without GANT58 co-intervention. Western blot results of DNMT1 and DNMT3a protein expression and grayscale value ratio to β-actin of western blot results. n = 3. f RAW264.7 cells were stimulated by RANKL for 3 days, with or without GANT58 co-intervention. Western blot results of DNMT1 and DNMT3a protein expression and grayscale value ratio to β-actin of western blot results. n = 3. Statistical analysis was performed using two-way ANOVA test. g, h Co-IP detection of protein binding between GLI1 and DNMT1/DNMT3a. n = 3. i Protein–protein interface interaction of GLI1 and DNMT1 with PyMOL. j Micro-CT scanning and 3D reconstruction of mouse paws. k Bone parameters of BV/TV, BMD, Tb.N, Tb.Th. n = 6. Statistical analysis was performed using one-way ANOVA test. Data shown represent the mean ± SD. *p

Comment 5. In Figure S8, the numbers between the western blots are not explained.

__Reply: __Many thanks for your careful reading and comment. The numbers between the blots represent the ratio of the gray value of DNMT1 and DNMT3a immunoblot to the gray value of β-actin immunoblot, so as to reflect the relative expression of proteins. In order to avoid confusion, we made a statistical chart of the results and added it to revised Figure S8.

Comment 6. In Figure S9 there are references to asterisks which do not appear in the figure.

__Reply: __We are sorry for the mistake. We have deleted the relevant information in the revised Supplementary information. Thanks again.

Reviewer #2 (Significance (Required)):

The paper presented by Ge et al present interesting data suggesting that a GLI1 inhibitor (GANT58) has a strong impact on inflammatory arthritis in a murine model. Interesting data are presented whose novelty need better contextualization with other published studies, as previously published studies which are not cited in this manuscript include the finding that GLI1 is upregulated in patients with rheumatoid arthritis, that other GLI inhibitors have been utilized in murine models of rheumatoid arthritis, and that GLI1 has been shown to regulate DNMT expression in cancer settings. The authors connect GLI1 inhibition with DNMT activation in limiting M1 macrophage and osteoclast differentiation. However, several important controls are needed to in the in vitro studies as outlined above.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary

The manuscript by Ge et al. describes the possible roles of GLI1 in macrophage and osteoclast activation in rheumatoid arthritis via its functional interaction with DNA methyltransferases. The authors found that the GLI1 expression was elevated in RA synovial tissues and GLI1-specific inhibitor, GANT58, ameliorated arthritis in CIA mice. GLI1 expression in F4/80-positive macrophages in CIA synovial tissues led the authors to assess the roles of GLI1 in macrophages and osteoclasts. GANT58 suppressed M1 macrophage polarization by IFNg+LPS and osteoclastogenesis by RANKL. RNA-seq analysis of GANT58-treated macrophages revealed that DNA methyltransferases, DNMT1 and DNMT3a were possible targets of GLI1, and the studies with small inhibitors or overexpression of DNMTs suggest that GLI1 enhanced M1 polarization and osteoclastogenesis through DNMTs. The manuscript is well-written, the methods are accurate, and the results and data interpretation are consistent and clearly presented. This work deserves publication in Research Commons after addressing the following questions:

Major comments

Comment 1. GANT58 may inhibit GLI2 in addition to GLI1 and have off-target effects. Major findings with GANT58 in vitro, the suppressive effects on M1 polarization, osteoclastogenesis, and DNMT3a expression should be assessed with siRNA/shRNA knockdown or CRISPR/Cas9 knockout of GLI1.

Reply: Many thanks for your careful reading and constructive comment. According to your comment, we have constructed Gli1 knock-down cells and carried out related experiments. The results have been added in the revised manuscript, which are shown in the Response to Reviewer File.

Comment 2. In CIA with GANT58, the author performed only preventive treatment, not therapeutic treatment. Does GANT58 suppress adaptive immune responses via inhibiting APC function (ex. anti-CII IgG production)? Alternatively, the inhibitory effects of GANT58 on the effecter phase of RA (M1 macrophage and osteoclast activation) can be assessed using the serum-transfer arthritis models.

__Reply: __Many thanks for your constructive comments. Your question is indeed a direction worthy of attention. In our study, GANT58 was given during the stage of model establishment, showing a good effect of relieving arthritis, which was proved to come from the direct inhibition of inflammatory phenotype macrophages and osteoclasts. However, as autoimmune diseases, the enhancement of antigen presenting function and anti-Col II IgG production can enhance the immune response of the body [1]. The regulatory effect of GANT58 on macrophages suggests that it may have a potential impact on APC function. Despite this, whether GANT58 can regulate the pathological process of RA by influencing this pathway is inconclusive. Therefore, according to your suggestion, we will improve the relevant experiments in our follow-up research, and apply GANT58 to various animal models of RA to further explore the possible mechanism of GANT58 in the treatment of RA and provide more reliable theoretical support for its transformation and application.

Reference:

[1] Tsark EC, Wang W, Teng YC, Arkfeld D, Dodge GR, Kovats S, Differential MHC class II-mediated presentation of rheumatoid arthritis autoantigens by human dendritic cells and macrophages, J Immunol 1;169(11) (2002) 6625-33.

Minor comments

Comment 1. GANT58 is a water insoluble agent. Can you please include how to dissolve GANT58, administration route, and rationale of 20 mg/kg, for CIA?

__Reply: __Thank you for your professional comment. In this work, GANT58 was ordered from MedChemExpress (MCE; Cat. No.: HY-13282) Company. According to the instructions for use, we prepared 20 mg/ml ethanol solution of GANT58 into 2 mg/ml working solution for injection in vivo according to the following ratio: 10% EtOH + 90% (20% SBE-β-CD in PBS); Clear solution; Need ultrasonic. During the experiment, GANT58 was injected i.p. at a dose of 20 mg/kg daily for 28 days. With regard to the choice of drug injection concentration, according to the previous literature, most studies used a dose of 50 mg/kg for daily injection [1, 2]. Hereby, we set up concentration gradient intervention (0, 10, 20, and 50 mg/kg) in the preliminary experiment and found that 20 and 50 both had good therapeutic effects. Therefore, according to the consideration of economy and safety, we chose 20 mg/kg as our final intervention concentration.

Reference:

[1] Li G, Deng Y, Li K, Liu Y, Wang L, Wu Z, Chen C, Zhang K, Yu B, Hedgehog Signalling Contributes to Trauma-Induced Tendon Heterotopic Ossification and Regulates Osteogenesis through Antioxidant Pathway in Tendon-Derived Stem Cells, Antioxidants (Basel) 16;11(11) (2022) 2265.

[2] Lauth M, Bergström A, Shimokawa T, Toftgård R, Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A. 15;104(20) (2007) 8455-60.

Comment 2. Zoom photos in Fig 1j are not clear. Is GLI1 exclusively expressed in F4/80+ macrophages in synovial tissues?

__Reply: __Many Thanks for your comment. In the revised manuscript, we have improved the resolution of the image for better observation. According to the results, although GLI1 is more expressed in F4/80 positive cells, not all GLI1 proteins are expressed in macrophages, and we can find that some GLI1 positive staining is expressed in other cells. In the follow-up study, we will continue to explore this phenomenon and study the relationship between GLI1 and cells like synovial fibroblasts in RA.

Comment 3. In Fig 2 and 3, the treatment of macrophages with IFNg+LPS and RANKL enhanced the nuclear translocation of GLI1, suggesting that these stimuli may activate hedgehog signals. Recent studies, however, suggest various non-canonical activation pathways of GLI1. Does hedgehog inhibitor (ex. SMO inhibitor) also suppress M1 polarization and osteoclastogenesis?

__Reply: __Thank you for your constructive comment. We agree with that the activation of GLI1 is regulated by many various pathways. According to your comment, we additionally used Cyclopamine, a selective inhibitor of SMO, to intervene during the polarization of M1 macrophages and the activation of osteoclasts. The results are shown in the Response to Reviewer File: Cyclopamine could also inhibit the proinflammatory polarization of macrophages to a certain extent, and a significant inhibition of the osteoclast formation could be observed as well. These results may further confirm the important role of HH/GLI1 in regulating macrophage caused inflammation and osteoclast activation.

Comment 4. In Fig 6, the overexpression of DNMT3a reversed the inhibitory effects of GANT58 in osteoclastogenesis. This supports the author's conclusion that GLI1 may enhance osteoclastogenesis via DNMT3a upregulation. However, this conclusion should be carefully evaluated by examining effects of the overexpression of DNMT3a without GANT58. Does the overexpression of DNMT3a by itself enhance osteoclastogenesis or just reverse the GANT58-mediated suppression?

Reply: Thanks for your constructive comment. According to your comment, we observed and compared the differences in the activation of osteoclasts between the DNMT3a overexpression group and the control group. The results showed that at day 4 of induction, the TRAP positive stained osteoclast number seemed to be no significance between WT group and Dnmt3aOE group. However, at day 3, there was more osteoclast in Dnmt3aOE group, which suggested that overexpression of Dnmt3a might accelerate the activation of osteoclasts to some extent. The results are shown in the Response to Reviewer File.

Comment 5. Is RNA-seq data with GANT58 compatible with known target genes of GLI1 reported in previous studies?

Reply: Thanks for your constructive comment. By consulting and comparing with other research articles, most of the data trends in RNA sequencing results are the same as those in other studies. In addition, the expression of some genes is different from other studies (MMP13 increased in our data but decreased in other study [1]), which may be caused by different cell lines and different intervention methods.

Reference:

[1] Akhtar N, Makki MS, Haqqi TM, MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes, Arthritis Rheumatol 67(2) (2015) 423-34.

Reviewer #3 (Significance (Required)):

Significance

The main limitation of this paper is the lack of siRNA knockdown study of GLI1 and DNMTs. Another limitation of this paper is that the direct in vivo data demonstrating the inhibitory effects of GANT58 on M1 macrophage and osteoclast activation in CIA is lacking. The strength is the promising activity of GLI inhibitor, GANT58 as an anti-rheumatic drug on monocyte/macrophage-associated inflammation and bone destruction. The roles of hedgehog/GLI signals in macrophage function are largely unknown, and the findings of this study may contribute to this research field. This study will be interesting to rheumatologists and immunologists.

Reply: Thanks again for your constructive comments, which helped us to improve the quality of the manuscript.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Reply to the Reviewers

I thank the Referees for their...

Referee #1

1. The authors should provide more information when...

Responses + The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). + Though this is not stated in the MS 2. Figure 6: Why has only...

Response: We expanded the comparison

Minor comments:

1. The text contains several...

Response: We added...

Referee #2

cytoframe vs flowFrame

, e agora?

Acha que devemos mencionar o cadastro da arena? Sei que temos uma FAQ específica, mas pontuar só porque é necessário fazer o cadastro novamente

h não tem s*

So, paying 1000 pesetas daily, I took M.J. out of th eSanmariana jail: I could watch her running around the streets with her friends, down as far as the Rambla-- with fog and emerging sun, with the fisherman's boats, the most fragile of the rowing club--, not completely happy, because she wasn't with me wondering what intrusion in her life stopped me from writing to her or imagining my last letter of tempered optimism that the promise of our reuniting could be read between the lines.

devido à falta

Aqui podemos listas os headliners?

Author Response

Reviewer #1 (Public Review):

Specifically, the authors define "efficacy" (eta) of a ligand as the fractional change in binding free energy between the open and the closed states of the channel.

We assume that the word in quotes is a typo; ղ is efficiency, not efficacy (now given the symbol λ). We now emphasize the distinction immediately after Eq. 2.

1) One concern regards the clustering of the data sets in Fig. 5 into exactly 5 eta-classes. First, two clusters contain only two data points each. Second, the proposed "catch&hold LFER model" (Fig. 2) does not predict the existence of a discrete number of such eta-classes. How strong is the evidence that there are exactly 5 classes as opposed to a continuum of possible eta values.

Statistical (x means cluster) analysis indicates that the 23 agonists segregate into 5 ղ classes. Groups with only 2 members (plus the intercept) are less well defined (Fig 4) but are supported by the 5 mutational ղ classes (Fig. 7). (see above)

2) The authors do not discuss the uniqueness of the proposed model.

see above. Ln 405 Induced fits are common.

In fact, it seems to me that the existence of eta-classes might be explained just as well by an alternative model which assumes a single gating mechanism for the receptor,

We are not sure what a “single gating mechanism” means. Does non-single refer to i) the2 stage induced fits (catch-hold LFER)? … ղ classes makes this conclusion unavoidable. ii) our conjecture that are there are 5 different C versus O binding site structural pairs…? Energy derives from structure, so we the 5 energy ratios indicate 5 structural pairs. iii) multiple steps inside gating (ϕ)? …So far there have not been any alternative explanations for the organized map of ϕ. iv) catch itself?... Evidence for this induced fit is given in Fig 2 and 7 SI, and on Ln 528-547 we discuss the implications of kon to C versus O. Ln 405 Local ‘Induced fit’ rearrangements in enzymes are common. We think the evidence is strong for the bottom scheme in Fig 2A.

but distinct patterns of ligand-protein interactions for the different agonists.

ղ classes derive from distinct interactions for different agonists, but what these are and whether the ‘contact number’ idea is useful are uncertain (see above).

The pore opening-associated increase in agonist affinity is typically caused by a tightening of the substrate binding site (often called clamshell closure) …

Ln 379-386 In the Discussion we now relate catch-hold to induced fit

Ln 455, 461-463, 471-474 Fig 2SI and the induced fit to clamshell closure

Reviewer #2 (Public Review):

This is an interesting manuscript with a worthwhile approach to receptor mechanisms. The paper contains an impressive amount of new data. These single molecule concentration response curves have been compiled with care and the authors deserve great credit for obtaining these data.

Ln 233 ղ can be estimated from a CRC built from whole-cell currents…

Ln 150 …or indeed any method that estimates KdC and KdO (for example binding assays, or perhaps in silico simulations of AC and AO structures)

I judge the main result to be that there are different values of the recently-proposed agonist-related quantity "efficiency".

Ln 21, 26-27, 535-547 OK, but to us the most interesting insight is that in AChRs binding IS gating.

These values are clustered into 5 quite closely spaced groups. The authors propose that these groups are the same whether considering mutations in the binding site or different agonists.

see above

It was unclear to me in several places, what new data and what old data are included in each figure. Therefore readers may have difficulty judging the claimed advance. This difficulty is not helped by the discussion, which includes some previous findings as "results".

see above.

A further weakness is that it is unclear how general or how specific these concepts are. The authors assert that they are, by definition, completely universal. However, we do not have reference to previous work or current data on any other receptor than the muscle nicotinic. I could not square the concept that "every receptor works like this" with the evident lack of desire to demonstrate this for any other receptor.

Ln 132-136 There are reasons to think that receptors in general work according to Figure 1A. A thermalized ligand (for instance TriMA, MW 60) has the momentum of only ~3 water molecules. A momentum sensor would have terrible signal/noise.

Reviewer #3 (Public Review):

This work attempts to introduce a new attribute of the receptor- efficiency, a fraction of an agonist binding energy consumed by conformational transition of the receptor from resting to active (open) states. Furthermore, the authors use an impressive set of experimental data (single channel recordings with 23 agonists and 53 mutations) to measure the efficiency for each agonist and mutant receptor. All the estimated efficiencies fall into a few groups and inside each of the efficiency groups there is a strong correlation between agonist affinity and receptor opening efficacy.

The main finding in this study is that estimated efficiencies fall into 5 groups.

see above.

There is no clear description of the method how the efficiencies were allocated into different groups. Most importantly, it is not clear if the method used takes into account the uncertainty of the efficiency estimate. The study does not show any statistical metrics of the efficiency estimates as well as any other calculated variable such as dissociation equilibrium constants to resting or open states. Surely, the uncertainty of the efficiency should matter especially considering how near the efficiency group values are (eg. difference about 10% between 0.51 and 0.56 or 0.41 and 0.45).

see above

All the tested agonists fell into groups according to the efficiency value attributed to them. It is difficult to see why some of the agonists belong to the same group. For example, it is not obvious at all why such agonists as epibatidine, decamethonium and TMP are in the same group. The question, I guess, arises if this grouping based on efficiency has any predictability value. Furthermore, if a series of mutations with the same agonist fall into different groups, the prediction power of this approach is very limited if one attempts to design a new agonist or look for a new mutation.

see above and Ln 548-561 (last para of text). Efficiency is a relatively new idea. This report is one of only a few on the subject. More experiments with different receptors by more labs using other approaches are needed to ascertain whether ղ is general.

Reviewer #1 (Public Review):

In this work Indurthy and Auerbach investigate the fundamental concept of how the energy of agonist binding is converted into the energy of the conformational change that opens the pore of the nicotinic acetylcholine receptor (nAChR). The conclusions are based on a very large pool of experimental data that are interpreted with great mechanistic insight.

Specifically, the authors define "efficacy" (eta) of a ligand as the fractional change in binding free energy between the open and the closed states of the channel. They construct a log-log scatter plot of efficacy vs. affinity which represents 23 different agonists acting on the WT receptor, plus a subset of the same agonists acting on various nAChR mutants. They go on to show that these largely scattered dots can be partitioned into 5 distinct clusters ("eta-classes") within which the dots are linearly arranged. They interpret these clusters in terms of a mechanistic gating model (the "catch&hold LFER model"), and suggest that a different model accounts for each different eta-class. Put in simple terms, the interpretation is that 5 different subtypes of gating isomerization exist for the nAChR, the choice among which depends on the agonist used.

These types of study are necessary to advance conceptual understanding in biophysics. I have some reservations regarding the mechanistic interpretation of the data set and the uniqueness of the proposed model.

1. One concern regards the clustering of the data sets in Fig. 5 into exactly 5 eta-classes. First, two clusters contain only two data points each. Second, the proposed "catch&hold LFER model" (Fig. 2) does not predict the existence of a discrete number of such eta-classes. How strong is the evidence that there are exactly 5 classes as opposed to a continuum of possible eta values.

2. The authors do not discuss the uniqueness of the proposed model. In fact, it seems to me that the existence of eta-classes might be explained just as well by an alternative model which assumes a single gating mechanism for the receptor, but distinct patterns of ligand-protein interactions for the different agonists. The pore opening-associated increase in agonist affinity is typically caused by a tightening of the substrate binding site (often called clamshell closure) which brings further protein side chains into the vicinity of the ligand, thereby allowing further ligand-protein interactions to form (or further strengthening interactions that exist also in the closed-pore state). Thus, at a first approximation, the ratio between binding free energies in the open- and closed-pore states reflects the ratio of the numbers (and strengths) of ligand-protein bonds in those two states.

As an illustration, consider the following simplified model for a channel and a given ligand. In the open-pore state the number of ligand-protein interactions is n(o), and all those interactions are comparably strong. Out of those interactions only a subset is formed in the closed-pore state, their number is n(c) (where n(c)<br /> The maximal possible values of n(c) and n(o) are determined by the number and spatial arrangement of protein chemical groups that surround the substrate binding site. On the other hand, depending on the number and arrangement of matching chemical groups on the ligand, different ligands will be able to "exploit" different subsets of these possible ligand-protein interactions, resulting in different values of eta. Furthermore, ligands for which the absolute values of n(o) are different, but the ratio n(c)/n(o) is similar, will form apparent "eta-classes", i.e., will be arranged on a "eta-plot" along a straight line. (See attached image file for a graphical representation of the model.)

This model would suggest that there is a single gating mechanism (i.e., the actual protein conformational change is similar regardless of which agonist is bound), but the relative stabilities of the ligand-bound closed and open states are agonist-dependent. Wouldn't such a mechanism equally well explain all the data shown? The authors should either acknowledge this possibility or discuss available structural or functional evidence to exclude it.

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Author Response

Reviewer #1 (Public Review):

This study by Park et al. describes an interesting approach to disentangle gene-environment pathways to cognitive development and psychotic-like experiences in children. They have used data from the ABCD study and have included PGS of EA and cognition, environmental exposure data, cognitive performance data and self-reported PLEs. Although the study has several strengths, including its large sample size, interesting approach and comprehensive statistical model, I have several concerns:

• The authors have included follow-up data from the ABCD Study. However, it is not very clear from the beginning that longitudinal paths are being explored. It would be very helpful if the authors would make their (analysis) approach clearer from the introduction. Now, they describe many different things, which makes the paper more difficult to read. It would be of great help to see the proposed path model in a Figure and refer to that in the Method.

We clarified the specific longitudinal paths explored in our study in the end of the Introduction section (line 149~160). We also added a figure of the proposed path model (Figure 1) and refer to it in the Method section (line 232~239).

• There is quite a lot of causal language in the paper, particularly in the Discussion. My advice would be to tone this down.

We corrected and tone-downed all causal languages used in our manuscript. Per your suggestion, we deleted statements like ‘unbiased estimates’ and used expressions such as ‘adjustment for observed/unobserved confounding’ instead.

• I feel that the limitation section is a bit brief, and can be developed further.

We specified additional potential constraints of our study, including limited representativeness, limited periods of follow-up data, possible sample selection bias, and the use of non-randomized, observational data. These corrections can be found in line 518~538.

• I like that the assessment of CP and self-reports PEs is of good quality. However, I was wondering which 4 items from the parent-reported CBCL were used and how did they correlate with the child-reported PEs? And how was distress taken into account in the child self-reported PEs measurement? Which PEs measures were used?

We believe that the Reviewer #1’s comment for the correlations between PLEs derived from PQ-BC (total score and distress score PLEs) and from CBCL (parent-rated PLEs) might have been due to the fact that she/he was referring to the prior version of our manuscript submitted to a different journal. We obtained Pearson’s correlation coefficients between the PLEs (baseline year: r = 0.095~0.0989, p<0.0001; 1-year follow-up: r = 0.1322~0.1327, p<0.0001; 2-year follow-up: r = 0.1569~0.1632, p<0.0001) and added this information in the Method section for PLEs (line 198~201).

• What was the correlation between CP and EA PGSs?

We also added the Pearson’s correlation between the two PGSs (r =0.4331, p<0.0001) in the Methods section for PGS (line 214~215).

• Regarding the PGS: why focus on cognitive performance and EA? It should be made clearer from the introduction that EA is not only measuring cognitive ability, but is also a (genetic) marker of social factors/inequalities. I'm guessing this is one of the reasons why the EA PGS was so much more strongly correlated with PEs than the CP PGS. See the work bij Abdellaoui and the work by Nivard.

We thank the reviewer for the feedback to clarify that educational attainment (EA) is not only a genetic marker of cognitive ability but also that of socioeconomic outcomes. Per your suggestion, we included the associations of EA PGS with multiple biological and socioeconomic outcomes found in prior studies (e.g., Abdellaoui et al., 2022) in the Introduction (line 131~142).

Abdellaoui, A., Dolan, C. V., Verweij, K. J. H., & Nivard, M. G. (2022). Gene–environment correlations across geographic regions affect genome-wide association studies. Nature Genetics. doi:10.1038/s41588-022-01158-0

• Considering previous work on this topic, including analyses in the ABCD Study, I'm not surprised that the correlation was not very high. Therefore, I don't think it makes a whole of sense to adjust for the schizophrenia PGS in the sensitivity analyses, in other words, it's not really 'a more direct genetic predictor of PLEs'.

We conducted this adjustment considering that PLEs often precede the onset of schizophrenia. In addition, prior studies found that schizophrenia PGS is significantly associated with cognitive intelligence within psychosis patients (Shafee et al., 2018) and individuals at-risk of psychosis (He et al., 2021), and that significant distress psychotic-like experiences had greater positive correlation with schizophrenia PGS than PGS for psychotic-like experiences (Karcher et al., 2018).

For these reasons, we thought that it is necessary to assess whether the effects of cognitive phenotypes PGS (i.e., CP PGS and EA PGS) in the linear mixed model are significant after adjusting for schizophrenia PGS. We believe our results from the mixed linear model showed the sensitivity and specificity of the association between cognitive phenotype PGS and PLEs.

He, Q., Jantac Mam-Lam-Fook, C., Chaignaud, J., Danset-Alexandre, C., Iftimovici, A., Gradels Hauguel, J., . . . Chaumette, B. (2021). Influence of polygenic risk scores for schizophrenia and resilience on the cognition of individuals at-risk for psychosis. Translational Psychiatry, 11(1). doi:10.1038/s41398-021-01624-z

Karcher, N. R., Paul, S. E., Johnson, E. C., Hatoum, A. S., Baranger, D. A. A., Agrawal, A., . . . Bogdan, R. (2021). Psychotic-like Experiences and Polygenic Liability in the Adolescent Brain Cognitive Development Study. Biological Psychiatry: Cognitive Neuroscience and Neuroimaging. doi:https://doi.org/10.1016/j.bpsc.2021.06.012

Shafee, R., Nanda, P., Padmanabhan, J. L., Tandon, N., Alliey-Rodriguez, N., Kalapurakkel, S., . . . Robinson, E. B. (2018). Polygenic risk for schizophrenia and measured domains of cognition in individuals with psychosis and controls. Translational Psychiatry, 8(1). doi:10.1038/s41398-018-0124-8

• How did the FDR correction for multiple testing affect the results?

For all analysis results presented in our study, False Discovery Rate (FDR) correction for multiple testing compared p-values of nine key study variables: PGS (cognitive performance or educational attainment), family income, parental education, family’s financial adversity, Area Deprivation Index, years of residence, proportion of population below -125% of the poverty line, positive parenting behavior, and positive school environment. An exception was the sensitivity analysis that included schizophrenia PGS in the linear mixed model for adjustment: with another PGS variable added, FDR correction compared p-values of ten key variables. Overall, the effects of FDR correction on the results were limited; i.e., the majority of associations between the key variables and the outcomes, which were deemed highly significant, remained unchanged after the FDR correction.

Overall, I feel that this paper has the potential to present some very interesting findings. However, at the moment the paper misses direction and a clear focus. It would be a great improvement if the readers would be guided through the steps and approach, as I think the authors have undertaken important work and conducted relevant analyses.

We express our appreciation to the reviewer for the constructive feedback and guidance, which has significantly contributed to the improvement of our manuscript. As addressed in the preceding sections, we have implemented the necessary corrections and clarifications in response to the reviewer's suggestions. We remain open to making further amendments as needed, and thus invite any additional comments should any aspect of our revisions be deemed inadequate or inappropriate.

Reviewer #2 (Public Review):

This paper tried to assess the link between genetic and environmental factors on psychotic-like experiences, and the potential mediation through cognitive ability. This study was based on data from the ABCD cohort, including 6,602 children aged 9-10y. The authors report a mediating effect, suggesting that cognitive ability is a key mediating pathway in the link between several genetic and environmental (risk and protective) factors on psychotic-like experiences.

While these findings could be potentially significant, a range of methodological unclarities and ambiguities make it difficult to assess the strength of evidence provided.

Strengths of the methods:

The authors use a wide range of validated (genetic, self- and parent-reported, as well as cognitive) measures in a large dataset with a 2-year follow-up period. The statistical methods have the potential to address key limitations of previous research.

We sincerely thank the reviewer for recognizing these methodological strengths of our study. The reviewer’s positive comments are highly supportive and encouraging for us.

Weaknesses of the methods:

The rationale for the study is not completely clear. Cognitive ability is probably a more likely mediator of traits related to negative symptoms in schizophrenia, rather than positive symptoms (e.g., psychosis, psychotic-like symptom). The suggestion that cognitive ability might lead to psychotic-like symptoms in the general population needs further justification.

We sincerely thank and highly appreciate the concerns that the reviewer has raised regarding our proposal that cognitive ability may serve as a mediator of psychotic-like experiences. To the best of our knowledge, it has been proposed that cognitive ability can be a mediator of positive symptoms in schizophrenia (including psychotic-like experiences), as well as negative symptoms. This mediating role of cognitive ability was proposed in several prior studies on cognitive model of schizophrenia/psychosis. Per your suggestion, we included further justification in the Introduction section of our study (line 104~107). Specifically, we highlighted that cognitive ability has been theoretically proposed as a potential mediator of genetic & environmental influence on positive symptoms of schizophrenia such as psychotic-like experiences. We refer to studies conducted by Howes & Murray (2014) and Garety et al. (2001).

Howes, O. D., & Murray, R. M. (2014). Schizophrenia: an integrated sociodevelopmental-cognitive model. The Lancet, 383(9929), 1677-1687. doi:https://doi.org/10.1016/S0140-6736(13)62036-X

Garety, P. A., Kuipers, E., Fowler, D., Freeman, D., & Bebbington, P. E. (2001). A cognitive model of the positive symptoms of psychosis. Psychological Medicine, 31(2), 189-195. doi:10.1017/S0033291701003312

Terms are used inconsistently throughout (e.g., cognitive development, cognitive capacity, cognitive intelligence, intelligence, educational attainment...). It is overall not clear what construct exactly the authors investigated.

Thank you for your comment. We corrected the term ‘cognitive capacity’ to ‘cognitive phenotypes’ throughout our manuscript. We also added in the Introduction (line 141~143) that we will collectively refer to these two PGSs of focus as ‘cognitive phenotypes PGSs’, which is similar to the terms used in prior research (Joo et al., 2022; Okbay et al., 2022; Selzam et al., 2019).

Joo, Y. Y., Cha, J., Freese, J., & Hayes, M. G. (2022). Cognitive Capacity Genome-Wide Polygenic Scores Identify Individuals with Slower Cognitive Decline in Aging. Genes, 13(8), 1320. doi:10.3390/genes13081320

Okbay, A., Wu, Y., Wang, N., Jayashankar, H., Bennett, M., Nehzati, S. M., . . . Young, A. I. (2022). Polygenic prediction of educational attainment within and between families from genome-wide association analyses in 3 million individuals. Nature Genetics, 54(4), 437-449. doi:10.1038/s41588-022-01016-z

Selzam, S., Ritchie, S. J., Pingault, J.-B., Reynolds, C. A., O’Reilly, P. F., & Plomin, R. (2019). Comparing Within- and Between-Family Polygenic Score Prediction. The American Journal of Human Genetics, 105(2), 351-363. doi:https://doi.org/10.1016/j.ajhg.2019.06.006

Not the largest or most recent GWASes were used to generate PGSes.

Thank you for mentioning this point. The reason why we were not able to use the largest GWAS for cognitive intelligence, educational attainment and schizophrenia is because (unfortunately) our study started earlier than the point when the GWAS studies by Okbay et al. (2022) and Trubetskoy et al. (2022) were published. We corrected that our study used ‘a GWAS of European-descent individuals for educational attainment and cognitive performance’ instead of the largest GWAS (line 206~208).

It is not fully clear how neighbourhood SES was coded (higher or lower values = risk?). The rationale, strengths, and assumptions of the applied methods are not fully clear. It is also not clear how/if variables were combined into latent factors or summed (weighted by what). It is not always clear when genetic and when self-reported ethnicity was used. Some statements might be overly optimistic (e.g., providing unbiased estimates, free even of unmeasured confounding; use of representative data).

Consistent with the illustration of neighborhood SES in the Methods section, higher values of neighborhood SES indicate risk. In the original Figure 2, higher values of neighborhood SES links to lower intelligence (direct effects: β=-0.1121) and higher PLEs (indirect effects: β=-0.0126~ -0.0162). We think such confusion might have been caused by the difference between family SES (higher values = lower risk) neighborhood SES (higher values = higher risk). Thus, we changed the terms to ‘High Family SES’ and ‘Low Neighborhood SES’ in the corrected figure (Figure 3) for clarification.

Considering that shorter duration of residence may be associated with instability of residency, it may indicate neighborhood adversity (i.e., higher risk). This definition of the ‘years of residence’ variable is in line with the previous study by Karcher et al. (2021).

We represented PGSs, family SES, neighborhood SES, positive family and school environment, and PLEs as composite indicators (derived from a weighted sum of relevant observed variables). To the best of our knowledge, it has been suggested from prior studies that these variables are less likely to share a common factor and were assessed as a composite index during analyses. For instance, Judd et al. (2020) and Martin et al. (2015) analyze genetic influence of educational attainment and ADHD as composite indicators. Also, as mentioned in Judd et al. (2020), socioenvironmental influences are often analyzed as composite indicators. Studies on psychosis continuum (e.g., van Os et al., 2009) suggest that psychotic disorders are likely to have multiple background factors instead of having a common factor, and notes that numerous prior research uses composite indices to measure psychotic symptoms. These are the reasons why we used components for these constructs instead of generating latent factors (which is done in the standard SEM method). On the contrary, we represented general intelligence as a common factor that determines the underlying covariance pattern of fluid and crystallized intelligence, based on the classical g theory of intelligence. We added this explanation in line 269~285.

Moreover, during estimation, the IGSCA determines weights of each observed variable in such a way as to maximize the variances of all endogenous indicators and components. We added this explanation in the description about the IGSCA method (line 266~268).

We deleted overly optimistic statements like ‘unbiased estimates’ and used expressions such as ‘adjustment for observed/unobserved confounding’ instead, throughout our manuscript.

Judd, N., Sauce, B., Wiedenhoeft, J., Tromp, J., Chaarani, B., Schliep, A., ... & Klingberg, T. (2020). Cognitive and brain development is independently influenced by socioeconomic status and polygenic scores for educational attainment. Proceedings of the National Academy of Sciences, 117(22), 12411-12418.

Karcher, N. R., Schiffman, J., & Barch, D. M. (2021). Environmental Risk Factors and Psychotic-like Experiences in Children Aged 9–10. Journal of the American Academy of Child & Adolescent Psychiatry, 60(4), 490-500. doi:10.1016/j.jaac.2020.07.003

Martin, J., Hamshere, M. L., Stergiakouli, E., O'Donovan, M. C., & Thapar, A. (2015). Neurocognitive abilities in the general population and composite genetic risk scores for attention‐deficit hyperactivity disorder. Journal of Child Psychology and Psychiatry, 56(6), 648-656.

van Os, J., Linscott, R., Myin-Germeys, I., Delespaul, P., & Krabbendam, L. (2009). A systematic review and meta-analysis of the psychosis continuum: Evidence for a psychosis proneness–persistence–impairment model of psychotic disorder. Psychological Medicine, 39(2), 179-195. doi:10.1017/S0033291708003814

It appears that citations and references are not always used correctly.

We thoroughly checked all citations and specified the references for each statement. We deleted Plomin & von Stumm (2018) and Harden & Koellinger (2020) and cited relevant primary studies (e.g., Lee et al., 2018; Okbay et al., 2022; Abdellaoui et al., 2022) instead. We also specified the references supporting the statement that educational attainment PGS links to brain morphometry (Judd et al., 2020; Karcher et al., 2021). As Okbay et al. (2022) use PGS of cognitive intelligence (which mentions the analyses results in their supplementary materials) as well as educational attainment, we decided to continue citing this reference. These corrections can be found in line 131~141.

Strengths of the results:

The authors included a comprehensive array of analyses.

We thank the reviewer for the positive comment.

Weaknesses of the results:

Many results, which are presented in the supplemental materials, are not referenced in the main text and are so comprehensive that it can be difficult to match tables to results. Some of the methodological questions make it challenging to assess the strength of the evidence provided in the results.

As you rightly identified, we inadvertently failed to reference Table S2 in the main text. We have since corrected this omission in the Results section for the IGSCA (SEM) analysis (line 375). The remainder of the supplementary tables (Table S1, S3~S7) have been appropriately cited in the main manuscript. We recognize that the quantity of tables provided in the supplementary materials is substantial. However, given the comprehensiveness and complexity of our analyses, which encompass a wide array of study variables, these tables offer intricate results from each analysis. We deem these results, which include valuable findings from sensitivity analyses and confound testing, too significant to exclude from the supplementary materials. That said, we are open to, and would greatly welcome, any further suggestions on how to present our supplementary results in a more accessible and digestible format. We are ready and willing to implement any necessary modifications to ensure clarity and ease of comprehension. Your guidance in this matter is highly valued.

Appraisal:

The authors suggest that their findings provide evidence for policy reforms (e.g., targeting residential environment, family SES, parenting, and schooling). While this is probably correct, a range of methodological unclarities and ambiguities make it difficult to assess whether the current study provides evidence for that claim.

Impact:

The immediate impact is limited given the short follow-up period (2y), possibly concerns for selection bias and attrition in the data, and some methodological concerns.

We added as study limitations (line 518~538) that the impact of our findings for understanding cognitive and psychiatric development during later childhood may be limited due to the relatively short follow-up period, the possibility of sample selection bias, and the problems of interpreting analyses results from an observational study as causality (despite the novel causal inference methods, designed for non-randomized, observational data, that we used).

As responded above, we made necessary corrections and clarifications for the points suggested by the reviewer. As we are willing to make additional revisions, please feel free to give comments if you feel that our corrections are insufficient or inappropriate.

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Referee #1

Evidence, reproducibility and clarity

Summary:

This manuscript shows the involvement of both the proteasome and autophagy pathways in the turnover and therefore regulation of ARF7, an auxin-responsive factor involved in lateral root formation. The authors bring crucial information for the understanding of how autophagy is involved in auxin-signaling.

Major comments:

The key conclusions appear overall convincing yet this reviewer would strongly advise to take into account the following remarks for a clearer and more convincing line of inquiry. This reviewer also believes that the additional experiments could be performed relatively fast apart for the point 9) where the establishment of a homozygous line could take 6 months or more.

1. Figure 1 & Figure EV1: The nature of the loading control should be stated as it appears to be a specific protein detected by immunoblotting. Furthermore, if the authors wish to make a stronger point as to whether ARF7 is degraded by the proteasome (considering the reserves mentioned in the Discussion section), I would recommend to perform the same assays as in Figure 1 but using an alternative proteasome inhibitor such as Bortezomib and to include a proteasome subunit KO mutant such as rpt2a-2.
2. The statement "The experiment revealed that both NBR1 (Fig 2A) and ATG8a (Fig 2B), but not free YFP, co-immunoprecipitated with ARF7-Venus." Is false as the authors did not try to co-immunoprecipitate free YFP with ARF7-Venus, they used a free YFP expressing line as a negative control for their GFP-immunoprecipitation (IP). It should further be noted that although NBR1 is detected in their free YFP IP, ATG8 is at very low levels so it should be stated that they see an enrichment of ATG8 in their ARF7-Venus IP.
3. Authors state "we were unable to detect ARF7-Venus in the input of both Co-IPs which can likely be explained by the fact that ARF7-Venus is under the control of its native promoter and thus lowly expressed.", yet putative degradation products (i.e. a smear) can be observed in the input of Figure 2A, similarly to the bands observed in both IP blots. It would be interesting to repeat these co-IPs with proteolysis inhibitors such as MG132 or Pepstatin & E64-d to pinpoint the proteolytic machinery at the origin of ARF7-Venus degradation in the IPs.
4. Figure 2: The use of multicolor BiFC "mcBiFC" should be stated as such for an easier understanding of the reader. It would be helpful for the reader if the "GFP" signal resulting from the complementation would be highlights thanks to some arrows. Moreover, a western blot to verify the expression levels should be performed since every construct has an epitope tag as stated in Gehl et al. 2009.
5. General remark: all drug/chemical treatments performed in this study use a "non-treated" negative control, yet it should be pointed out that the correct corresponding negative controls should have the solvent used to dissolve the respective drug/chemical in order to exclude any effect of the solvent or vehicle.
6. Figure 4, Figure EV4: Considering the variability in size and staining of the Rubisco large-subunit in the 4 immunoblot panels, I would suggest blotting with another antibody such as anti-tubulin or anti-histone 3 as a loading control for a more convincing quantification. Moreover, the nature of the staining used to stain the Rubisco large-subunit should be stated. The authors also state "differences in ARF7 accumulation in atg5 compared to Col-0" yet no immunoblot is shown where both genotypes are present on the same membrane, in order to verify this statement.
7. Figure 5: In regards to LR density measurements, I recommend reading "Quantitative Analysis of Lateral Root Development: Pitfalls and How to Avoid Them" by Dubrovsky & Forde (Plant Cell, 2012) for a more robust method of evaluating lateral root density.
8. Discussion: The authors state that "autophagy blockage leads to increased ARF7 cytoplasmic condensates". To support this statement, I recommend crossing pARF7::gARF7-Venus into atg mutants and analysing the localization and the fluorescence intensity of ARF7-Venus in specific parts of the root, as well as performing immunoblotting in order to assess overall ARF7 accumulation in autophagy deficient genetic backgrounds.

Minor comments

1. The following statement: « In contrast, plants are able to tolerate disruption of autophagy activity without major penalties" holds true to A. thaliana of some other plants but it must be noted that in O. sativa, autophagy-deficiency may lead to male sterility, which should be considered a major penalty for evolutionary fitness. For review see Norizuki et al. 2020 (Front. Plant Sci.).
2. Figure 2: The molecular weights appear to be potentially misannotated as free YFP aligns with the 35 kDa marks although it should appear around 27 kDa.
3. Figure EV3: There are 2 merged image columns, the furthest one to the right appears to include a DIC or Trans image on top of both fluorescence channels. It would be more helpful for the reader if the DIC or Trans image was shown with the overlay of fluorescent channels in order to assess the effect of 10% 1,6-hexandiol on the plant tissue. Moreover, demonstrating the absence of tissue damage or cell-death after 1,6-hexandiol treatment would be a plus.
4. There is a typo throughout the manuscript: ZT should be "Zeitgeber" not "zeitberg".

Significance

This manuscript has the quality of describing the proteolytic balance of ARF7 and thereby, the involvement of the autophagy pathway in regulating auxin-signaling components. This research adds on to the growing interest in how autophagy participates in developmental cues, and how hormonal signaling is regulated throughout the plant.

Charcoal,

DOI: 10.12688/f1000research.130587.1

Resource: (R and D Systems Cat# MAB2582, RRID:AB_2893288)

Curator: @scibot

SciCrunch record: RRID:AB_2893288

What is this?

DOI: 10.1016/j.celrep.2023.112637

Resource: (BD Biosciences Cat# 562391, RRID:AB_11153664)

Curator: @scibot

SciCrunch record: RRID:AB_11153664

What is this?

DOI: 10.1126/sciadv.abq0712

Resource: RRID:Addgene_45797

Curator: @scibot

SciCrunch record: RRID:Addgene_45797

What is this?

La diferencia entre el modelo de Leland y el de Leland y Toft es que el odelo de Leland asume que VB NO depende ni del tiempo de la deuda (T) o el trayecto temporal (t)

Author Response

Reviewer #1 (Public Review):

The manuscript by Huang, Li, et al. describes the identification of variants in the gene coding for p31 comet, a protein required for silencing the spindle assembly checkpoint or SAC, in women with recurrent pregnancy loss upon IVF. In three families mutations affecting splicing or expression of full-length protein were identified. The authors show that oocytes of the patients arrest in meiosis I, are most likely to fail to inactivate the SAC without a fully functional p31 comet. Indeed, the metaphase I arrest occurring in mouse oocytes upon overexpression of Mad2 can be rescued by overexpression of wild-type p31 comet, but not a truncated version. Injection of wt p31 comet into 6 human oocytes from one patient rescued the meiosis I arrest.

Main points:

The fact that inactivation of the SAC is required for anaphase I onset in human oocytes is not novel. Biallelic mutations of TRIP13 were shown to lead to the same phenotype (Zhang et al. Am J. Hum Gen., 2020).

As pointed out by the editors and both other reviewers, the strength of this study is highlighted by the identification of genetic variants responsible for oocyte meiosis I arrest in human patients. As a fact, very few genetic variants that cause female oocyte meiotic failure are identified (Ref: Qing Sang, et al. Understanding the genetics of human infertility. Science. 2023). In this study, we for the first time reported the novel deleterious p31comet variants causing human oocyte MI arrest. Without exploring the etiological landscape of human genetic variants, it is impossible to comprehensively invent diagnostic and therapeutic approaches for female patients.

No new mechanistic insights are obtained.

To gain the molecular mechanism, we have optimized and performed a modified Smart-seq2 protocol using frozen single-cell human oocytes (Page 11 and Figure 4-figure supplement 1). These data were in well agreement with the phenotypes as reported.

The authors propose a role for female fertility, however, also a male patient with a p31 comet variant is sterile.

This manuscript focuses on screening the genetics variants responsible for the oocyte failure in female patients, rather than male patients. In addition, we had difficulties with collection of more detailed information from this male patient because he rejected to provide the consent to us. We currently only have limited information after we tried every effort to get in touch with the male patient. We have added more discussion in the MS. Certainly, further exploration of the roles of MAD2L1BP variants in the male meiosis, for example, by collection of a cohort of male patients’ samples with meiotic defects, would be an interesting direction in the future, but this is beyond the scope of this study.

The fact that the C-terminus of p31 comet is required for interaction with Mad2 and hence, turning off the SAC, is already known.

The interaction between p31comet and Mad2 is known in somatic cells, but not in oocytes. As it is widely known that the oocytes are distinct from somatic cells in that the SAC in oocytes is not effective because oocytes can proceed to anaphase I in the presence of even one unattached kinetochore, as compared with somatic cells. We provided evidence that the overexpression of Mad2 can only be rescued by overexpression of wild-type p31comet, but not the truncated p31comet variant in both mouse and human oocytes (Fig.3 and 4), which sufficiently characterized the causative roles of p31comet variants underlying female infertility.

Reviewer #2 (Public Review):

In this manuscript by Huang et al. the authors explore the genetic underpinnings that may cause human oocyte meiotic arrest. The meiotic arrest of oocytes can cause female infertility leading patients to seek treatment at IVF clinics to assist in having genetically related babies. However, because oocytes fail to develop to MII, oocytes from these patients cannot be fertilized, leaving no current options for genetically related babies for patients with this pathology. Huang et al identified 50 IVF patients with this phenotype, and after the whole exome sequence, 3 patients had mutations in a spindle assembly checkpoint regulator, Mad1bp1. This study describes these mutations in detail, shows how these mutations affect Mad1bp1 expression, evaluates gross function in mouse oocytes, and explores therapeutic treatment in human oocytes. Overall, this is an important translational study that adds to the growing body of literature that genetic mutations impact oocyte quality and fertility.

Thank you for your favorable comments.

In its current form, I find that the strengths exist in the analysis of the patients' genomes and pedigree information. This is unique data and is important for the field. The expression in oocytes, structure modeling, and conservation in evolution, while not essential for this study, add interesting information for the reader to consider. I sometimes find these distracting in manuscripts, but appreciate them here in this context. The conclusion using human oocytes to propose possible treatment takes the study to completion and is not an easy approach to carry out.

Thank you for your positive comments on this manuscript.

I do find some weaknesses that weaken the conclusions. The conclusion described is that the SAC is not satisfied in oocytes from these patients. The authors attempt to show this by analysis of mouse oocytes using polar body extrusion and its timing as an assay. There could be many reasons contributing to arrest, therefore a singular assay is not ideal to justify the conclusions. While I do suspect the authors are correct, an intact SAC should be shown at the molecular level to fully justify this conclusion. There are many assays routinely performed in mouse oocytes that the authors can consider (check papers by authors from Wassmann, FitzHarris, and Schindler labs for example).

Thanks for your good comments. Following your advice, we have performed the immunofluorescence assay to evaluate the SAC integrity using mouse oocytes by microinjection of WT and Mut Mad2l1bp cRNA, which clearly validated the intact SAC activation with Mut Mad2l1bp cRNA injection. Please see the reply as detailed below.

Reviewer #3 (Public Review):

The spindle checkpoint ensures the accuracy of chromosome segregation by sensing unattached kinetochores during mitosis and meiosis and delays the onset of anaphase. Unattached kinetochores catalyze the conformational activation of the latent open MAD2 (O-MAD2) to the active closed MAD2 (C-MAD2). C-MAD2 is then incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C) to delay anaphase. When all kinetochores are properly unattached, the MAD2-binding protein p31comet and the ATPase TRIP13 extract C-MAD2 from the MCC, leading to MCC disassembly and the conversion of C-MAD2 back to O-MAD2. This action turns off the spindle checkpoint, resulting in APC/C activation and anaphase onset. Cells deficient in p31comet exhibit mitotic delays.

In the current study, Huang et al. have linked p31comet mutations to female infertility. Biallelic loss-of-function alleles of p31comet cause delays in the exiting metaphase of meiosis I and polar body extrusion. The p31comet mutant proteins contain C-terminal truncations and fail to bind to MAD2. Reintroducing full-length p31comet into patient oocytes can bypass the metaphase arrest. Together with a previous study that showed biallelic mutations of TRIP13 caused female infertility, this work established a critical role of the p31comet-TRIP13 module in regulating meiotic progression during oogenesis. As such, this is a significant study.

Thank you for the very positive comments on this manuscript.

Reviewer #3 (Public Review):

The spindle checkpoint ensures the accuracy of chromosome segregation by sensing unattached kinetochores during mitosis and meiosis and delays the onset of anaphase. Unattached kinetochores catalyze the conformational activation of the latent open MAD2 (O-MAD2) to the active closed MAD2 (C-MAD2). C-MAD2 is then incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex or cyclosome (APC/C) to delay anaphase. When all kinetochores are properly unattached, the MAD2-binding protein p31comet and the ATPase TRIP13 extract C-MAD2 from the MCC, leading to MCC disassembly and the conversion of C-MAD2 back to O-MAD2. This action turns off the spindle checkpoint, resulting in APC/C activation and anaphase onset. Cells deficient in p31comet exhibit mitotic delays.

In the current study, Huang et al. have linked p31comet mutations to female infertility. Biallelic loss-of-function alleles of p31comet cause delays in the exiting metaphase of meiosis I and polar body extrusion. The p31comet mutant proteins contain C-terminal truncations and fail to bind to MAD2. Reintroducing full-length p31comet into patient oocytes can bypass the metaphase arrest. Together with a previous study that showed biallelic mutations of TRIP13 caused female infertility, this work established a critical role of the p31comet-TRIP13 module in regulating meiotic progression during oogenesis. As such, this is a significant study.

Reviewer #1 (Public Review):

Wang et al., present a paper aiming to identify NALCN and TRPC6 channels as key mechanisms regulating VTA dopaminergic neuron spontaneous firing and investigating whether these mechanisms are disrupted in a chronic unpredictable stress model mouse.

Major strengths:

-This paper uses multiple approaches to investigate the role of NALCN and TRPC6 channels in VTA dopaminergic neurons.

Major weaknesses:

-The pharmacological tools used in this study are highly non-selective. Gd3+, used here to block NALCN is actually more commonly used to block TRP channels. 2-APB inhibits not only TRPC channels, but also TRPM and IP3 receptors while stimulating TRPV channels (Bon and Beech, 2013), while FFA actually stimulates TRPC6 channels while inhibiting other TRPCs (Foster et al., 2009).

Are the author's claims supported by the data?

-The multimodal approach including shRNA knockdown experiments alleviates much of the concern about the non-specific pharmacological agents. Therefore, the author's claim that NALCN is involved in VTA dopaminergic neuron pacemaking is well-supported.

-However, the claim that TRPC6 is the key TRPC channel in VTA spontaneous firing is somewhat, but not completely supported. As with NALCN above, the pharmacology alone is much too non-specific to support the claim that TRPC6 is the TRP channel responsible for pacemaking. However, unlike the NALCN condition, there is an issue with interpreting the shRNA knockdown experiments. The issue is that TRPC channels often form heteromers with TRPC channels of other types (Goel, Sinkins and Schilling, 2002; Strübing et al., 2003). Therefore, it is possible that knocking down TRPC6 is interfering with the normal function of another TRPC channel, such as TRPC7 or TRPC4.

-The claim that TRPC6 channels in the VTA are involved in the depressive-like symptoms of CMUS is supported.

- However, the connection between the mPFC-projecting VTA neurons, TRPC6 channels, and the chronic unpredictable stress model (CMUS) of depression is not well supported. In Figure 2, it appears that the mPFC-projecting VTA neurons have very low TRPC6 expression compared to VTA neurons projecting to other targets. However, in figure 6, the authors focus on the mPFC-projecting neurons in their CMUS model and show that it is these neurons that are no longer sensitive to pharmacological agents non-specifically blocking TRPC channels (2-APB, see above comment). Finally, in figure 7, the authors show that shRNA knockdown of TRPC6 channels (in all VTA dopaminergic neurons) results in depressive-like symptoms in CMUS mice. Due to the low expression of TRPC6 in mPFC-projecting VTA neurons, the author's claims of "broad and strong expression of TRPC6 channels across VTA DA neurons" is not fully supported. Because of the messy pharmacological tools used, it cannot be clamed that TRPC6 in the mPFC-projecting VTA neurons is altered after CMUS. And because the knockdown experiments are not specific to mPFC-projecting VTA neurons, it cannot be claimed that reducing TRPC6 in these specific neurons is causing depressive symptoms.

Impact:

It is valuable to compare pacemaking mechanisms in VTA and SNc neurons and this paper convincingly shows that NALCN contributes to VTA pacemaking, as it is known to contribute to SNc pacemaking. It also shows that TRPC6 channels in VTA dopamine neurons contribute to the depressive-like symptoms associated with CMUS.

It is important to note that the experiments presented in Figure 1 have all been previously performed in VTA dopaminergic neurons (Khaliq and Bean, 2010) including showing that low calcium increases VTA neuron spontaneous firing frequency and that replacement of sodium with NMDG hyperpolarizes the membrane potential.

Additional context:

-The authors explanation for the increase in firing frequency in 0 calcium conditions is that calcium-activated potassium channels would no longer be activated. However, there is a highly relevant finding that low calcium enhances the NALCN conductance through the calcium sensing receptor from Dejian Ren's lab (Lu et al., 2010) which is not cited in this paper. This increase in NALCN conductance with low calcium has been shown in SNc dopaminergic neurons (Philippart and Khaliq, 2018), and is likely a factor contributing to the low-calcium-mediated increase in spontaneous VTA neuron firing.

-One of the only demonstrations of the expression and physiological significance of TRPCs in VTA DA neurons was published by (Rasmus et al., 2011; Klipec et al., 2016) which are not cited in this paper. In their study, TRPC4 expression was detected in a uniformly distributed subset of VTA DA neurons, and TRPC4 KO rats showed decreased VTA DA neuron tonic firing and deficits in cocaine reward and social behaviors.

- Out of all seven TRPCs, TRPC5 is the only one reported to have basal/constitutive activity in heterologous expression systems (Schaefer et al., 2000; Jeon et al., 2012). Others TRPCs such as TRPC6 are typically activated by Gq-coupled GPCRs. Why would TRPC6 be spontaneously/constitutively active in VTA DA neurons?

-A new paper from the group of Myoung Kyu Park (Hahn et al., 2023) shows in great detail the interactions between NALCN and TRPC3 channels in pacemaking of SNc DA neurons.

References

Bon, R.S. and Beech, D.J. (2013) 'In pursuit of small molecule chemistry for calcium-permeable non-selective TRPC channels -- mirage or pot of gold?', British Journal of Pharmacology, 170(3), pp. 459-474. Available at: https://doi.org/10.1111/bph.12274.

Foster, R.R. et al. (2009) 'Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells', Cell Calcium, 45(4), pp. 384-390. Available at: https://doi.org/10.1016/j.ceca.2009.01.003.

Goel, M., Sinkins, W.G. and Schilling, W.P. (2002) 'Selective association of TRPC channel subunits in rat brain synaptosomes', The Journal of Biological Chemistry, 277(50), pp. 48303-48310. Available at: https://doi.org/10.1074/jbc.M207882200.

Hahn, S. et al. (2023) 'Proximal dendritic localization of NALCN channels underlies tonic and burst firing in nigral dopaminergic neurons', The Journal of Physiology, 601(1), pp. 171-193. Available at: https://doi.org/10.1113/JP283716.

Jeon, J.-P. et al. (2012) 'Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels', The Journal of Biological Chemistry, 287(21), pp. 17029-17039. Available at: https://doi.org/10.1074/jbc.M111.326553.

Khaliq, Z.M. and Bean, B.P. (2010) 'Pacemaking in dopaminergic ventral tegmental area neurons: depolarizing drive from background and voltage-dependent sodium conductances', The Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 30(21), pp. 7401-7413. Available at: https://doi.org/10.1523/JNEUROSCI.0143-10.2010.

Klipec, W.D. et al. (2016) 'Loss of the trpc4 gene is associated with a reduction in cocaine self-administration and reduced spontaneous ventral tegmental area dopamine neuronal activity, without deficits in learning for natural rewards', Behavioural Brain Research, 306, pp. 117-127. Available at: https://doi.org/10.1016/j.bbr.2016.03.027.

Lu, B. et al. (2010) 'Extracellular calcium controls background current and neuronal excitability via an UNC79-UNC80-NALCN cation channel complex', Neuron, 68(3), pp. 488-499. Available at: https://doi.org/10.1016/j.neuron.2010.09.014.

Philippart, F. and Khaliq, Z.M. (2018) 'Gi/o protein-coupled receptors in dopamine neurons inhibit the sodium leak channel NALCN', eLife, 7. Available at: https://doi.org/10.7554/eLife.40984.

Rasmus, K. et al. (2011) 'Sociability is decreased following deletion of the trpc4 gene', Nature Precedings, pp. 1-1. Available at: https://doi.org/10.1038/npre.2011.6367.1.

Schaefer, M. et al. (2000) 'Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5', The Journal of Biological Chemistry, 275(23), pp. 17517-17526. Available at: https://doi.org/10.1074/jbc.275.23.17517.

Strübing, C. et al. (2003) 'Formation of novel TRPC channels by complex subunit interactions in embryonic brain', The Journal of Biological Chemistry, 278(40), pp. 39014-39019. Available at: https://doi.org/10.1074/jbc.M306705200.

Conversion chart of AC voltage based calculations

Essto me ayuda un montón!

são estruturas que abrigam o material genético dentro da célula

Scholars tend consistently if not quite unanimously to emphasise the ambiguity of Levi’s term ‘gigantesco’. The discussion of Dante’s Ulysses is ‘broken off’, as Hayden White puts it, before Levi can tell us ‘what we are supposed to conclude’ (2015, 12). As a result, ‘there is no final manifestation of the message, of the meaning that Levi is so desperately trying to grasp and communicate’, argues Giuseppe Stellardi (2019, 715). ‘Nessuno potrà mai affermare nulla con sicurezza’, assert Alberto Cavaglion and Paolo Valabrega (515). The lack of certainty regarding the term’s meaning is perhaps responsible for the substantial divergence between the critical interpretations that this passage has inspired.

There are those who argue that Levi’s ‘gigantic’ discovery when reciting Dante in Auschwitz is an unconquerable ‘faith in his culture’ (Hartman 1996, 52), and those who claim, conversely, that Levi instead recognises how ‘behind the gas chambers, the ovens, the starvation rations, and the astonishing otherworldly everyday viciousness and cruelty can be found at the heart of Western culture’ (Feinstein 2003, 365). In other words, while some hold that Levi finds in Dante the antidote to Auschwitz, others argue that he finds the cause.

Levi’s subsequent glosses on this passage have done little to alleviate the uncertainty. In the version of SQ that he prepared for a 1964 radio broadcast, he clarified the meaning of the quoted phrase ‘come altrui piacque’ (OC I, 1237); for the 1973 Schools edition of SQ, intended for an audience of Italian students, he provided footnotes explaining the term ‘anacronismo’ and the phrase ‘il perché del nostro destino’ (OC I, 1417-18). In both instances, Levi left ‘gigantesco’ undefined. This apparent authorial reticence should inspire some restraint in our critical exegesis. There is no need to pursue false certainty where the text offers legitimate ambiguity.

What we may note, however, is that elsewhere in SQ, and with significant frequency in Levi’s subsequent writing, the term ‘gigantesco’ serves to identify the monstrosity of the Lager. In the chapter ‘I sommersi e i salvati’, he describes Auschwitz as ‘una gigantesca esperienza biologica e sociale’ (OC I, 217). In the aforementioned school edition of SQ, he explains the historical shift in Nazi policies that transformed concentration camps into ‘gigantesche macchine di morte’ (OC I, 292). In a 1955 article celebrating the tenth anniversary of Italy’s liberation from Fascism, he describes how the Nazis ‘[h]anno lavorato con tenacia a creare la loro gigantesca macchina generatrice di morte e di corruzione’ (OC II, 1293). In a 1968 preface to a book on Auschwitz, he argues that the vital question remains ‘per quali ragioni e cause, prossime o lontane, abbia potuto nascere in questo civile continente una gigantesca fabbrica di morte’ (OC II, 1357). In the 1975 article ‘Così fu Auschwitz’, he describes what he calls the Nazis’ ‘costituzione di un gigantesco esercito di schiavi, non retribuiti e costretti a lavorare fino alla morte’ (OC II, 1374). In a 1979 response to the broadcast of the TV mini series Holocaust, he notes how the public seemed to focus on the question of why the genocide of the European Jews had occurred, ‘e questo è un perché gigantesco, ed antico quanto il genere umano: è il perché del male nel mondo’ (OC II, 1456). The accretion of these examples is by no means definitive; the ‘qualcosa di gigantesco’ that Levi discovered in discussing Dante with Jean may well differ from the ‘perché gigantesco’, the ‘perché del male nel mondo’, on which he deliberated decades later. Nevertheless, there would appear to be a pattern.

It is a pattern, moreover, that obtains well beyond Levi’s own work. To cite just one relevant example, the Italian anti-Fascist expatriate Giuseppe Antonio Borgese entitled his 1937 study of the totalitarian movement in Italy Goliath: The March of Fascism. And in that text Borgese lay the ultimate blame for the enormity of Fascism not on the Duce—‘it is futile […] to explain Fascism as if it were the creation of a single man, Mussolini’—but rather on another ‘gigantic individuality’ in the Italian national pantheon: Dante, who ‘distorted the soul of his people’, giving rise to ‘Nationalism and Racialism’ (46). Many Fascists, too, claimed Dante as the founder of their movement (Albertini 2013). Not for nothing did ‘Giovinezza’, the Fascist anthem, boast that ‘la vision dell’Alighieri, | oggi brilla in tutti i cuor’ (Pugliese 2001, 55). A 1927 study of Dante Alighieri e Benito Mussolini argued that the Duce’s Italy was closer than ever to Dante’s ideal (7-8). The Fascist Party’s own 1940 Dizionario di Politica made the same claim (735). ‘Che Dante sia Fascista lo dimostrano tutte le sue opere’, insisted one of the regime’s intellectuals; ‘solo oggi possiamo riconoscere in Dante il profeta del nostro destino’, maintained another (Scorrano 2001, 92-93, 198).

It is perhaps tempting to hear the echo of such sentiments in Levi’s epiphany that in Dante’s ‘Canto di Ulisse’ is to be found ‘il perché del nostro destino’. After all, Levi had learned his Dante in an Italian school system that had been turned to Fascism’s totalitarian aims. If he is indeed suggesting to Jean that abuses of Dante - the appropriation of tradition to support the arrogation of power in the present; the fraudulent claims to a divine warrant for violence - bear responsibility for the Häftlinge’s tragic fate, he has good reason.

Yet those who interpret Levi’s Dante lesson in a more liberatory manner have good reason as well. If the inhuman contrapasso of eternal damnation ‘come altrui piacque’ is to be found in Dante, so, too, is Ulysses’ call for an innate and inviolable human dignity: ‘considerate la vostra semenza’. Borgese in Goliath recognised this duality (12). So, too, I suspect, does Primo Levi in ‘Il canto di Ulisse’. Perhaps this is the ‘gigantic’ discovery that Levi has made in his meditation on Dante’s Inferno: that in our cultural inheritance are to be found both the roots of Fascism and the seeds of resistance.

CLL

Very often, when we think about ‘Il canto di Ulisse’, we tend to recall only the most famous pages in which Levi tries to remember Dante’s canto. The depth and sense of urgency of the Ulyssean passages are so overwhelming and passionate that they may distract us from other elements in the chapter. However, if we go back to the text and read it closely, we cannot avoid noticing that, after a brief opening in which Levi introduces Pikolo and narrates how he came to be Pikolo’s ‘fortunate’ chaperone to collect the soup for the day, ‘Il canto di Ulisse’ also dwells quite significantly on a moment of domestic memories. While going to the kitchens, Levi writes: ‘Si vedevano i Carpazi coperti di neve. Respirai l’aria fresca, mi sentivo insolitamente leggero’. This is the first moment in the chapter in which Levi refers to the mountains as something that revitalises him and makes him feel fresh and light, both physically and mentally.

This moment foreshadows another, also in this chapter, when Levi goes back to his mountains, those close to Turin, and compares them to the mountain that the protagonist of Dante’s canto, Ulysses, encounters just before his shipwreck with his companions:

... Quando mi apparve una montagna, bruna

Per la distanza, e parvemi alta tanto

Che mai veduta non ne avevo alcuna.

Sì, sì, ‘alta tanto’, non ‘molto alta’, proposizione consecutiva. E le montagne, quando si vedono di lontano... le montagne... oh Pikolo, Pikolo, di’ qualcosa, parla, non lasciarmi pensare alle mie montagne, che comparivano nel bruno della sera quando tornavo in treno da Milano a Torino! Basta, bisogna proseguire, queste sono cose che si pensano ma non si dicono. Pikolo attende e mi guarda. Darei la zuppa di oggi per saper saldare ‘non ne avevo alcuna’ col finale.

The significance of the mountains in Levi’s narration is confirmed in this passage. For him, the mountains represent his experience of belonging, his youthful years, and his work as a chemist – the job he was doing when he commuted by train from Turin to Milan. At the same time, Levi’s own memories of the mountains intertwine and overlap with another mountain, Dante’s Mount Purgatory. Here, a deep and perhaps not fully conscious intertextual game starts to emerge and to characterise Levi’s writing. The lines that Levi does not remember are these:

Noi ci allegrammo, e tosto tornò in pianto,

ché de la nova terra un turbo nacque,

e percosse del legno il primo canto.

For Dante’s Ulysses, Mount Purgatory signifies the final moment of his adventure and his desire for knowledge. The marvel and enthusiasm that Ulysses and his company feel when they see the mountain is suddenly transformed into its contrary. From the mountain, a storm originates that will destroy the ship and swallow its crew: ‘Tre volte il fe’ girar con tutte l’acque, | Alla quarta levar la poppa in suso | E la prora ire in giù, come altrui piacque’. Dante’s Mount Purgatory, so majestic and spectacular, represents the end of any desire for knowledge that aims to find new answers to and interpretations of human existence in the world without God’s word.

Going back to Levi’s text, we find that, instead, in a kind of reverse overlapping between his image and that of Ulysses, the image of the mountain of Purgatory suggests to Levi a very different set of thoughts that, although seemingly and similarly overwhelming, opens up new interpretations: ‘altro ancora, qualcosa di gigantesco che io stesso ho visto ora soltanto, nell’intuizione di un attimo, forse il perché del nostro destino, del nostro essere oggi qui’. For a moment, it is almost as if Levi, a new Dantean Ulysses in a new Inferno, stands in front of Mount Purgatory and forgets the terzine and the shipwreck. Maybe Levi cannot or does not want to remember those terzine because the mountain in Purgatory represents something very different for him than for Dante’s Ulysses. Levi’s view of the mountain does not lead to a moment of recognition of sin, as it does in Dante’s Ulysses. For him, the mountain, like his mountain range, is the gateway to knowledge, enrichment, and illumination and to a world that lies beyond the imposed limits of traditional, constricting, and distorted views and that awaits discovery (‘qualcosa di gigantesco che io stesso ho visto ora soltanto’). Something about and beyond the Lager.

To better understand how the mountains are central in ‘Il canto di Ulisse’, we have to remember that Levi’s view of the mountains strongly depends on his anti-Fascism, which he expressed particularly vigorously in two moments of his life: during his months in the Resistance, just before he was captured and sent to Fossoli, and, even more intensely, during the adventures of his youth, when he was a free young man who enjoyed climbing the mountains surrounding Turin. As Alberto Papuzzi has suggested, ‘le radici del suo rapporto con la montagna sono ben piantate in quella stagione più lontana: radici intellettuali di cittadino che cercava sulla montagna, nella montagna, suggestioni e risposte che non trovava nella vita, o meglio nell’atmosfera ispessita di quella vita torinese, senza passato e senza futuro’ (OC III, 426-27). Indeed, reports Papuzzi, Levi confirms that:

Avevo anche provato a quel tempo a scrivere un racconto di montagna […]. C’era tutta l’epica della montagna, e la metafisica dell’alpinismo. La montagna come chiave di tutto. Volevo rappresentare la sensazione che si prova quando si sale avendo di fronte la linea della montagna che chiude l’orizzonte: tu sali, non vedi che questa linea, non vedi altro, poi improvvisamente la valichi, ti trovi dall’altra parte, e in pochi secondi vedi un mondo nuovo, sei in un mondo nuovo. Ecco, avevo cercato di esprimere questo: il valico.

The heart of that epic story made its way into the chapter ‘Ferro’ in Il sistema periodico. The discovery of this (brave) new world, ‘mondo nuovo’, is an integral part and a direct achievement of Levi’s experience in the mountains. The mountains open a new understanding and a new perspective on the world.

Something that escapes common understanding is revealed through the experience of the mountains, both in Levi’s memories of his youth and in his literary recounting of Auschwitz. Reciting Dante in ‘Il canto di Ulisse’ is therefore not only an intertextual exercise for Levi. Only by inserting Levi’s literary references in the complexity of his own experience – before, during, and after Auschwitz – can we fully capture the depth of his reflections. Levi mentally and metaphorically brought to Auschwitz not only Dante but also his ‘metafisica dell’alpinismo’. Together, they contributed to his attempt to come to terms with that reality.

VG

This is not the only porcupine to appear in Levi’s writing. In The Truce, we learn that Levi’s companion Cesare, disappointed after a misadventure on the black market, spent two days ‘huddled on his bed, bristling like a porcupine’ (CW I, 338). In The Wrench, Faussone identifies a clearing in which ‘a porcupine was advancing cautiously, with brief stops and starts’ (CW II, 1025). These English translations suggest a possible connection to SQ that is less obvious in the original Italian, where the text refers not to an ‘istrice’ but rather to a ‘porcospino’. In La tregua, Cesare is described as ‘ispido come un porcospino’ (OC I, 417), and in La chiave a stella, Faussone points out that ‘un porcospino avanzava cauto, con brevi arresti e riprese’ (OC I, 1099).

The terms ‘istrice’ and ‘porcospino’ refer to animals of the same family, Hystricidae, and identify the same species, Hystrix cristata, the crested porcupine, which is native to Italy. In the Grande dizionario italiano dell’uso, ‘porcospino’ is listed as a synonym of ‘istrice’, which is defined scientifically as a ‘piccolo mammifero con il corpo coperto di aculei appuntiti ed erettili’, with a second figurative meaning as a ‘persona intrattabile, scontrosa’ (805).

Despite their similarity, there is a notable difference between the two synonyms with regard to their literary resonances. As the Tesoro della lingua Italiana delle Origini demonstrates, ‘istrice’ was the preferred term for medieval philosophers, historians, and poets, including Boccaccio, who used it in his Caccia di Diana and Ameto, where the husband’s beard is described as being ‘né più né meno pugnente che le penne d’uno istrice’ (Tutte le opera di Giovanni Boccaccio, 774). The Grande dizionario della lingua italiana attests subsequent citations from Parini, D’Annunzio, De Amicis, and Foscolo, with the latter two adopting the term metaphorically to refer to a person who is taciturn and cagey (615).

I suspect that Levi had another literary reference in mind when he opted for ‘istrice’ rather than ‘porcospino’ in SQ. Here are the words with which the Ghost in Shakespeare’s Hamlet reveals both his identity and the infernal torments he suffers in the afterlife:

I am thy father’s spirit,

Doom’d for a certain term to walk the night,

And for the day confined to fast in fires,

Till the foul crimes done in my days of nature

Are burnt and purged away. But that I am forbid

To tell the secrets of my prison-house,

I could a tale unfold whose lightest word

Would harrow up thy soul, freeze thy young blood,

Make thy two eyes, like stars, start from their spheres,

Thy knotted and combined locks to part

And each particular hair to stand on end,

Like quills upon the fretful porpentine:

But this eternal blazon must not be

To ears of flesh and blood. List, list, O, list! (Hamlet, Act I, Scene 5, vv. 14-28)

In standard Italian translations dating back at least to the early nineteenth century, Shakepeare’s ‘fretful porpentine’ is rendered as a ‘pauroso istrice’ (Amleto, 59). This word, and these lines, would seem to resonate remarkably well with Levi’s description of the hell of Auschwitz, which is the context for his invocation of ‘la difesa dell’istrice’.

After all, Hamlet’s Ghost is compelled to speak quickly, in the brief interval he has been granted in his eternal suffering:

My hour is almost come,

When I to sulphurous and tormenting flames

Must render up myself (Hamlet, Act I, Scene 5, vv. 5-7)

Cannot Levi and Jean say the same thing? The ‘lungo giro’ that Jean has arranged buys them a brief respite, but this precious time has begun to disappear as soon as it arrives: ‘quest’ora già non è più un’ora’. Cannot Hamlet’s Ghost say the same thing?

The moment of connection and communication at the heart of ‘Il canto di Ulisse’ can be said to have begun with Jean’s clever strategy to curry favour with cruel Alex, the Kapo Levi describes as ‘un bestione violento e infido’, who is won over by Jean’s ‘opera lenta cauta e sottile’, finally ceding to him the coveted role of Pikolo. It is this victory that Levi describes as penetrating ‘the porcupine’s defence’. And it is this victory that frees Jean to choose Levi for the task of fetching the daily soup ration, enabling the disquisition on Dante that gives the chapter its title.

If I am correct that the reference to ‘la difesa dell’istrice’ is thus evidence that Levi and Jean’s Dantean voyage begins under the sign of Hamlet, this would be a particularly elegant literary manoeuvre, since the voyage concludes under the very same sign. As their hour runs out once they have reached the kitchen, Levi finds himself unable to say all that needs to be said and is forced to concede that ‘il resto è silenzio’, an unmistakable echo of Hamlet’s final words: ‘the rest is silence’ (Hamlet, Act V, Scene 2, v. 395).

If further evidence for a Shakespearean source text is warranted, I would note that Levi included in Ad ora incerta a poem that explicitly references Hamlet’s Ghost, who is referred to as an ‘old mole’ because he continues to speak from beneath the floorboards (Hamlet, Act I, Scene 5, v. 183). Italian translations render this line as ‘vecchia talpa’, words that Levi borrowed for the title of a 1982 poem, which literalises the reference - the poem is in fact written from the perspective of an old mole - while nevertheless conveying the sense of the original, with its profound intimations of the latent power of buried knowledge.

In altri tempi seguivo le femmine,

E quando ne sentivo una grattare

Mi scavavo la via verso di lei:

Ora non più; se capita, cambio strada.

Ma a luna nuova mi prende il morbino,

E allora qualche volta mi diverto

A sbucare improvviso per spaventare i cani. (OC II, 727)

The reference to an ‘istrice’ in ‘Il canto di Ulisse’ similarly suggests hidden depths.

CLL

‘The Canto of Ulysses’ records a truly remarkable feat of cultural memory, as Levi recalls and recites Dante amidst the ruin of Auschwitz. He does so to teach Italian to his campmate Jean. But as he recites the words of Dante, Levi also feels his past come back to him. He begins to recover something of his identity and his humanity in the degraded world of the Lager, to feel again that he is ‘a man’.

Dante is not, however, the only canonical writer to appear in ‘The Canto of Ulysses’. So too does Shakespeare. <br /> Levi admits to ‘gaps’, ‘holes’, and ‘lacuna[e]’ in his memory as he recites Dante, some of which would seem to be ‘irreparable’. Unable to recall his Dante, Levi is forced to confront ‘silence’:

I would give today’s soup to know how to connect ‘the like on any day’ to the last lines. I try to reconstruct it through rhymes, I close my eyes, I bite my fingers – but it is no use, the rest is silence [il resto è silenzio]. Other verses dance in my head: ‘…The sodden ground belched wind …’, no, it is something else. [Woolf’s translation.]

‘[T]he rest is silence’: Levi is quoting the last words that are spoken by Hamlet, before he dies:

O, I die, Horatio!

The potent poison quite o’ercrows my spirit.

I cannot live to hear the news from England.

But I do prophesy th’election lights

On Fortinbras; he has my dying voice.

So tell him, with th’occurrents more and less

Which have solicited – the rest is silence.

With his ‘spirit’ succumbing to the ‘potent poison’ Claudius and Laertes have used to kill him, Hamlet undertakes the impossible: to testify to his own death, to ‘tell’ in words his fall into deathly silence. This is his ‘dying voice’.

What is Hamlet doing in ‘The Canto of Ulysses’? Why does Levi place it where he does, in ‘the caesura’, the abyssal gap between Auschwitz and his cultural identity and memory, embodied by Dante? It is certainly ironic that Levi draws once again on the Western canonical literary tradition to record the moment of its ostensible breakdown. What emerges from the lapse, the silence that Levi testifies to, is another tie to his compromised past, and the literary culture that would seem to have been obliterated by the Holocaust – even if Levi does not choose to bring obvious notice to his allusion by using quotation marks or by writing, ‘as Hamlet says’. The poetry of Hamlet appears to be among the ‘other verses’ Levi has confusedly ‘dancing in his head’ while he tries to fill the gaps in his memory.

By quoting Hamlet, Levi would appear to again testify to the power of literature as a mainstay of culture and humanity, evincing his commitment to humanist ideals. This is certainly the positive interpretation of Hamlet in ‘The Canto of Ulysses’. I would make the case, however, that Levi is using Shakespeare for an altogether more challenging purpose. By appropriating the ‘dying voice’ of Hamlet, Levi records the place where his memory and his identity collapse, testifying to the reduction of the camp inmate to silence and oblivion. Bryan Cheyette writes that ‘even when his memory self-consciously fails him’, Levi is always ‘at pains to bear witness to those moments of failure’ (Cheyette 1999, 64). Levi seeks to testify to the silence, to show that ‘something has occurred even if it cannot be understood’ (Druker 2009, 64). This is the role played by Hamlet in ‘The Canto of Ulysses’. Levi does not use the play to testify to the endurance of the human spirit in the camps, but to silence, to a ‘world of negation’ (OC I, 235). Hamlet, perhaps ironically given its near-unrivalled canonical and cultural status, marks the space beyond language and culture, into which Levi is at risk of falling. This, as Levi called it, is the ‘black hole’ of Auschwitz (OC II, 1663).

Through his allusion to Hamlet, Levi rewrites Shakespeare from the perspective of Auschwitz, endowing the play with new meanings as he confronts the lacunae and voids produced by the world of the concentration camp. Levi uses the play to record the disintegration of humane values in Auschwitz, as memory and language are brought to the point of collapse. Jacques Derrida does much the same in his own work on the play. He draws Hamlet into conversation with Holocaust testimony when he compares the play to the poetry of survivor Paul Celan in his 1995 piece ‘The Time is Out of Joint’. Derrida contends that the paradox of testimony is that the witness must uncannily ‘outlive his life’ (3.2.117) – or ‘survive’ that which is not ‘survivable’: the collapse of all meaning and death (Derrida 1995). This is the sense in which Hamlet is a play about the ‘impossible possibility of testimony’, as Derrida calls it. Hamlet has ‘seen the worst’ and is ‘the witness of the worst disorder, of absolute injustice’, writes Derrida. Hamlet has witnessed too much for words – but testimony, ‘though it hath no tongue, will speak’ (2.2.546).

Levi must also confront and testify to the painful death of memory, the destruction of human identity and culture, before the event of physical death itself – or as Jacques Lacan would call it, ‘symbolic’ before ‘actual’ death, the death of the self before physical death. Lawrence Langer uses the phrase ‘deathlife’ to name the same phenomenon, of ‘dying while one is living’ (Langer 2021, 13). Not unlike the melancholic prince, Levi attempts the impossible of testifying to his own demise. He deploys Hamlet to record his fall into a place beyond humanity and beyond culture – even beyond language. It is, to adopt the words of Jean Améry, an act of both resignation and revolt: resignation to silence, and a determined revolt against oblivion, by testifying to it. It is an astonishing moment.

RA

Among many other layers to this chapter and its revelations, the ‘qualcosa di piú’ is also a reference to poetry as a means to escape Auschwitz. Poetry itself becomes the means to resist the process of bestialisation and reification.

MJ

DOI: 10.1210/endocr/bqad072

Resource: (Thermo Fisher Scientific Cat# 31430, RRID:AB_228307)

Curator: @scibot

SciCrunch record: RRID:AB_228307

What is this?

Entre el 2009 y 2016, OSINFOR emitió 3706 resoluciones de inicio de procedimientos sancionadores (PAU),a partir de los indicios de infracciones identificados en la evaluación legal de los informes de supervisión. Entre el 2013 y 2014, se emitieron la mayor cantidad de resoluciones, ya que fueron 677 y 664 respectivamente. Posteriormente, esas cifras disminuyeron, ya que en el 2015 se emitieron 497 resoluciones y en 2016 solo 567 resoluciones.

Por otro lado, también se tiene información respecto a la cantidad de resoluciones directorales de finalización de PAU emitidas entre el 2009 y 2016. Estas resoluciones se emiten cuando se archivan expedientes al no confirmarse alguna infracción o cuando se aplican sanciones al verificarse la afectación al bosque.

Entre el 2009 y 2014, se observa un crecimiento progresivo de la cantidad de resoluciones emitidas, pasando de 2 resoluciones en el 2009 a 1100 resoluciones en el 2014. Posteriormente, en el 2015 a cantidad de resoluciones decayó a 796 y finalmente en el 2016 solo se emitieron 547 resoluciones.

because living in the present moment w/o an exteriorization of the psychological and emotional anguish/turmoil inside is too unbearable—physical pain is cathartic in that way?

also, if restriction is moralized and thinness is valorized as the ideal aesthetic for health and desirability, positive affect may result from the embodied practice of ED-related beliefs —> positive reinforcement as it ameliorates cognitive and emotional hardship

Author Response

Reviewer #1 (Public Review):

This article describes the development and refinement of an open-source software framework that is used to track how the COVID-19 pandemic impacted healthcare use in England over a range of key healthcare use indicators.

Important strengths of this study include the high coverage of 99% of practices in England, the development of health care indicators with the input of a clinical advisory group, extensive online documentation, and rigorous safeguards for the protection of patient confidentiality.

Perhaps the largest limitation is that only high-level descriptive data on the monthly volume of health outcomes are presented. It is not clear whether the system could be used to generate more fine-grained or stratified information, ex. weekly or daily data, or data stratified by important characteristics of practices or of patient characteristics. As such, the utility of the system for answering new scientific questions is unclear, and also what the utility and long-term potential uses of this system will be past the COVID-19 pandemic.

OpenSAFELY allows access to the full primary care record for patients registered with a TPP or EMIS practice in England.This includes medical diagnoses, clinical tests, prescriptions, as well as demographic details such as age, sex, ethnicity. Dates attached to these records allow for daily analyses to be performed. This data is updated weekly. Through linkage of other data sources, it also provides information such as hospital admissions, registered deaths or COVID-19 testing data. Detailed subgroup analysis is possible; OpenSAFELY has already been used to understand disease risk 1, monitor vaccination coverage 2,3 and novel treatments 4, assess patient safety 5, inform public health guidance and policy and much more6. These are all widely applicable beyond the COVID-19 pandemic.

Reviewer #3 (Public Review):

This manuscript by Fisher and colleagues documents the change in clinical activity in English general practices during the COVID-19 pandemic according to a set of indicators of clinical activity. The indicators include measures of clinical reviews (e.g. blood pressure, asthma, chronic obstructive pulmonary disease, medication, and cardiovascular risk reviews), blood tests (e.g. cholesterol, liver function, thyroid function, full blood counts, diabetes monitoring blood tests, and kidney function). All these measures saw a drop during the pandemic, to a varying degree, and some recovered afterwards but others did not.

Clinical activity was measured using SNOMED CT codes, which are standard codes used for recording clinical events in UK GP records.

Strengths:

This is a large and comprehensive study including data from 99% of general practices in England. The indicators are clinically relevant, cover a broad range of disease areas, and have been chosen in a sensible manner, involving relevant stakeholders such as GPs, pharmacists, and pathologists.

The OpenSAFELY platform has the ability to enable federated analyses to be run on raw coded data of almost all patients registered with a GP in England.

The study demonstrates the value of OpenSAFELY in being able to monitor clinical activity in general practice at a detailed level, which is essential for planning and improving health services. The statistical methodology is broadly sound.

Weaknesses:

The measures are all related to chronic physical diseases in adults, with a particular focus on cardiometabolic and respiratory conditions. There are no measures related to mental health, maternal or child health.

Results from preliminary analyses of a wider range of clinical conditions can be found in our previous work7. This includes mental health and female and reproductive health with details on why these were not covered by the initial key measures described.

The description of the measures does not distinguish between different types of clinical activity e.g. lab tests, clinical measurements, or diagnoses, and all are lumped together as 'codes'. This is a peculiarity of the way that information is recorded in GP systems - many different types of clinical information (such as diagnoses and lab tests) are recorded using a SNOMED CT 'code', and only the exact code differentiates what type of information is in the record.

Multiple codes of different types can arise from a single encounter, all of which could be indicative of a clinical event of interest. The codelists for each key measure, available at opencodelists.org shows the type of clinical activity (e.g procedure or observable entity) captured by each code within the codelist (see e.g.https://www.opencodelists.org/codelist/opensafely/red-blood-cell-rbc-tests/576a859e/#tree).

The codelists were broad and comprehensive, but it is unclear how necessary this is because for some measures e.g. lab tests, laboratories typically record a particular type of test using a single standardised code. Instead of using a broad set of codes in the analysis, the authors could have initially verified which codes are associated with the clinical activity being measured (e.g. a numerical value of a blood pressure measurement) in all practices, as I would expect the same single or small number of codes would be used in all practices. This would have provided a smaller and simpler final codelist.

Supplementary table 1 shows up to 5 of the most common codes for each key measure across the two electronic health record (EHR) systems used in this analysis. This shows that whilst a single code is often used for many of the clinical activities assessed here, there are exceptions and there can be variation in coded activity between different EHR systems. We have previously described how design features of EHR systems can impact clinical practice 8. Broad codelists allow us to capture activity across multiple EHR systems.

1. Williamson, E. J. et al. Factors associated with COVID-19-related death using OpenSAFELY. Nature 584, 430–436 (2020).
2. Trends and clinical characteristics of 57.9 million COVID-19 vaccine recipients: a federated analysis of patients’ primary care records in situ using OpenSAFELY | British Journal of General Practice. https://bjgp.org/content/early/2021/11/08/BJGP.2021.0376.
3. Parker, E. P. et al. Factors associated with COVID-19 vaccine uptake in people with kidney disease: an OpenSAFELY cohort study. BMJ Open 13, e066164 (2023).
4. Green, A. C. A. et al. Trends, variation, and clinical characteristics of recipients of antiviral drugs and neutralising monoclonal antibodies for covid-19 in community settings: retrospective, descriptive cohort study of 23.4 million people in OpenSAFELY. BMJ Med. 2, (2023).
5. Collaborative, T. O. et al. Potentially inappropriate prescribing of DOACs to people with mechanical heart valves: a federated analysis of 57.9 million patients’ primary care records in situ using OpenSAFELY. 2021.07.27.21261136 https://www.medrxiv.org/content/10.1101/2021.07.27.21261136v1 (2021) doi:10.1101/2021.07.27.21261136.
6. OpenSAFELY Pubmed search results. PubMed https://pubmed.ncbi.nlm.nih.gov/?term=OpenSAFELY.
7. OpenSAFELY NHS Service Restoration Observatory 2: changes in primary care activity across six clinical areas during the COVID-19 pandemic | medRxiv. https://www.medrxiv.org/content/10.1101/2022.06.01.22275674v1.
8. Suboptimal prescribing behaviour associated with clinical software design features: a retrospective cohort study in English NHS primary care | British Journal of General Practice. https://bjgp.org/content/70/698/e636.

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Reply to the reviewers

Reply to the reviewers

Dear Editor and reviewers,

We would like to thank the three reviewers for their thorough review of our manuscript and their detailed comments and very helpful suggestions to improve the manuscript. Overall, we thought the reviews were very positive with the reviewers commenting that our discovery of a novel genetic code variant is a “cause for celebration” and that our study is “technically solid” and “rigorous”. All three reviewers agree that our manuscript would “stimulate new discussions in the field of genetic code evolution” and also be of broad interest to evolutionary cell biologists, protistologists and the translation/protein synthesis community at large. The reviewers highlight the particular novelty of the genetic code variant described here due to it being an exception to the wobble hypothesis which adds a new level of complexity to stop-codon reassignment. The reviewers share our frustration about the lack of proteomics data due to being unable to establish a stable culture but acknowledge that we address this limitation frankly in our discussion and agree that it is “frustrating but it's not a limitation”.

We present an updated and improved version of the manuscript after taking on board the reviewers’ suggestions. Our point-by-point responses to their comments and our modifications are detailed below in bold.

Point-by-point description of the revisions

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

Summary

This study by J. McGowan and colleagues reports the discovery of a ciliate species that uses a variant genetic code where the codons UAA and UAG, which are stop codons in the canonical code, instead code for lysine and glutamate respectively. The primary data are genomic and transcriptomic sequence libraries from single cells. The genetic code was predicted by aligning coding sequences to references from other species and examining the most frequent amino acids in positions homologous to putative coding-UAA/UAGs. They also identified suppressor tRNAs for UAA and UAG, and tandem in-frame stop UGAs (but not UAA/UAG) in the 3'-UTR, which further support the recoding of UAA and UAG.

A limitation of this study (and several other recent studies on variant genetic codes) is that the predictions are based on nucleic acid sequencing, without confirmation from proteomics. The authors acknowledge and briefly but frankly discuss the limitations in their manuscript (lines 258-261).

Major comments

Controls against contamination and sequence chimeras

The ciliate species studied here was an environmental isolate, and sequence libraries were prepared by amplification from small pools of cells sorted by FACS. The genome assembly was produced by co-assembly of multiple amplified libraries. Given the potential for contamination and amplification artefacts (such as sequence chimeras) associated with these methods, I think it is important to demonstrate that the data truly originate from one species, so as to rule out the possibility that the co-assembly may be chimeric, i.e. representing two or more organisms with different genetic codes (one with UAA recoded and the other with UAG recoded, for instance). Even if the cell sorting was accurate, contamination could still enter down the line during library preparation so it would be important to show internal evidence from the sequence data too.

We understand the reviewer's concerns about the possibility of contamination as it can be a major issue in environmental single cell sequencing experiments. We have addressed the individual points below in detail to demonstrate that we have generated a clean genome assembly of a single ciliate species but also summarise here:

• The cells we sequenced originated from the same clonally isolated cell propagated in culture
• We have manually curated the assembly
• The assembly has a unimodal GC content peak with a low BUSCO duplication score
• Most genes (95.9 %) contain both in-frame UAA and UAG codons
• We recovered a single identical ciliate 18S rRNA gene across all 10 samples
• De novo assemblies of the 10 individual gDNA libraries are virtually identical in terms of average nucleotide identity
• We also predicted the genetic code for each of the genome and transcriptome samples individually

• 85% of the final assembly is taxonomically classified as Ciliophora. The remainder is either unclassified (i.e. no hits) or has spurious/inconsistent hits

  Specifically:

(a) From the description in Methods under "Sampling, Ciliate isolation, culturing, and cell-sorting", it is not clear whether all the cells that were ultimately sequenced originated from the same clone (i.e. the same well in the 96-well plate described in line 389). Could the authors confirm whether this was the case?

Yes. All the sorted cells originated from the same ciliate clone. A single-cell was isolated and cleaned (without removing all the environmental bacteria). The ciliate single-cell divided and we established a mono-clonal ciliate culture that we used for the cell sorting and sequencing. This culture grew but only for a relatively short period. We could not establish a long term culture.

(b) What % of genes have in-frame coding UAA, UAG, or both? How per gene on average? Counts are given for the conserved genes/domains identified by PhyloFisher or Codetta (lines 192-207), and overall frequencies per codon are addressed later in lines 263 onward, but how often do they occur together in the same genes?

My reasoning behind this is that if genes with both in-frame coding UAA and UAGs are common then it is very unlikely to be the result of chimeric sequence artefacts from whole-genome amplification.

We have updated the text to include this information. From the PhyloFisher analysis, we had reported that 58 genes contained in-frame UAA codons and 46 genes contained in-frame UAG codons. We have now added the text “Amongst the genes identified by PhyloFisher, 27 contained both an in-frame UAA codon and an in-frame UAG codon.”

Additionally, from our annotated gene set, we had reported that 98.6% of genes contain at least one UAA codon and 96.4% of genes contain at least one UAG codon. We have now added text to report how many genes contain both codons “The reassigned codons are widely used across genes with 95.9% of genes containing both a UAA codon and a UAG codon”.

The example gene (tubulin gamma chain protein) shown in Figure 1 contains both in-frame UAA codons and in-frame UAG codons, with the UAA codons aligning to lysine and the UAG codons to glutamic acid.

(c) What is the sequence identity of conserved marker sequences between the individual amplified replicate libraries?

I would naively expect that individual replicates may not have the full set of markers because of uneven amplification, but if the sequences originate from the same clone they should have overlapping coverage of the conserved markers, and these should be +/- identical between replicates (save for allele variants). If so this would support the claim that contaminant sequences were mostly removed during sequence QC and that the cells were clonal.

We generated an individual assembly for each of the 10 gDNA libraries and calculated average nucleotide identity at the whole assembly level. On average, the 10 assemblies are 99.43% identical to each other, with the least similar pair being 99.37% identical to each other. This level of variation includes not only allelic variants but also sequencing/assembly errors as the individual libraries are relatively low coverage. In terms of assembly alignment coverage (i.e. the fraction of each assembly that is aligned to another assembly), the average value is 76.5% and the value for the lowest pair is 59.1%. We have now also made the individual 10 assemblies available in the Zenodo repository (10.5281/zenodo.7944379) and updated the methods section.

Furthermore, as an additional quality control step, we predicted the genetic code for each of the 10 individual genome assemblies and obtained the same predictions that UAA encodes lysine and UAG encodes glutamic acid for all 10 individual assemblies. We also predicted the genetic code for each individual RNA-Seq sample based on individual transcriptome assemblies which yielded consistent predictions.

(d) Line 392: "Non-axenic" presumably refers to environmental prokaryotes. This also appears to contradict the statement that the cells were "free of any other contaminant" (line 387). Could authors confirm whether they mean "non-axenic but monoeukaryotic"?

In line 387, when we say "free of any other contaminant” we mean that we isolated a ciliate single-cell from the environmental sample, and the picked ciliate cell was washed 3 times until it was free of any other eukaryotes, but still containing environmental bacteria. In line 392, when we say non-axenic, we mean that the mono-clonal ciliate culture contained environmental bacteria and was monoeukaryotic.

We have modified the text in the methods section to say “free from any other eukaryote” and “non-axenic but monoeukaryotic”.

(e) Lines 448-451: More details should be given on the criteria used to identify and bin out contaminants. MetaBAT typically bins prokaryotic genomes quite well, but not eukaryotic ones. What did the bins look like and how were the eukaryotic ones chosen?

We routinely use MetaBAT2 to assist with separating bacterial contigs from protist genomes. From our experience we find that it generally performs well but requires careful manual curation. We only use tetranucleotide frequencies when binning single-cell assemblies and not coverage variance as this is heavily skewed due to amplification bias from single-cell amplification. We integrated the binning results from MetaBAT2 with taxonomic classification from tools such as CAT, Blobtools and Tiara, and manually curated the assembly.

We have modified both the results and methods section to clarify that the assembly was manually curated to remove contaminant contigs.

For example, using CAT, which taxonomically classifies contigs based on blast/diamond hits to open reading frames:

The final curated assembly is 69.7 Mb in length.

59.5 Mb (85.4%) is classified as Ciliophora.

9.7 Mb (13.9%) is unclassified.

The remaining 0.5 Mb (0.7%) have inconsistent, low-identity hits to 22 different Eukaryotic and Bacterial phyla (due to lack of closely related species in public databases).

Furthermore, we recovered only a single ciliate 18S rRNA gene and the final curated assembly has a unimodal GC content peak with a low BUSCO duplication score and high cDNA mapping rate.

__Minor comments __

Line 52: Not strictly true, some germline-limited segments contain mobile elements with coding sequences, e.g. TBE elements in Oxytricha (doi:10.1371/journal.pgen.1003659)

Thank you for pointing this out. We have rephrased “excision of non-coding sequences” to “excision of micronucleus-limited sequences” to describe the process of macronuclear development more generally.

Lines 229-231, Supplementary Table 1: Presenting the identity matrix as a distance tree may make it easier to see the pattern of similarity between the tRNAs

We have added a phylogenetic network of tRNA genes as a supplementary figure to better visualise the relationships between tRNA genes.

Lines 274-275: Suggest stating the criterion for classifying genes as "highly expressed" on the first mention of this in the Results, although it's explained later on in the Methods.

We have clarified this in the results section by adding the text: ‘We defined a subset of genes as “highly expressed” based on the 10% of genes with the highest transcripts per million (TPM) values for comparison below.’

Lines 298-299: What is the frequency of tandem UGA stops in the 3'-UTR in genes with coding-UAA/UAG vs. genes without, and is there a significant difference? The argument in this paragraph is that UAA+UAG reassignment increases selective pressure to minimize translational readthrough. Therefore I think that it would make sense to compare the frequency in genes with and without these codons.

Following the reviewer’s suggestion, we have looked at tandem UGA stop codons in the 3’-UTR of genes that don’t use UAA and genes that don’t use UAG. We found similar enrichment for in-frame UGA codons at the beginning of the 3’-UTR in these small subsets of genes.

To clarify, the hypothesis from the literature is that there may be stronger selective pressure to maintain tandem stop codons in ciliates with reassigned genetic codes, particularly those that use only UGA as a stop codon. Within a genome, we wouldn’t expect a difference if a gene contains UAA/UAG codons.

Lines 353-354, Figure 5: Suggest marking the internal nodes where genetic code changes likely occurred. At the moment only the leaves of the tree are annotated with the genetic codes of the respective species. This would make it clearer how one counts the numbers of independent origins as reported in the text (e.g. "... a fourth independent origin of UGA being translated as tryptophan").

We have decided not to label the internal nodes on the phylogeny. We think that deeper sampling will reveal that some of these genetic code changes occurred independently, so we don’t want the figure to be misleading. Also, for the species with the genetic code UAA=Q, UAG=Q and UGA=W, we can’t determine the order of events.

Lines 371-372: Question out of curiosity (not necessary to address for the manuscript at hand): Do the authors think the recoding of UAA and UAG happened simultaneously in both codons or stepwise, or is there insufficient information to speculate?

An initial guess would be that it happened as a stepwise process but without deeper sampling of this lineage it is not possible to determine the order of events.

This highlights the need for deeper sampling and sequencing across undersampled lineages of ciliates and demonstrates the utility of single-cell OMICs approaches for species that are not yet amenable to culturing.

Line 395: "10uL" should use the actual symbol for "micro" prefix. Also, the choice of spacing or no spacing between numerical figure and units should be made consistent in manuscript.

Fixed

Line 403: "Biotynilated" should be "Biotinylated"

Fixed

Line 414 and elsewhere: "2" in MgCl2 should be subscripted

Fixed

Lines 419-420: Clarify whether the "r" and "+" symbols are to be read as prefixes or suffixes, i.e. is the modified base the preceding or succeeding one.

We have clarified in the text that these symbols are to be read as prefixes.

Table 1: What is the difference between the two sets of BUSCO completeness scores reported? One is given under "Genome assembly" and the other under "Genome annotation", but the annotation is based on the same assembly, right? I'm assuming this has to do with different modes in which BUSCO can be run, but this should be explained in the Methods (lines 452-453, 496-497) and briefly explained in the Table caption.

Yes this is because we ran BUSCO in two different modes. BUSCO is run in genome mode on the genome assembly and in protein mode on the genome annotation. In genome mode gene prediction is performed by Augustus guided by amino acid BUSCO group block-profiles while in protein mode the gene set described in our methods is the input to BUSCO classification. The superior BUSCO results for the protein mode reflect the superiority of our final annotation over that generated by BUSCO Augustus. We have added text to the methods section and to the table caption to clarify which mode was used.

**Referee Cross-commenting** I generally agree with the other reviewers' comments. Specifically I like reviewer #3's suggestion #3 to have a more detailed summary of the codon frequencies, perhaps as a graphic, and to compare the tandem stop frequencies with other ciliate species, especially those with all three canonical stops.

Reviewer #1 (Significance (Required)):

Any new genetic code variant discovered is a cause for celebration! This is a basic biological fact with inherent significance and should be generally interesting to biologists because the rarity of variant codes stands in contrast to the diversity of most biological systems.

This variant code would also stimulate new discussions in the field of genetic code evolution specifically because, as the authors point out, when both UAA and UAG are recoded they both usually encode same amino acid, but here they are recoded to different ones. This is an apparent exception to the "wobble" hypothesis for why these codons often evolve in concert, which was well explained with relevant citations in the Introduction.

For context: My expertise is in genomics and environmental microbiology.

END reviewer 1

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

This study reports the reassignment of the UAA and UAG stop codons to lysine and glutamic acid, respectively, in the ciliate Oligohymenophorea sp PL0344. The paper is nicely written, easy to read and the experimental approach, ideas and questions are easy to follow. The work is technically solid both at the NGS - in house library preparation, sequencing and data interpretation - as well as phylogeny levels. The conclusions are consistent with the comparative genomic and transcriptomic data obtained by the study.

__Reviewer #2 (Significance (Required)): __

The work extends current knowledge on codon reassignment in ciliates, confirming previous discoveries of existence of very high stop codon assignment flexibility in these organisms. The assignment of UAA and UAG to two different amino acids by two different tRNAs is very interesting and reinforces the idea that stop codon reassignment in ciliates is rather common. It also raises important questions about the parallel evolution of the release factor-1 (eRF1), Lysine and Glutamine tRNAs, as the reassignment requires loss of recognition of both UAA and UAG by eRF1 with parallel appearance of the new Lysine and Glutamic Acid suppressor tRNAs.

The main issue of this work is the inability to cultivate the ciliate Oligohymenophorea sp PL0344 in the laboratory to prepare protein extracts for direct analysis of the amino acids inserted at UAA and UAG sites by Mass Spectrometry. The comparative genomic and transcriptomic data, as well as the identification of cognate tRNA anticodons for UAA and UAG, are likely correct, but provide indirect evidence for the assignment of UAA to Lysine and UAG to Glutamic Acid. This issue is relevant because one cannot exclude the possibility of insertion of other amino acids at UAA and UAG sites beyond Lysine and Glutamic acid, respectively; nor can one exclude the possibility that such amino acids are inserted at high level. The authors do acknowledge the limitations of the unavailability of protein extracts for direct MS analysis of the reassignment, but should consider, in particular in the discussion, the possibility of multiple amino acid insertions in a context where Lysine and Glutamine Acid are the major but not the only amino acid species being inserted at those sites.

Based on my expertise of studying codon reassignments in fungi of the CTG clade, I believe this work is very interesting and appealing to the genetic code community, and is of relevance to the evolution and protein synthesis research communities at large.

We thank the reviewer for their positive review. They raise an important point about the possibility of amino acids other than lysine and glutamic acid being inserted for UAA/UAG codons which we hadn’t considered. We have added text and relevant references to our discussion to highlight this possibility:

“Additionally, while the genomic and transcriptomic data provide strong evidence that lysine and glutamic acid are the major translation products of UAA and UAG codons, respectively, we cannot rule out the possibility that other amino acids are (mis)incorporated at these sites which could be detected using mass-spectrometry [38, 39].”

Krassowski T, Coughlan AY, Shen X-X, Zhou X, Kominek J, Opulente DA, et al. Evolutionary instability of CUG-Leu in the genetic code of budding yeasts. Nat Commun. 2018;9:1887. Mordret E, Dahan O, Asraf O, Rak R, Yehonadav A, Barnabas GD, et al. Systematic Detection of Amino Acid Substitutions in Proteomes Reveals Mechanistic Basis of Ribosome Errors and Selection for Translation Fidelity. Molecular Cell. 2019;75:427-441.e5.

END reviewer 2

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

Summary: from genome and transcriptome sequencing of what appears to be a novel ciliate from the class Oligohymenophorea, McGowan et al provide convincing evidence of a protist in which the stop codons UAA and UAG have almost certainly been recoded to specify incorporation of different amino acids (UAA = K; UAG = E) during translation. Several ciliates from different classes use a non-standard genetic code (as do a narrow variety of other protists), but this is an unusual observation in that stop codons which differ only in the wobble position code for different amino acids in the ciliate identified here.

I say 'almost certainly' the stop codons have been recoded in Oligohymenophorea sp. PL0344 because in the absence of being able to retain the ciliate in culture the authors have not been able to complete the proteomics which would unequivocally (a) show stop codons now code for amino acids and (b) confirm the identity of the amino acids now encoded (the authors discuss this issue on p12).

Comments: overall this manuscript is straightforward to read and the analyses realistically taken as far as is realistic in the absence of a continuous culture method. My suggested revisions should be straightforward for the authors to address.

1) The manuscript appears to report the identification and genome/transcriptome sequencing of a novel ciliate species - clarity should be provided by the authors. However, it disappointed me that this manuscript was crafted entirely from nucleotide sequencing. I would have welcomed seeing the morphology of the ciliate identified here and would have anticipated that there was sufficient material to perform microscopy at the light level (for DIC images) and by scanning or transmission electron microscopy.

Yes, based on the 18S rRNA sequence and phylogenies of protein-coding genes, this is a novel species that hasn’t been described before. The most similar hits to the 18S rRNA gene are to other unnamed/environmental sequences. We haven’t attempted to name or describe this species as we weren’t able to establish a culture, so have referred to it as Oligohymenophorea sp. PL0344. We have clarified in the text that this is a novel, unnamed ciliate species.

The genomic and transcriptomic data was generated from a single cell isolate propagated into micro-cultures of 10’s of cells. These were done in the strictest conditions in an attempt to minimise contamination. Consistent with this approach it was not possible to obtain useful SEM/TEM as it would be very hard to recover EM imaging from 10’s of cells (a process that would have drastically reduced our ability to do replete genome sampling). Similarly, our approach to culturing limited our ability to acquire useful DIC images. After discovering that this ciliate uses a novel genetic code, we attempted on a number of occasions to re-isolate the same species from the same and surrounding water bodies but failed.

2) It is unfortunate that the ciliate could not be maintained in culture (or cryopreserved). Coordinates for the University Parks pond are provided, but I got the impression that this ciliate could be repeatedly isolated. Thus, in the absence of culture methods could the authors indicate the points in the year when the ciliate could be isolated (i.e. is there a season element to when PL0344 could be isolated) and how frequently when sampling was performed could PL0344 be seen? From the environmental sequence data that is publicly available is there any evidence for the presence of PL0344 anywhere else in the world? I'd be surprised if this was a UK-specific ciliate.

The water sample from which this ciliate was isolated was collected in April 2021. After having sequenced its genome and identifying the genetic code change, we made several attempts to reisolate it from the same pond but were unsuccessful. Regarding the geographic distribution of this ciliate, in the text we mention that the most similar 18S rRNA sequence in GenBank is to an unnamed species recovered in a metabarcoding study in France with 99.81% identity. We assume that this is the same species. We also examined other publicly available environmental datasets such as the PR2/metaPR2 database. The most similar match in the metaPR2 database was to a sequence “OLIGO4_XX_sp”. In the metaPR2 database this sequence is unique to Lake Garda in Italy (sample name: “Lake_Garda-LTER-euphotic-water”). However, this hit was only 98% identical with a partial alignment so we did not discuss it in the text. We agree that it is very unlikely that this is a UK-specific ciliate but cannot determine its geographic range based on the publicly available environmental sequence data, other than the single hit to a sequence from France. We think it is important to stress that it was not the aim of our paper to describe the taxonomy and biogeographical range of this ciliate but rather to report the exciting shift in codon usage.

3) I felt the statistics presented on pages 13-14 (lines 277-301) for codon usage were a little superficial. It would be helpful to see how frequently other E and K codons are used in PL0344 and ideally to see how similar codon usage differs in the more model ciliates Paramecium, Tetrahymena or Stentor. To complete an analysis and justify/confirm conclusions drawn, I would also like to see how frequently in-frame, downstream stop codons are seen in ciliates where stop codons have NOT been reassigned - although the data in Fig 5 indicates genome/transcriptome sequences are not necessarily complete for many ciliate species (where stop codons are not reassigned), there is certainly more varied data to look at than when Fleming and Cavalcanti published their PLoS One work (which is cited in the manuscript).

We have shortened this section about UAA and UAG usage, with supplementary table 3 showing usage of all codons in all genes compared to our subset of highly expressed genes.

We have also added a sentence stating how many genes contain both in-frame UAA and UAG codons based on the point from Reviewer 1: “The reassigned codons are widely used across genes with 95.9% of genes containing both a UAA codon and a UAG codon.“

According to our knowledge, there are no new genome assemblies available for ciliates that use the canonical genetic code since the Fleming and Cavalcanti publication from 2019, certainly not any with annotated gene sets available for comparison. The species in Fig 5 which use the canonical genetic code are all from transcriptome data (other than Stentor) that have generally low completeness. We do not think comparison with low-quality transcriptome assemblies would make a fair comparison as they would be biased towards transcripts with higher expression. Furthermore, they likely include many fragmented transcripts which are not suitable for detailed comparisons of the stop codon/3-UTR region.

4) Given the presence of just one stop codon in PL0344 have the authors looked genome-wide at nucleotide composition 5' and 3' to UGA. The nucleotide sequences 5' and 3' to a stop can influence whether read through is and thus potentially limits the frequency of or tendency for unwanted readthrough?

We thank the reviewer for this suggestion which is something we did not investigate initially but have now added a short section in the manuscript to address. Many studies in model organisms have demonstrated that UGA is the least robust stop codon and the most prone to read through. As the reviewer alludes to, this is particularly interesting for ciliates with reassigned genetic codes that use only UGA as a stop codon. Experimental data from model organisms have shown that the sequence composition surrounding a stop codon can influence the frequency of read through, with the nucleotide immediately downstream of the stop codon (“+4 position”) being particularly important.

We have now looked at the sequence composition around stop codons for Oligohymenophorea sp. PL0344 and our results show that cytosine tends to be avoided following the UGA stop codon. From the literature, presence of a cytosine following UGA (i.e., UGAC) leads to a substantial increase in translational read through. Furthermore, when examining the subset of highly expressed genes, there are significantly fewer cases of UGAC when compared to all genes. This trend has previously been reported in Paramecium and Tetrahymena based on EST data (Salim, Ring and Cavalcanti; 2008).

We have added a short section to the text reporting this and a supplementary figure showing a sequence frequency logo around the stop codon for all genes and for the subset of highly expressed genes. We are very cautious, however, that there is a paucity of experimental studies investigating stop codon robustness in ciliates. While several publications hypothesise that read through may happen at higher rates in ciliates due to a combination of factors (e.g., ERF-1 mutations, presence of tandem stop codons, competition from suppressor/near-cognate tRNA genes, etc..) we are careful not to speculate without experimental evidence.

__Reviewer #3 (Significance (Required)): __

Strengths - I found this a straightforward manuscript to read - aside from the interesting and unexpected observation about genetic code use in PL0344, Fig 5 draws together a lot of earlier published information into an easily accessible form - I felt this a particularly useful part of the manuscript.

I don't feel the absence of proteomics to back up the genome/transcriptome analysis is a notable limitation - it's perhaps frustrating but it's not a limitation. However, the work does perhaps inevitably feel a little bit observational - there's not really a lot of insight or new insight into why the genetic code can be revised in some microbial eukaryotes - in contrast, for instance, to a recently published study of the aptly named Blastocrithidia nonstop. McGowan et al's manuscript, however, will be of interest and should be formally published.

Descriptions of organisms that have tweaked the standard genetic code are not new; coupled to the limited insight into why the genetic code can be rewritten so readily in ciliates, this limits the general appeal of the work. However, the study executed is rigorous and it should be of interest to a wide variety of protistologists, evolutionary cell biologists, and researchers in the translation field.

END reviewer 3

Câu hỏi: Theo quy định của TT số 11/2021/TT-BXD hướng dẫn lập dự toán thì đối với các loại vật tư, vật liệu không có trong CBG, đơn vị tư vấn có trách nhiệm thu thập báo giá của NCC, NSX phổ biến trên thị trường. Vậy cho hỏi: + Phải lấy tối thiểu bao nhiêu báo giá? Báo giá do các đơn vị khác nhau cấp cho 1 sản phẩm của 1 nhà sản xuất hay các sản phẩm tương tự nhau (nhà sản xuất khác nhau) đều được? + Có quy định nào bắt buộc phải lấy giá thấp nhất trong các báo giá không (trừ sản phẩm nhập khẩu);

Trả lời: - Xác định chi phí vật liệu để xác định đơn giá xây dựng chi tiết phục vụ lập dự toán quy định tại mục 1.2.1 Phụ lục IV của Thông tư 11/2021/TT-BXD. NĐ, TT về quản lý chi phí của BXD không quy định lấy tối thiểu bao nhiêu báo giá. Mục tiêu là để lấy đúng mặt bằng giá thị trường, khách quan, chống thất thoát, tham nhũng. Cứ lấy báo giá như nào phản ánh thực tế khách quan của thị trường là được. Tương tự như bạn khảo giá thị trường khi đi mua cái điện thoại, cái ti vi, xe máy, ô tô… - Điểm a khoản 2 Điều 11 của Thông tư 58/2016/TT-BTC (được sửa đổi, bổ sung bởi Thông tư 68/2022/TT-BTC) có quy định về lấy 3 báo giá. Mọi người thường tham khảo, vận dụng từ đây - để cố gắng chứng minh cho việc lấy thông tin lập - thẩm - duyệt chi phí là khách quan.

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Reviewer #1:

Major comments:

1. • The relevance of these findings to human biology remains unclear. In Figures 1-4, the authors present data showing that AATBC is enriched in thermogenic fat, and they argue that it regulates thermogenesis and mitochondrial biology. However, in Figures 6-7, where the authors look at AATBC in different human cohorts, they actually find that it is enriched in visceral fat, which is thought of as being the least thermogenic fat depot. The authors do not explain this seeming paradox, and thus, the role of AATBC in fat remains uncertain. *

RESPONSE: We thank the reviewer for this comment and have clarified the discussion to address this point. It has been recently shown (PMID: 28529941) that the pattern of browning genes in human white adipose tissue depots is actually inverted to mice, making visceral adipose tissue in humans actually more thermogenic than subcutaneous. This aligns well with our findings of AATBC is predominantly expressed in thermogenic adipose tissue.

• In many of the experiments, insufficient controls are provided, or the data are not at all convincing. For example:*

(a) The first four figures rely on in vitro adipocyte models, but the authors do not present data to show these cells differentiate properly and equally. This is especially relevant for the gain and loss of function studies.

RESPONSE: We agree with the reviewer that equal differentiation is necessary for in vitro adipocyte models. Therefore, we added Oil-red-O stainings and the corresponding quantifications to Supp. Fig. 4 (see below) for the differentiation of hMADS in the absence of AATBC. We also want to emphasize, that the expression levels of PLIN1, a surrogate marker for differentiation was unchanged in our experiments, as already shown in the initial draft of the manuscript. On top of that, in all experiments presented in the original draft of the manuscript, AATBC gene expression was only altered in mature adipocytes.

(b) Some of the experiments in Figure 1 (K-L) seem to only show an N of 1.

RESPONSE: Figure 1 highlights a screening process to find new lncRNA regulated during thermogenesis. The forskolin sample was included to achieve an additional dimension in the filtering process. The displayed values in K&L demonstrate the validity of the sample. The validation of AATBC as a target was performed with statistical power in the work displayed in the following figures.

(c) The RNAscope data in Figure 2 is not at all convincing for nuclear localization

RESPONSE: We respectfully disagree. In our opinion, the RNAScope is convincing for nuclear localization of the lncRNA. However, we have repeated the experiments with different probes that strengthen our data (see figure for the reviewer)

(d) The ASO mediated knockdown of AATBC in Figure 3 only reduced expression slightly. A more complete knockdown or deletion may elicit a stronger phenotype.

RESPONSE: We thank the reviewer for the feedback. We have repeated the knockdown experiments but were not able to reduce the expression further, even after designing additional ASOs. However, already with current approach, the reduction in AATBC expression elicited a phenotype, highlighting the importance of AATBC in a dose-dependent manner.

(e) In Figure 4, OPA1 is shown as a single band in panel E and a doublet in panel N. Based on this, are the authors certain they are detecting OPA,1 or could this be a nonspecific band?

RESPONSE: We thank the reviewer for this comment. Protein extraction has been performed at different research institutes with slightly different buffers. Multiple bands (cleaved/uncleaved) have been described for OPA1 in the past, therefore we are certain that the correct protein has been detected.

*(f) The correlations in Figure 6 I-L and Figure 7 do not include any statistical analysis. *

REPONSE: For better readability, the statistical analysis is being mentioned in the figure legend. The reviewer might have overlooked this information.

• The gain of function studies in mice are problematic. The authors have performed a large amount of invasive studies in a short period of time. The animals will undoubtedly lose weight after each study and with insufficient time to recover, this could influence the subsequent studies.*

RESPONSE: These general concerns are valid, but all controls are in place and the animals gained weight during the experiments, as one would have been expected with animals of that age (see below).

*In addition, since the authors present data in Figures 1-4 arguing that AATBC overexpression is associated with increased thermogenesis, it is surprising that the authors never looked at this in Figure 5 (aside from measuring Ucp1 mRNA). It would be interesting to measure energy expenditure by indirect calorimetry and cold tolerance. *

RESPONSE: We agree with the reviewer on this point but are due to animal protocol limitations in conjunction with the viral approach are unable to perform these experiments.

• The authors do not provide any mechanistic insights into how AATBC may be acting.*

RESPONSE: Certainly, more mechanistic insight into the direct mode of action of AATBC would be interesting. To address this point, over the past year we performed multiple attempts to perform pulldown of AATBC using the ChIRP technology. However, we were unable to achieve a sufficient enrichment, which would have allowed us to give further information about direct interaction partners of AATBC. However, we believe that our data regarding mitochondrial dynamics, which we now also have confirmed in in vivo experiments, explain the connection of AATBC and thermogenicity. In future, we aim to work on this point further but for multiple reasons have decided to close this chapter here.

Minor comments:

• The introduction is rather long and would benefit from being condensed.*

RESPONSE: We have edited the text for better readability.

Reviewer #2:

Major Comments:

1. • The key conclusion that AATBC is a novel obesity-linked regulator of adipocyte plasticity is made relatively clear with the comparison between various stages of adipocytes and the loss and gain of function with AATBC. - Figure 1 H and J do not seem to be consistent with the data in Figure 1F in LINC00473 level-There is no difference in Control vs NE in the heatmap but in Figure1J, the difference seems to be quite obvious; Figure 1K does not seem to be consistent with AATBC level-The measurement in Control VS Fsk group showed no difference in AATBC in heatmap, but in Figure K, there seem to be a dramatic increase. Therefore, the claims that there is a difference in these two lncRNA expression in these cell groups needs further clarification. *

RESPONSE: To combine the different approaches to identify novel lncRNA into one heatmap the data need to be normalized over experiments. As the fold change of the expression of AATBC in BAT compared to WAT (on average ~100x) is higher than with forskolin (~4x), this will stand out in the heatmap and will to some extent overshadow the smaller fold changes. The same holds true for LINC00473, which is drastically induced with forskolin, which to some extent masks the higher expression in the other approaches. Therefore, we decided to show both the heatmap to represent the general approach and the “zoomed in” versions to show the consistent increases. We are confident this clarifies the issue.

• Figure 4H and I, the difference in the representative immunoblot seem to be minimal and inconsistent with the decrease shown in the bar graph. *

RESPONSE: We agree with the reviewer and have removed the claim from our manuscript.

• In Figure 5, after overexpressing human AATBC in murine adipose tissue , is it possible to look at the mitochondria changes that were seen before in cell lines? If there are similar changes in murine adipose tissue, then it would prove the changes in vitro hold up with the in vivo model. But if the mitochondria changes were not seen, then it would indicate the changes in leptin, triglyceride levels may due to other mechanisms. The length of the suggested experiment to look into the mitochondrial differences in mice may vary depending on whether there are preserved samples from previous experiments. If there are, then the time period would be couple of weeks for immunblot and analysis. If there are no samples preserved, then the estimated period for the suggested experiments may be around 1.5 to 2 months at least .*

RESPONSE: We thank the reviewer for the suggestion. We performed Western Blot analysis on the tissues from the in vivo study and have included them in Fig. 5, further strengthening the link between AATBC and mitochondrial dynamics (please see figure on the right).

• The data are convincing overall in that the replicates are clearly marked with dots in many figures. Some immune blot and expression level are inconsistent with other data showing the same results however. *

RESPONSE: We thank the reviewer and have removed the necessary quantifications.

• Figure 6 and 7 are provocative and significant, reporting strong associations of AATBC with well-known markers of metabolism in adipocytes. The sex difference for adiponectin and AATBC expression is particularly intriguing. Further discussion of this point would be interesting. However, there is no information provided about the medication status of the obese subjects that were consented for samples used in the analysis. Specifically, many of the obese subjects (mean BMI 45 or more with a range going up to 97.3) would be expected also to have metabolic diagnoses and to be treated with numerous medications, including Metformin, GLP1 agonists, Orlistat, Liraglutide, Bupropion/Naltrexone and combinations. It is unreasonable to ignore possible effects of major medications on AATBC expression. Please comment on the strengths and weaknesses of the analysis that ignores medications, or if some annotations of clinical data are available, perhaps to explain outliers in the plots, please discuss. *

RESPONSE: We thank the reviewer for this suggestion. Unfortunately, we are unable to exclude additional diagnoses and medication of our patients due to the points the reviewer stated. However, given the large size of the cohorts we are confident that such effects are being compensated for. We have added a part on weaknesses of the study in the discussion.

Minor Comments:

• The labeling of figure 2 A-K is not clear because the use of the same color of bars is easily misunderstood as the same source of cells, but it is in fact not. For example, the grey color that appeared in 2B and 2C are not the same source but can be misunderstood. *

RESPONSE: The coloring of Fig.2A&G has been changed.

• Figure 3 ASO-AATBC has two repeats #1 and #2, and over-expression of AATBC has one, even though there are enough repeats. It would be less confusing to present all of the repeats in ASO_AATBC together in one bar.*

RESPONSE: The two different ASO target different areas of AATBC. In line with general guidelines for ASO use, those are not pooled but used separately, which is why the results are also split up. As the overexpression is additional genomic information of AATBC, it is impossible to use different variants in this case, therefore only one bar for overexpression is shown.

• The experimental outline can be a bit more detailed and explain some of the words like Thermo versus Browning.*

RESPONSE: The manuscript has been revised regarding this point.

• Some of the panels in Figure 7 could be put into supplementary if space is at a premium, and present the representative graph would be enough*

RESPONSE: We think that all our data of Fig. 7 warrants enough attention to be considered in a main figure, but if space is sparse, we are very happy to oblige. We would kindly ask the editors for input on this matter.

Reviewer #3:

1. • Throughout the study, the data provided are mainly correlative and in some cases not robust. In Fig. 2, AATBC expression is described to be elevated in the so-called "thermogenic condition", which contained prolonged PPARg agonist treatment (rosiglitazone) known to promote adipogenesis. Consistent with this notion, adipogenic markers, such as PLIN1 and FABP4, are higher in "thermogenic adipocytes" (Suppl Fig. 2). As such, the result may only suggest that AATBC has higher expression in mature adipocytes vs pre-adipocytes. *

RESPONSE: We thank the reviewer for the suggestion. We have added Oil-Red-O-Stainings to Suppl. Fig. 2 to show unchanged lipid content upon modulation of AATBC gene expression, which can be seen as a surrogate for differentiation. Concerning the use of rosiglitazone as a browning agent, we want to emphasize that rosiglitazone was used during the entirety of differentiation until day 9, where it was removed in the “non-thermogenic” group. At this point we already observe fully differentiated adipocytes. This is an established protocol. Furthermore, the data is in line with using norepinephrine or forskolin as a short-term inducer of browning, making it very likely that the effect seen is due to the “more thermogenic” character of the adipocytes.

• Along the same vein, whether and how AATBC affects adipogenesis is unclear. Suppl Fig. 3H and 3L (misplaced as Suppl Fig. 4) show the adipocyte differentiation marker FABP4 is down-regulated by both ASO- and AV-AATBC. Since mitochondrial respiration (and other parameters including UCP1 expression) is tightly linked to adipogenic efficiency, the authors need to address whether these manipulations affect adipocyte differentiation. *

RESPONSE: We agree with the reviewer that differences in differentiation capacity would falsify our data on mitochondrial dynamics. We have added Oil-Red-O-Staining to Suppl. Fig. 2 to show that no significant difference in lipid content exists during modulation of AATBC gene expression, which can be seen as a surrogate for differentiation. Furthermore, in all experiments presented in the manuscript, the modulation of AATBC occurs in already fully differentiated adipocytes. Accordingly, we are confident that AATBC does not influence differentiation but mainly acts through the modulation of mitochondrial dynamics.

• The data in Fig. 4 supporting a role for AATBC in regulating mitochondrial dynamics are superficial and not robust. Fig. 4A/4J do not have high enough resolution to provide accurate assessment of the mitochondrial network.*

RESPONSE: We respectfully disagree with the reviewer on this point. State of the art methods and algorithms were used to image and analyze the mitochondrial network. Furthermore, we have used multiple established markers of mitochondrial dynamics in western blot analysis to further strengthen our assessments of the immunofluorescence. In summary, we feel like have given enough evidence for an accurate assessment of the mitochondrial network.

• The level of loading control TUBB is clearly lower in siAATBC in Fig. 4H. In addition, OPA1 should have multiple isoforms and Fig. 4E/4N show inconsistent patterns. As such, mitochondrial dynamics is not likely an underlying mechanism. *

RESPONSE: We agree with the reviewer on the assessment of the expression of complex 5 and have removed this claim from the manuscript. Regarding the expression of OPA1, protein extraction has been performed at different research institutes with slightly different buffers. Multiple bands (cleaved/uncleaved) have been described for OPA1 in the past, therefore we are certain that the correct protein has been detected.

• Notably, RNAseq data in Suppl Fig. 4 (misplaced as Suppl Fig. 3) seem to indicate that AATBC over-expression promotes TG synthesis, while AATBC knockdown modulates cell death. The authors should consider exploring the leads from RNAseq analysis?*

RESPONSE: We thank the reviewer for the feedback. The small number of altered genes in the RNASeq make us believe in a rather post-transcriptional role of AATBC. We investigated cell death and oxidative stress response as GO terms were highlighted in the analysis, but we were unable to detect any differences in the absence of AATBC, pointing to a minimal effect on transcriptional level (See figure below for the reviewers).

• In Fig. 5, the AV-AATBC transduction in WAT/BAT is localized, transient and not homogeneous. Not surprisingly, this manipulation does not produce any robust effects. The difference in circulating leptin/leptin expression appears to be driven by 4-5 mice in the control group (Fig. 5H/5N). The correlation data in Fig. 6 and Fig. 7, although relevant, do not provide additional mechanistic insights. Unfortunately, the efforts in Fig. 5-7 fail to lead to information related to the biological function of adipose AATBC.*

RESPONSE: We agree with the reviewer on the limitations of the AV model, but we have performed these experiments with the highest technical standard. As the reviewer states, the overexpression, especially in WAT, has different magnitudes depending on the individual mouse, but the overexpression is present and consistently high in every animal. We would expect even bigger alterations in a genetic model, which, however, is beyond the scope of this first manuscript on AATBC in adipocytes. We are disappointed that the reviewer does not value the human data presented, as it very strongly hints to a relevant function of our human lncRNA in vivo by robust correlations with established biomarkers mirroring the effects seen in vitro and in the mouse model. A limitation of human studies is in virtually every case that it is based on correlations, as manipulation of gene expression, which would be necessary to delineate a biological process as requested by the reviewer, is not possible in humans. We do not concur on dismissing our human data on that behalf.

1

La autopoiesis en la teoría de sistemas de Niklas Luhmann es un concepto fascinante que ofrece una perspectiva interesante sobre la naturaleza de los sistemas y su capacidad para mantener su propia organización. La autopoiesis se refiere a la capacidad de un sistema para generar y mantener sus propios componentes y estructuras a través de la interacción continua con su entorno.

En el contexto de la teoría de sistemas de Luhmann, la autopoiesis se relaciona con la idea de que los sistemas sociales, como las organizaciones o las sociedades, son sistemas cerrados que se autorreferencian y se reproducen a sí mismos. Estos sistemas se caracterizan por su capacidad para comunicarse y procesar información de manera interna, generando así su propia realidad y manteniendo su propia identidad.

Wouldn't each element have at most constant added? Doesn't this add O(N)?

powerful and true!! no matter the positions or roles. We are working with the same objective our students and school community.

This year my coach and I managed to connect and since this happened our trust and relationship as coach and teacher has improved significantly. I feel appreciated and open to deep conversations about my training and improvements without any resentment or fear.

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

*The current manuscript by Shiryaev et al describes their observation of the new function of zika NS2B-NS3 proteases. They have shown that NS2B-NS3 protease lacking the helicase domain binds to RNA and the interaction can be affected by protease inhibitors. Main two new findings are presented in the manuscript: super open conformation of the protease; RNA binding activity of the protease region. Although the manuscript is interesting, the design of the experiments is not convincing. *

Major issues:

1. • the claim of a super open confirmation is problematic. Using an artificial construct lacking the C-terminal portion of NS2B will of course generate the open conformation. This is a wrong definition unless you observe such a conformation in living cells.*

2. We understand the skepticism towards a less known super-open confutation of flavivirus NS2B-NS3pro complex. In addition to our own structure of ZIKV NS2B-NS3pro (PDB ID 7M1V), the crystal structure of another orthologous flavivirus Japanese encephalitis virus (JEV) NS2B-NS3pro (PDB ID 4R8T) was discovered in 2015 1. However, no functional analysis was provided for this crystal structure resulting in the lack of attention paid by the research community. We computed the overlay of the ZIKV NS2B-NS3 protease structures in the super-open conformation (PDB ID 7M1V, deposited by us in 2021) with the crystal structure of JEV protease (PDB ID 7M1V ) (Rebuttal Figure 1). We observed an almost identical organization of the critical NS3pro C-terminal loop between these two structures (RMSD 0.6A). Polypeptides with over 35% identity are very likely to have a similar fold2. Given over 50% identity(!) between flaviviral proteases across the family3,4, we posit that the super-open conformation demonstrated for JEV and ZIKV NS2B-NS3pro is a common feature of the Flaviviridae family. Further, NS2B peptide is always tightly associated with NS3pro via a three-strand beta-barrel (aa 49-58 of NS2B), which remains intact in all NS3Pro conformations. The C-terminal portion of NS2B progressively loses association with NS3pro, being mostly associated in the closed conformation, less so in the open, and even less in the super-open conformation. The G4SG4 linker between NS2B and NS3pro remains unstructured in all conformations. The native C-terminal portion of NS2B (TGKR) is equally unstructured when competed out of the protease active site by another substrate. It is unclear to us why “lacking the C-terminal portion of NS2B will of course generate the open conformation”.

3. It is odd that authors made homology model to generate open conformation structures. the authors did not cite the two papers of eZiPro (Phoo et al 2016 NC) and bZiPro (Zhang et al 2016, Science). these two structures show the closed conformation of protease in the absence and presence of a natural substrate.*

4. We agree with the reviewer that in both constructs eZiPro5 and bZiPro6 of ZIKV NS2B-NS3pro are likely to exist in the closed conformation as documented by the crystal structures. However, in both cases, the active center of ZIKV NS2B-NS3pro is occupied with a short peptide fragment, which is sufficient to induce the closed conformation of NS2B-NS3 protease. We superimposed eZiPro (PDB ID 5GJ4) with bZiPro (PDB ID 5GPI) to better demonstrate that the active center in both structures is occupied either by tetrapeptide TGKR (T127-G128-K129-R130 ) originating from the NS2B C-terminus (eZiPro) or by a tetrapeptide KKGE (K14-K15-G16-E17) originating from a neighboring NS3 molecule (bZiPro) (Rebuttal Figure 2). Indeed, Zheng et al., 2016 6 stated that: “the structure (bZiPro) does capture the protease in complex with a reverse peptide. The tetrapeptide K14K15G16E17 folds into a small hairpin loop to occupy the active site.” Further, Phoo et al., 2016 5 stated that “binding of the ‘TGKR’ peptide to the catalytic site stabilizes the protease (eZiPro)”. To the best of our knowledge, so far there are no crystal structures of flaviviral NS2B-NS3 proteases in the closed conformation without peptide/inhibitor in the active center. We take it as a hint that the closed conformation is always induced by a substrate present in the active center.

Finally, we would like to draw the attention of this reviewer to the fact that the 15N R2 NMR signal from NS2B residues 65-85 is missing in bZiPro alone but re-appears when AcKR is added. This is consistent with the idea that without AcKR, bZiPro exists in the open conformation where much of the C-terminal part of NS2B is dissociated from NS3Pro and remains unstructured, thus resulting in the lack of NMR signal.

• RNA binding is novel, but is it observed in cells? only one method was used for testing the interactions, not other biophysical methods are used.*

• Given a complex network of protein-RNA interactions and the fact that NS3pro and NS3hel are connected by a single polypeptide, separating dynamically bound 11kB RNA to NS3pro from that to NS3hel in a native cell is a major technical challenge beyond the scope of this work. We employed a fluorescent polarization assay to demonstrate ssDNA and ssDNA binding to ZIKV NS2B-NS3pro. Subsequently, we employed a proteolytic activity assay with labeled peptide mimicking natural substrate for protease to demonstrate that the presence of ssRNA and ssDNA can efficiently inhibit proteolytic activity. To the best of our knowledge, this is the first indication that ssRNA or ssDNA could block proteolytic activity for any serine proteases, let alone a viral protease. Therefore, we consider the proteolytic activity assay used in the current work an orthogonal biochemical method supporting ssRNA binding to ZIKV NS2B-NS3pro.

• binding studies with RNA used artificial construct, how about the one with KTGR present like eZiPro. Keep in mind that the P1-P4 residues are present under native conditions.*

__- __As mentioned by the reviewer, TGKR peptide was found in the active center in the eZiPro crystal. Indeed, the junction region between NS2B and NS3 protease contains native cleavage sites for the NS2B-NS3Pro and is naturally cleaved by protease during the viral polyprotein processing. However, the TGKR peptide representing P1-P4 positions will have to leave the active center after the cleavage to ensure enzyme processivity/cleaving additional targets (otherwise, the protease would get stacked after the first cleavage). Proteolytic activity assay utilizes the fluorogenic peptide labeled with FAM (such as TGKR-FAM; where FAM is a group representing P1’ position in this case). TGKR-FAM peptide will compete and easily replace cleaved TGKR peptide from the active center in proteolytic activity assay. In sum, the C-terminal end of NS2B will be competed out of the protease active center by the next substrate, and there is no evidence that it will be naturally placed back in the active center after each round of protease proteolytic activity. Indeed, several crystal structures of flaviviral NS2B-NS3Pro in open conformation lack the C-terminal part of NS2B in the active center. Our unpublished NMR studies demonstrated that the C-terminal part of NS2B is unstructured in solution if the substrate peptide or small molecule inhibitor are not present in the active center of the protease.

• authors built up nice models, it is great to consider the full length NS2B, but authors haven't taken into account the effect of NS2B on the open or closed conformation of the protease. *

- __ All crystal structures of flavivirus NS2B-NS3pro in the closed, open, or super-open conformations have NS2B associated withNS3pro via a beta-barrel (__Rebuttal Figure 3), which is located at the opposite side from the RNA binding site. The transition from the closed to the open and to the super-open conformation is associated with the progressive dissociation of NS2B from NS3pro. Therefore, the effect of NS2B on NS3Pro is progressively diminished. In the closed conformation of NS3Pro, the negatively charged C-terminal part of NS2B is associated with the same positively charged grove as the RNA in the open conformation of NS3Pro. The C-terminal part of NS2B is dissociated from NS3Pro in the open conformation.

Minor issues:

*This manuscript shows the novel function of zika protease and conclude that protease binds to RNA. This is a novel finding, but the conclusion needs to be further confirmed, to avoid misinterpretations by future readers *

• closed, and super open conformations. But the definition was not carefully compared with current literatures. I am surprised that the two important papers are not cited. It is well known the G4SG4 linker affect the conformation of the protease.*

• The crystal structures and the proteolytic activities of gZiPro, eZiPro, and bZiPro are rather similar. In fact, Km (μM) are 2.86 ± 0.90 for gZiPro, 6.332 ± 2.41 for bZiPro, and the IC 50s of BPTI inhibition for gZiPro, eZiPro and bZiPro are 350, 76 and 12 nM respectively. NS2B and NS3pro have a large binding area in the closed conformation. Upon changing the conformation to the open conformation (and even more so to the super-open conformation), the C-terminal part of NS2B is progressively dissociated from NS3Pro. Therefore, possible minor effects introduced by the G4SG4 linker is unlikely to affect any of the conclusions in our work.

• Authors need to show super open conformation is present in nature e.g. the model in which full length NS2B and NS3pro.*

• A full-length NS2B has 2 transmembrane domains, which tether the NS2B-NS3pro complex to the cell membrane (we have modeled the presence of such transmembrane domains to account for the orientation of NS2B-NS3pro with respect to the cell membrane). The full-length complex has never been crystallized or tested in any assay due to the major technical challenges associated with the modeling of complex transmembrane proteins.

• RNA is a charged molecule under some conditions, NS3 also have charged residues, it is important to show whether the binding between RNA-protease is relevant to the function{Luo, 2010 #9270;Chernov, 2008 #9275;Xu, 2019 #10006}, or is this due to the application of the artificial constructs used in this study. Why so many mutants are used? *

• The requirement of NS3pro for the helicase function was shown by several investigators 7–9. Given the structural independence of NS3pro and NS3hel, which mostly rules out the allosteric effect, RNA binding by NS3pro is a newly proposed function of NS3pro for the helicase activity. We demonstrated biochemically that RNA-bound to NS3pro inhibits its protease function. A variety of mutants were used to constrain the conformations of NS2B-NS3pro (e.g. enforce the super-open confirmation) for crystallization studies.

• Using a construct close to the native protease, at least the P1-P4 residues should be present. Using a peptide in the assay is also useful.*

• We were unable to interpret this critique.

• Test binding of RNA with protease using another method such as biophysical methods, or even gel shift assay*

• We thank the reviewer for this suggestion. Although the gel-shift assay seems to be a reasonable method to test the binding, given the ease of spontaneous conformational change (i.e. into the super-open conformation), this assay could result in a progressive loss of bound RNA during migration in the gel.

• I don't know the correlation between Figure 7 and Figure 6. The authors describe ploy A binding to protease, while Figure 7 is talking about Helicase binds to dsRNAs. *

• There is no correlation. Figure 6 describes the models for NS2B-NS3pro binding to ssRNA. Figure 7 describes a separate point, the direction of dsRNA processing by NS3hel.

• I am glad to see the consideration of full length NS2B, NS3 in the models Figure 8, 9 and 11, but there is no data to support any of the model proposed. *

• There is no experimental data. We have modeled the N-terminal and C-terminal parts of full NS2B, which are predicted to be inserted into the cell membrane due to their characteristic amphipathic helical structure.

• Is the linker a ploy G not G4SG4? *

The linker is GGGGSGGGG (G4SG4) as stated in Materials and Methods of the manuscript.

• Do the mutant sustain their protease activity? *

• All mutants with intact catalytic centers have protease activity, except the mutants with a disulfide bridge that fixes the polypeptides in the super-open conformation.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

*The manuscript by Shiryaev et al., submitted to BioRXiv is an exploration of the ability of NS2B-NS3protease to bind RNA and its subsequent role in NS3 helicase processivity. The authors first utilize fluorescence polarization assays to demonstrate that NS2B-NS3protease can bind ssRNA with a strong affinity (and also ssDNA with lower affinity). They subsequently utilize mutational and small molecule inhibitor strategies in these assays to force the NS2B-NS3protease into different conformations, with the associated results inferring that the "open" conformation is responsible for ssRNA binding affinity. Furthermore, they demonstrate that ssRNA binding impairs protease activity, suggesting these roles may be exclusive in the viral life cycle. They also identified a number of small molecule ligands that target the putative ssRNA binding channel, and demonstrate that these ligands inhibit ssRNA binding by NS2B-NS3protease, providing potential inhibitor candidates for ZIKV. Finally, the authors utilized their crystal structures and others for the various conformations of NS2B-NS3protease to model ssRNA binding by the domain and the full NS3 protein, and used these models to propose a reverse inchworm model for NS3 travelling along ssRNA as it unwinds the dsRNA duplex. Overall, the authors utilize a comprehensive approach to demonstrate a number of novel findings (ssRNA binding by NS2B-NS3protease, small molecule ligands that inhibit this interaction) that would be of interest to both virologists and structural biologists. However, there are some important experimental design limitations and viral life cycle considerations that the authors should address before acceptance of the manuscript. Major and minor comments intended to improve the manuscript are outlined in more detail below. *

Major Comments:

1. • While the quantity of indirect data (ruled out closed and super-open, inhibitors of ssRNA binding pocket) suggest that the open conformation of NS2B-NS3protease is associated with ssRNA binding, the argument would be greatly strengthened by direct experimental data. Is there a mutational or small molecule approach to locking the NS2B-NS3 protease in the open conformation? If so, the authors should perform such experiments to strengthen the foundation of their argument.*

2. Unfortunately, despite significant efforts, mutations or small molecules locking the NS2B-NS3 protease in the open conformation have not been identified for the ZIKV protease. However, several structures for NS2B-NS3 proteases have been documented in other flaviviruses (i.e., DENV PDB IDs 2FOM and 5T1V; WNV PDB ID 2GGV). Polypeptides with over 35% identity are very likely to have a similar fold2. Given over 50% identity(!) between flaviviral proteases across the family3,4, there is little doubt that ZIKV NS2-NS3 protease adopts an open conformation similar to all flaviviral proteases. Our modeling demonstrated that there are no sterically/structural problems in folding NS2B-NS3 protease into the open conformation.

3. A negative control should be used in Figure 4A to strengthen the claim that ssRNA binding in the open conformation impairs protease activity (ie. include a curve for dsRNA). Such an experiment would lend support to ssRNA inhibition being due to specific binding instead of some other non-specific effect of increasing local nucleic acid concentration.*

4. To address this critique, we have conducted the modeling of dsRNA binding to the open conformation of NS2B-NS3Pro. The model revealed that dsRNA could not be accommodated by the open conformation of the NS2B-NS3Pro complex (Rebuttal Figure 4). Indeed, dsRNA has a very different rigid structure compared to the extended form of the ssRNA chain. The dsRNA is unable to provide continuous interactions between negatively RNA backbone and positively charged side chain amino acids in NS3pro. The continuous interface on NS2B-NS3 protease interacting with ssRNA is an extension of the exit groove for one of the ssRNA strands exiting the NS3 Helicase after unwinding. Therefore, the ssRNA, but not dsRNA is naturally always present in close proximity of the NS2B-NS3Pro complex.

5. *

6. Due to the highly coupled roles of NS5 and NS3 in replication, the authors should include some more consideration of the role of NS5 in their complex. They very briefly address this interplay in the fifth paragraph of the discussion, but then neglect to discuss the implications any further. In particular (perhaps in a brief comparison to an NS3/NS5 modeling approach such as Brands et al., 2017; WIRES), the authors should consider some of the following questions: could the channel on protease domain lead to ssRNA entry site on RdRp?*

7. Indeed, our model suggests that the negative strand (-)ssRNA exits from NS2B-NS3protease facing the ER membrane in the area where the protease is anchored to the ER membrane via the NS2B transmembrane domains. It is possible that NS3pro interacts with NS5 polymerase and “handles” (-)ssRNA to the NS5 polymerase. This scenario would modify Brands et al., 2017 model to add NS2B-NS3Pro complex between NS3Hel and NS5. However, at present, the NS3-NS5 (or NS2B-NS3-NS5) complex together has not been crystallized. It would be logical for NS5 polymerase to access the (-)ssRNA strand after it is released from NS2B-NS3Pro since the (-)ssRNA strands are used as a template for the (+)ssRNA which is used for polyprotein synthesis and packaging into viral particles.

8. would NS5 interaction constrain or augment inchworm model of NS2B/NS3 translocation? *

9. Yes, integrating NS5 interaction with the NS2B-NS3pro handling (-)ssRNA will augment the utility of the suggested reverse inchworm model.

10. how does increased activity of NS3 when complexed with NS5 (**Xu et al. 2019) align with proposed inchworm model? *

11. We appreciate the reviewer's question. We think that NS2, NS3, NS4, and NS5 work in concert as one coordinated complex where various subunits of NS2 and NS4 may provide anchoring of the entire complex to the ER membrane. Indeed, such a complex has recently been proposed6. Also, see our response to the previous reviewer’s point (#4). We have incorporated this discussion into the revised manuscript.

Minor Comments: 1. Introduction, 4th paragraph, NS3-NS4 should read NS3-NS4A.

• We corrected this sentence.

* ** Throughout the manuscript, the authors should denote some key amino acid residues in each figure to help orient the reader better to the observed structural changes and rotations. Inclusion, at least in the supplement, of the crystal structures of mutants solved herein should **also be included. *

• We annotated the key residues in all figures (e.g. catalytic residues, loop interacting with the membrane, position of NS2B, and other elements) and kept the same orientation of complexes in all figures.

• Section: RNA binding inhibits the proteolytic activity of ZIKV NS2B-NS3pro, last sentence, NS2N-NS3pro should be NS2B-NS3pro*.

• We corrected this sentence.

• Section: Allosteric inhibitors of NS2B-NS3 protease interfere with RNA binding- first sentence: "The open conformation of NS2B-NS3pro is achieved by the rearrangement of NS2B cofactor (its dissociation from the C-terminal half of NS3pro) leading to a loss of proteolytic activity [32]. - the reference is not correct. I could not find the reference the authors refer to here and had not heard before that NS2B cofactor was able to disassociate from the C-terminal half of NS3pro; hence, this really needs to be appropriately referenced. *

• We have revised this sentence and added additional references. “The open conformation of NS2B-NS3pro is achieved by the rearrangement of NS2B cofactor (partial dissociation from NS3pro), leading to a loss of proteolytic activity4,11.”

• Section: Modeling RNA binding to ZIKV NS2B-NS3, first sentence - unwinds should be unwind*.

• We corrected this sentence.

• With respect to the results of Figure 3A, the authors should address that adding the linker alone to the NS3 protease may not be an accurate examination of its role/importance. The linker in this scenario is only constrained at its N-terminus, while it is always constrained at both termini during infection (and even more so by the interactions of those two linked domains [protease and helicase] with each other). As such, the authors statement that "observations suggests that the 12-aa linker region modulates RNA binding to NS2B-NS3pro" should be more strongly qualified to this effect. In addition, it would be interesting to see the effects of the linker mutations on ssRNA binding in the context of the full NS3 protein, albeit admittedly more complex due to the confounding ssRNA binding by the helicase domain.*

• We agree with this reviewer that the protease-helicase linker is also restrained at both termini. We have rephrased the statement in the revised manuscript. The goal of the experiment shown in Figure 3A was to examine whether a negatively charged linker is able to compete with ssRNA binding as we expected from the structural model. The mutational analysis of the protease helicase linker is, indeed, a very interesting subject that is, however, beyond the scope of this work.

7. The NS#hel should be changed to NS3hel in part (C) of figure legend for Figure 11. - We corrected this mishap.

• The authors data in Figure 4A (and even more so the nature of the viral life cycle where 1000s of viral polyproteins are created from the first genome during infection) disputes the depiction in the inchworm model of how NS3 protease cleaves the polyprotein while the helicase binds ssRNA. At minimum, the authors need to discuss this discrepancy, and it is recommended that they modify the cartoon in their model to not include the ssRNA binding on the protease side of the equation (or show as alternative on that side to the existing cartoon).*

• Indeed, as proposed by our reverse inchworm model, ssRNA is not bound to NS3Pro in the closed conformation, while NS2B-NS3pro has a protein substrate in the active center (Figure 11A). We agree that NS2B-NS3Pro in the closed conformation cannot bind ssRNA as we demonstrated in competitive cleavage assay. Only large amounts of ssRNA can shift the balance towards the open conformation which binds ssRNA. We think that most of the time NS2B-NS3Pro cycles between the open and the super conformations handling ssRNA (Figure 11(B-C_D), but as soon as protein substrate becomes available (typically a loop from a transmembrane viral polypeptide), NS2B-NS3Pro quickly switches to the closed proteolytically active conformation to act as protease.

• In the third paragraph of the discussion, the authors state "An alternative model of coupled transcription and translation where viral RNA is associated with ribosomes right after the release from NS2B-NS3 is also possible". Considering there is abundant evidence that translation and replication are exclusive and that translation does not take place in ROs, it would be prudent to remove such statements from the discussion. Without any supporting evidence, these statements will be misleading to readers by providing a false equivalency. The preceding discussion of RFs would be sufficient to contextualize your inchworm model in the broader viral life cycle (which was done quite well). *

• We have adjusted the discussion in the revised manuscript to avoid a false equivalency.

10. There were a number of aspects I appreciated about the manuscript and will briefly list a few here: ** i) the focus on how different non-structural proteins effect the structure and function of ** each other during the viral life cycle, which forms a more comprehensive and informative model ** ii) the use of structural and functional assays as complementary approaches to studying the intra- and inter-protein relationships of NS3 ** iii) the depiction of the forks in Figure 10, which effectively demonstrated the channels and oriented the reader to the conservation data ** *iv) the use of small molecule inhibitors to modify structure and function of NS3, which greatly deepened the richness of the story from both a basic and applied science view point *

• We are very grateful to the Reviewer for these kind remarks.

Reviewer #2 (Significance (Required)): ** Strengths and limitations: ** - provides some experimental and modeling data to provide a new model for RNA interactions with the NS3pro-hel; may help inform models for enzyme function, mostly consistent with previous literature ** - leaves out the NS5 RdRp, known to contribute to NS3 activity. ** - some suggestions are made which might strengthen the conclusions and inclusions of additional controls would improve the data. ** Advance ** - conceptual, perhaps may provide some insight into mechanism; although limited by the lack of NS5 RdRp which is crucial to helicase activity. It is unclear if the ssRNA would be oriented this way given interactions with NS5 RdRp and MT domains (is the ssRNA routed to NS5 or along NS3, or potentially are both happening?) ** Audience: ** - quite specialist, but may include structural biologists and virologist alike. ** Expertise of the reviewer(s): ** *- molecular virologists, RNA viruses - including flaviviruses; replication complex biogenesis, protein-RNA and RNA-RNA interactions. While comfortable with the concepts regarding complex formation, the appropriateness of computational modeling and RNA docking tools as well as structural biology is out of our area of expertise. *

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

*This paper investigates the nucleic acid binding properties of zika virus protease. In particular the data suggest that single stranded RNAs and DNAs are capable of binding to and inhibiting ZIKV protease at micromolar concentrations. With the use of active site inhibitors and mutants that lock the protease in closed and super-open conformation, the authors concluded that RNA binds to the open conformation. Through extensive modeling of the protease and helicase domains, this manuscript provides a model of how ssRNAs can bind to all conformations of the proteas, but the open conformation provides two positively charged forks that should be available to bind RNA. *

* SECTION A - Evidence, reproducibility, and clarity ** Major comments: **

*·The main conclusions of this paper rely on the existence of the super-open conformation, however this conformation has not been reported in the scientific literature previously. Structures deposited in the pdb are referenced in this manuscript, however no citation for an accompanying publication is provided. This calls into question the biological relevance of this super open conformation. This is of particular concern because in other highly-homologous flaviviral proteases, structures that have been observed crystallographically (e.g. the open conformation of dengue virus protease) appear to be only very sparsely populated in solution. What is the evidence that the super-open conformation exists in solution.

• Please, see our reply to question #1 from Reviewer 1.

• The activity of each of the constructs used was not reported making it impossible to directly compare the impact of these changes on intrinsic activity. In particular, the NS2B-NS3 long construct is predicted to exist in the super-open conformation. If this is correct, it should show no activity against a peptide substrate. *

• We appreciate these concerns. The NS2B-NS3pro-long construct is proteolytically active (only NS2B-NS3pro-short construct is proteolytically inactive because its NS3pro C-terminal part is too short to fold into the closed conformation). It is unconstrained and likely capable of adopting all possible conformations (closed, open, super open). As we suspected, the negatively charged linker interferes with RNA binding, potentially via direct competition. Investigating the role of the protease-helicase linker is an exciting subject of a separate manuscript in preparation.

• This paper reports that the IC50 is much weaker than the Kd for binding of ssRNA to ZIKV NS2B-NS3pro. Are orthogonal assays, such as thermal shift assay, available which could distinguish between the reported IC50 and the Kd. *

• Binding of ssRNA occurs in an area distinct from the protease active center. We think that there is a constant competition between C-terminal NS2B binding/release versus ssRNA binding/release from NS3pro. We think that ssRNA “catches” the moment when protease has the open conformation and freezes that conformation by blocking the C-terminal of NS2B from binding to NS3Pro. In terms of thermal shift assay, the structure of NS3Pro is changed, only the C-terminal of NS2B is affected. Note that the 15N R2 NMR signal from NS2B residues 65-85 is missing in bZiPro alone but re-appears when AcKR is added6. This is consistent with the idea that without AcKR, bZiPro exists in the open conformation where much of the C-terminal part of NS2B is dissociated from NS3Pro and remains unstructured, thus resulting in the lack of NMR signal. Taken together, these observations suggest that thermal shift assay is unlikely to be of much help.

• *This paper suggests that ssRNA binds to the open conformation of ZIKV NS2B-NS3pro, however no experimental evidence, only modeling has been used to suggest binding to the open conformation. In Dengue virus protease, the M84P variant has been reported to lock the protease into the open conformation. How does the F84P variant of ZIKV NS2B-NS3pro impact ssRNA binding? *

• We appreciate this question. Indeed, M84P mutation shifts Dengue NS3Pro to the open conformation, which is proteolytically inactive12, consistent with our reverse inchworm model. We have not investigated the effect of this mutation on ZIKV NS3pro. We expect this mutation has a similar effect in ZIKV NS3pro in Dengue NS3Pro.

• The relevance of the discussion on the co-crystallization of NSC86314 with the Mut7was not clear. What point was being made?

• We provide a proof-of-principle for a novel class of allosteric inhibitors that specifically target newly identified druggable pockets present in the open and super-open conformations of ZIKV NS2B-NS3pro. Our results suggest that such allosteric inhibitors can interfere with the RNA-binding activities of NS2B-NS3pro in addition to blocking the protease activity. The co-crystallization of NSC86314 with the Mut7 confirms a novel pocked bound by NSC86314.

*- These data show that both active site and allosteric inhibitors block binding of ssRNA to the protease. The paper also suggests that ssRNA only binds to the open conformation. What is the evidence that the allosteric inhibitors do not enable or promote formation of the open conformation? *

• We thank this reviewer for an interesting question. Indeed, we have no evidence of whether allosteric inhibitors enable or promote the formation of the open conformation. This is formally possible and will need to be investigated.

• This paper makes two claims about the function of the protease. The title should specify what those dual functions are (proteolytic activity and ssRNA-recruitment).*

• We appreciate this reviewer's suggestions for the title.

• The discussion of Figures 6 and 9 are highly similar. The main takeaway points for both figures seem to be nearly identical: the presence of two positively charged pitchfork on the open conformation. The distinction between these two figures should be more significantly and explicitly stated. *
• Figure 6 presents several models that provide evidence for the open conformation of ZIKV NS2B-NS3pro being uniquely suitable to bind RNA. Figure 9 presents several models of the entire RNA-NS2B-NS3pro-NS3hel complex anchored into the ER membrane. Figure 9 illustrates that the open conformation of NS2B-NS3pro provides two positively charged/polar forks, contiguous with the positively charged groove on NS3hel. Figure 6 does not illustrate that point.

*- Mention explicitly in the materials and methods if the 12-amino acid linker is present in all the mutants used. *

• This is mentioned explicitly and shown in Supplementary Figure 2A.

Minor comments: ** · Figure 1. The rotation that promotes the transitions from orientation in panel A to that in panel B should be drawn. ** · FAM should be defined in the legend of Figure 2. ** · The term Cold should be changed to unlabeled. ** · Please check labels for the supplementary Figure 2. For example one label states 1-1 but it ** should be 1-170. ** · Figure 1C does not exist and it is referenced in the results section under "NS2B-NS3pro substrate-mimicking inhibitors compete with RNA binding." ** · As discussed above, if the super open conformation is going to be addressed in this paper, then either a reference for the manuscript describing those structures should be included, or this manuscript should include in the materials and methods the procedure on crystallization, data collection, structure determination, refinement, and analysis as well as a table for crystallographic data and refinement statistics. ** · Adjust figure arrangement (ABCED to ABCDE) in Figure 11.

• We thank this reviewer for all minor comments. We corrected the above-mentioned errors in the manuscript.

Reviewer #3 (Significance (Required)): ** It is well established that the flaviviral proteases exist in different conformations but most of the structures published are concentrated on the closed conformation which is the one required for effective substrate processing. The open conformation has recently been the subject of increased interest, especially with the discovery of allosteric inhibitors for which modeling suggests that these compounds result in the dissociation of the C-terminal region of NS2B from the NS3. This paper adds important insights into the function of the open conformation and in general implicitly shows the importance of the dynamic nature of ZIKV NS2B-NS3pro. In addition to these insights, this paper aptly demonstrates that ssRNA can bind and inhibit these proteases as has not been shown previously. ** I am a senior graduate student working on characterizing and understanding the mechanism of action of allosteric compounds against viral proteases, specifically proteases from Zika and dengue viruses.

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https://docdrop.org/pdf/Senn-et-al.---2018---Groove-in-drum-patterns-as-a-function-o-6fcmc.pdf/

Groove in drum patterns: A Function of Both Rhythmic Properties and Listerners' Attitudes Senn et al 2018

Naïve implementation would be O(m x n)?

Super important to recognize and remember each of these premises, both teacher candidates and veteran teachers

https://docdrop.org/pdf/Nelias-et-al.---2022---Downbeat-delays-are-a-key-component-o-9vl4g.pdf/

Downbeat delays are a key component of swing in jazz Nelias et al 2022

Вот как этому научиться? Хотя, что-то похожее я испытываю, когда просыпаюсь утром или ночью со словами Господи помилуй… Это момент перехода из сна в бодрствование. Бывает страшно.

Cuándo se deben utilizar los términos multilingüe y plurilingüe? En pocas palabras, el término "multilingüe" se utiliza para describir un país, un lugar o una institución que utiliza varias lenguas. En cambio, el término "plurilingüe" lo utilizamos para describir a una persona que habla varias lenguas

https://www.alphatrad.es/noticias/diferencias-plurilinguismo-multilinguismo#:~:text=%C2%BFCu%C3%A1ndo%20se%20deben%20utilizar%20los,persona%20que%20habla%20varias%20lenguas.

https://docdrop.org/pdf/Collier-Lincoln-Collier---1994---An-Exploration-of-the-Use-o-8c8ft_ocr_force.pdf/?src=ocr

An exploration of the use of tempo in jazz Collier, G.L. & Collier, J.L. 1994

Boulder County Organizational Chart. The line items in each box (at least for DHHS) are the names of the divisions

DOI: 10.1016/j.molcel.2023.05.012

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This is intuitive, you wouldn't expect more deltas than that because >1000 deltas imply that numbers greater than 1000 could be a delta at which point I would stop calling it a pattern, especially when clustering comes into play.

Mujer que se dedica a mazamorrear o lavar la arena en batea para extraer oro

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Referee #3

Evidence, reproducibility and clarity

The authors ask the important question whether post-embryonic organ formation follows the same mechanisms as during embryonic development. They focus on neuromasts of the lateral line system in the caudal fin of medaka. They used live imaging to find that the post-embryonic caudal-neuromast-cluster develops from organ-founder neural stem cells as a bud (and not as individual migrating stem cells as in zebrafish) that detaches and migrates from the founding neuromast (P0). They show that the formation of post-embryonic neuromasts does not require the lateral line nerve, which establishes another difference to the process in zebrafish. Artificial reduction in Cxcr4b chemokine signalling slows down stem cell delamination, which invariably occurs at the anterior aspect of the P0-neuromast. They then show (via changes in cadherin-type gene expression and cellular imaging) that stem cell delamination from the P0 neuromast involves an epithelial-to-mesenchymal transition. Forcing this process in the entire neuromast accelerates new organ formation, but directionality is maintained. Finally they ask whether the stem cells required for this process are pre-specified or are generated from the neuromast by ablating the pre-bud stem cells. They find that other stem cells within the same organ rearrange and re-establish organ founder cells. The authors liken this new mechanism of organ formation to pathological metastases in humans and name it metastatic-like organogenesis.

Major comments

• In Fig. 2D both P0- and PE1-neuromasts appear with fewer hair cells, the reasons for this should be explained. Did the ablation also damage the primordium or is the pLL nerve required for complete neuromasts to form?
• p. 6: The reasoning behind generating a cxcr4b l-o-f mutant does not become clear, since a mutant already existed with a non-migratory primordium. Why did the authors expect their mutant would have a different phenotype?
• p. 7: The authors state that K15::cxcr7 larvae lack secondary embryonic neuromasts, but it seems from Figs. 3B,D that they might simply be delayed (note that the last one is missing in 3B). This delay may have been taken over from the delay in embryonic primordium formation of P0 and this result (as shown in Fig. 3) would not contradict the assumption that PE1 can form in the absence of cxcr4b signalling. I suggest that the authors actually show and quantify that K15::cxcr7 adults have fewer CNC neuromast numbers, because this seems to be the definitive proof that overexpressing the "sink" may be enough to reduce cxcr4b signaling to a level where its requirement for the formation of the PE1-neuromast can be assayed.
• Fig. 6A': The scheme is at least ambiguous and interpretation of it requires more supporting information: In A the stippled lines represent position within the neuromast, but what do they represent in A', numbers of BrdU+ cells? So what does e.g. the grey peak at the anterior mean - position or number of cells? Is the area inside the coloured lines important or the edge-points? Stage III is the only distribution with a clear left-bias, the others are centered (with a left-extreme for stage II), so what in the figure is the anterior proliferation peak? These are just some questions this reviewer had. Maybe the problem lies in the octagonal lines, meaning different things in both images? It is further unclear how the means given in the text can be derived from the figure. Maybe it would be best to try to represent the data with a heat map-overlay of the image in A, one for each stage?
• The authors propose two competing models regarding the origin of founder stem cells (p. 9, first sentence: early determination vs. in situ generation). In the third sentence again two scenarios are presented as to why experimentally prompting EMT does not trigger organ founder cell migration. This paragraph would benefit from stating more precisely which of these questions is addressed by the BrdU- and ablation experiments, together with a clearer statement at the end of that section as to which hypothesis in each case (origin and migration) is preferred.

Minor comments

• K15+ cells are described as neuromast stem cells and Fig. 1 suggests that these are the mantle cells: Please comment on the question whether all mantle cells are stem cells.
• p. 5: The reference (Seleit, Krämer et al., 2017) is ambiguous, as there are 2 references listed that fit the abbreviation.
• Fig.1B-F': Even though, as the authors state, there is variation in the timing of the budding process, it would be helpful to add an exemplay time frame to stages I-V.
• Fig. 4A'-D': The cell bodies of the support cells should have a distinct colour, otherwise they are easily confused with the nuclei of the other cell types. This would make it easier to understand the schematic at first sight.
• p. 9: T2A and H2A should be explained.
• Nuclei are shown protruding posteriorly in wildtype neuromasts (Fig. 5A-A'), while P0 neuromasts stem cells protrude anteriorly. Please explain the significance of the difference.
• Fig. 5 legend: Quantification is "E"; "and increased" should probably read "an increased"?
• p. 10: Unconventionally, Fig 7 is mentioned prior to Fig. 6B-C, I suggest combining both figures into one.

Significance

The manuscript describes a new mechanism of post-embryonic organ formation. Investigating how accesory neuromasts are formed during growth of juvenile medaka, the authors find that stem cells from a founder neuromast undergo epithelial-mesenchymal transition and migrate away directionally to form a complete new organ. This new mechanism is likened to that of cancer metastases.

Importantly, and different from zebrafish, this process is not dependent on innervation of the neuromast and is not a budding process, but relies on neural stem cells leaving the organ.

The interesting question posed by the authors, and answered here positively for accessory neuromasts in juvenile fish, is whether the mechanisms of organ formation differ between embryonic and post-embryonic development. The reported findings should be of interest to the stem cell communities and to researchers interested in post-embryonic development.

This review was written by a developmental biologist.

DOI: 10.1016/j.cell.2023.04.012

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DOI: 10.1016/j.celrep.2023.112591

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DOI: 10.1016/j.immuni.2023.05.004

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As a society we fail to make the education system a fun and welcoming places where anyone is welcomed and teachers believe students can be successful, if there were more administrators that cared and act on the issue more students would want to return to school and stay enrolled.

Brenez faz uma provocação interessante, mas me parece que a crise do cinema de autor tem mais relação com a diminuição do espaço destinado aos filmes de médio orçamento nos grandes estúdios, um tipo de investimento de alto risco. Tirando os figurões da indústria (e olhe lá, Martin Scorcese teve dificuldade de financiar O irlandês), a produção autoral teve que se adequar muito à lógica do baixo orçamento, o que reduz algumas possibilidades criativas e de contato com um público mais amplo. O digital, na verdade, facilitou o acesso à filmografia completa de inúmeros diretores, antigos e contemporâneos, num fluxo mais horizontalizado de revisitações e apropriações possíveis.

Há, aqui, uma preocupação óbvia com a questão da conservação da obra, mas é interessante como a resistência do analógico também revela algo da nossa própria experiência com a matéria, uma certa inclinação àquilo que pode ser tocado, pesado, cheirado, enfim.

Apesar de Brenez fechar um pouco a discussão em torno das imagens psíquicas (portanto, mentais), é possível convocar Vivian Sobchack para pensar essa circularidade em termos de "experiência (corpórea) da percepção" e "percepção da experiência", sendo o cinema "uma expressão da experiência por [meio da] experiência" própria do sujeito e mediada pela técnica.

Farocki foi um documentarista experimental. Junto com Andrei Ujică (citado mais pra frente), fez um documentário muito importante nessa intersecção da tecnologia das imagens com a política, o "Videogramme einer Revolution". Ele tá na íntegra no YouTube com legendas em inglês.

"Fellini disse certa vez que a televisão criou uma nova geração de espectadores que ele considerava arrogantes, autoritários, neuroticamente impacientes. A capacidade de mudar de canal a qualquer hora, o que aniquila a narrativa, de prosseguir à procura de uma coisa a mais (a ideia de que imagens que eu não estou vendo estão sendo exibidas em outro lugar é intolerável), originou espetáculos de fogos de artifícios que jorram das pontas dos dedos. O mágico controle remoto nos dá a ilusão de que essas imagens nos pertencem, de que temos poder total sobre elas, de que não existiriam sem nós. Essa impaciência, nunca poderia ser satisfeita ou mitigada, pois não conseguimos fixar nossos olhos numa imagem antes de eliminarmos o filme a que estávamos assistindo, assim como não podemos pronunciar duas palavras ao mesmo tempo." Jean-Claude Carrière