com o oferecimento da denúncia ou queixa (STF)

As the final statement made to the reader, I found it quite interesting that Eliot decides to further dimensionalize his already well-formed metaphor of drowning and water. In it, he utilizes rock—which was firstly represented as a physical representation of struggle and strife, but not death—as a parent of water, as rock and minerals filter water. But now, without the presence of water, what is left is sediment and "[T]he road winding above among the mountains/Which are/mountains of rock without water/If there were water we should stop and drink/Amongst the rock one cannot stop or think..." In this, a mental image of difficulty and great pain is forced onto the reader, dramatizing death further than it once was, which Eliot adds to his commentary on humanity in a post-World War I world, demonstrating the final moments of humans that live according to impulse and without the stronghold of faith and spirituality within them.

In “What The Thrush Said. Lines From A Letter To John Hamilton Reynolds, ” by John Keats, he assures the reader that through faith in God and trust in His word, "the spring will be a harvest-time," and good fortune is imminent. Not only this but the afterlife in the heavens is promised, so long as the Christian remains faithful: "O thou, whose only book has been the light Of supreme darkness which thou feddest on Night after night when Phoebus was away, To thee the Spring shall be a triple morn."

Keats supports Eliot's idea of peace through religion, representing the other man's possibility of tranquility, despite hardships that may seem to prevail.

In Historical Annotations 1, Lauren Sonneborn explains the differences between how fire is portrayed in Christianity vs. Buddhism, and the contradiction that occurs when considering Dante's Inferno, where the lowest, most torturous levels of hell are actually icy and cold. What strikes me about this contradiction is that it actually (ironically) bridges the previously mentioned gap between Buddhism and Christianity. In the translated The Ādittapariyāya Sutta (Pali, "Fire Sermon Discourse”), it is explained that "all things are on fire," as fire is an umbrella term that describes all indicators of human emotion–anything from passion to hatred. By the nature of these emotions, this fire is fueled by one's consciousness. Fire could be a metaphor for the ego (honestly, I use "the ego" for lack of a better word to describe the force that drives the human mind to be self-centered and subjective), because later on it is explained that the only way to avoid the fire is to avoid, or "conceive an aversion" to any feeling, perception, or earthly/mortal sensation entirely. Ultimately, once successful in creating such an aversion, one becomes "free". The text then seems to suggest that the tangible things and sensations of the earth are actually restrictive, and that any illusion of personal autonomy is false, since one is not free until their mind is completely sterile and removed from the world. The way that Pali Canon describes life is not "unsimilar" to how Dante pictures the last ring of Hell: complete lack of freedom, surrounded by reflections of yourself (ice is reflective), and burning (both fire and ice have the capacity to burn). Lauren then poses the question,

Is the waste land a space in the mind, extinguished and waterlogged?

After previously explaining that

Perhaps, this is why the next section is titled "Death by Water," as the same water that quenches a fire also extinguishes the human spirit.

She suggests that Eliot is skeptical of the sterility that Buddhism describes, and that he is actually in favor of maintaining the "human spirit", not quenching it. However, I see Elliot as aligned with the Buddhist philosophy. He writes "O Lord Thou pluckest me out," following his reference to the burning, seemingly begging to be liberated from the Earth–the use of the word "pluck" illustrates the "plucking" of a flower or plant from the ground. This image further solidifies the connection between human consciousness and vegetation, trapped by the land that sustains its life. So, instead of the wasteland being a fragment of the mind that is "waterlogged", which suggests that "waste" is a discrepancy, I wonder if it is actually the entirety of human consciousness.

Annie Xu’s annotation from two years ago explains how the hermit thrush embodies a multitude of contradictions, serving as “life and death, darkness and light, suffering and vitality simultaneously”. Building off of this idea, I think that the hermit thrush almost represents Eliot's conception of the ideal human attitude towards natural cycles of life and death, retreating during winter but predictably returning, in full rejuvenation, in the spring. The advantage of the hermit thrush’s paradoxical qualities is that it accepts fertility patterns without resistance. Humanity’s essential problem, as portrayed by Eliot in The Waste Land, is its struggle to relate to cycles of nature, hence why it perceives April as the “cruellest month” and is unable to see spring as the "harvest-time” that the hermit thrush does. This is exemplified by the proliferation of false prophets — such as Madame Sosostris — whose prophesying, often in the crude, sensationalist imagery of the tarot cards, has displaced ancient fertility rites as a means of telling the future. The repetition of “O fret not after knowledge” can also be read as a kind of indictment of the temerity of human fortune telling. The birds remind us: humans “have [no knowledge]”, and any seeming success of intelligibility and communication is owed to nature, which is the organising force of all human affairs: “And yet my song comes native with the warmth”. The song is “native” because of spring warmth. Annie highlights the contradiction implied in Keat’s line, “whose only book has been the light Of supreme darkness which thou feddest on Night after night when Phoebus was away”. In these words, the poem suggests that the hermit thrush is capable of nurturing himself and maintaining intellectual integrity when Phoebus, the mythological god of son, is absent. According to the habits of the hermit thrush, light can be found within the all-encompassing “supreme darkness” of winter. This line also reminded me of a quotation from the Book of John: [9] Jesus answered, Are there not twelve hours in the day? If any man walk in the day, he stumbleth not, because he seeth the light of this world. [10] But if a man walk in the night, he stumbleth, because there is no light in him. And later: [35] Then Jesus said unto them, Yet a little while is the light with you. Walk while ye have the light, lest darkness come upon you: for he that walketh in darkness knoweth not whither he goeth. These quotations represent the relatively banal notion that humans contain a capacity to act both rationally and, in the absence of light, irrationally and immorally. Furthermore, John advances the notion of an innate connection between human behaviour and external factors, including cosmological phenomena. When a man “[walks] in the night”, he stumbles not only because of the lack of light provided by the sun, but because “there is no light in him”. Similarly, the aimlessness of man’s walk at night is because he internalises the darkness of his surroundings, which have “come upon” him in both a physical and spiritual sense. The thrush engages in a similar act of absorption in the way he consumes the book of “supreme darkness”, feeding on it.

Eliot’s reference to the hermit thrush songs also epitomises the spoof scholarship of many of his footnotes. There is something quite nonsensical about the way he describes the bird with its latin title and claims that the thrush’s “'water-dripping song' is justly celebrated.” If the thrush acts as a paradigm of how humans ought to relate to fertility cycles, what is the purpose of his sarcasm?

Eluding to the described sensations of passionate human life and the overstimulation that is worldly attachments, Eliot references Buddha's "The Fire Sermon" in both this quote and the title of this poem. Utilizing Buddha's understanding of human life, Eliot's exclamation of burning and cry for the Lord's mercy synthesizes the tone of the poem and of Wasteland as a whole, making a final statement upon human life and its burdens and the crumbling society of Europe and London in a post-Great War world as a pessimistic realm of physical and spiritual agony in which the only escape is death.

In the eyes of Buddha, an escape from life constitutes a spiritual AHA, in which the subject releases all concerns of earthly attachments and "burnings." - "Whatever sensation, pleasant, unpleasant, or indifferent, originates in dependence of impressions received by the eye, which is also on fire,"- Thus, the only way to be released from these hindering shackles is a spiritual awakening, he claims, rather than Eliot's pessimistic perspective on escapism by way of death.

SÚMULA VINCULANTE 31 - É inconstitucional a incidência do Imposto sobre Serviços de Qualquer Natureza — ISS sobre operações de locação de bens móveis.

Fundamentos:

RE 446.003 AgR - imposto sobre serviços (iss). locação de veículo automotor. inadmissibilidade, em tal hipótese, da incidência desse tributo municipal. distinção necessária entre locação de bens móveis (obrigação de dar ou de entregar) e prestação de serviços (obrigação de fazer). impossibilidade de a legislação tributária municipal alterar a definição e o alcance de conceitos de direito privado (ctn/1966, art. 110). inconstitucionalidade do item 79 da antiga lista de serviços anexa ao dl 406/1968. precedentes do supremo tribunal federal. recurso improvido. Não se revela tributável, mediante ISS, a locação de veículos automotores (que consubstancia obrigação de dar ou de entregar), eis que esse tributo municipal somente pode incidir sobre obrigações de fazer, a cuja matriz conceitual não se ajusta a figura contratual da locação de bens móveis. [RE 446.003 AgR, rel. min. Celso de Mello, 2ª T, j. 30-5-2006, DJ de 4-8-2006.]

• Segundo entendimento desta Corte, o poder de tributar municipal não pode alterar o conceito de serviço consagrado pelo Direito Privado, consoante prevê o art. 110 do CTN/1966. Ademais, não há que se falar na superação do entendimento da Súmula Vinculante 31 pelo advento da edição da LC 116/2003. É certo que a LC 116/2003 revogou a lista de serviço da legislação anterior e estabeleceu um novo rol de materialidades para o imposto. Na lista atual, a locação de bens móveis seria o item 3.01 (Locação de bens móveis) da lista de serviços tributáveis. Entretanto, a intenção do legislador não se confirmou por força do veto presidencial, que foi motivado pela orientação jurisprudencial desta Corte (...). 3. Também não merece prosperar o argumento de que há fortes indícios da superação do entendimento deste Tribunal a respeito da matéria em exame, uma vez que a jurisprudência permanece afirmando que não incide ISS sobre locação de bens móveis e que a CF/1988 não concede aos entes municipais da Federação a competência para alterar a definição e o alcance de conceitos de Direito Privado para fins de instituição do tributo. [RE 602.295 AgR, voto do rel. min. Roberto Barroso, 1ª T, j. 7-4-2015]

Observar que não são taxativas as regras sobre limitações do poder de tributar.

Questão correlata:

PROCURADORIA GERAL DO ESTADO DO AMAZONAS CESPE (CEBRASPE) 2016

Considerando os limites ao exercício do poder de tributar, julgue o item seguinte.

As limitações ao poder de tributar são normas de restrição da competência tributária taxativamente previstas na CF.

Certo.

Errado.

Justificativa:

• ERRADA.

• "As limitações ao poder de tributar são normas de restrição da competência tributária taxativamente previstas na CF"

• A questão cobra conhecimentos acerca das limitações ao poder de tributar, contidas no Título VI da CF/88, em seu art. 150. As limitações ao poder de tributar instituídas pela CF são regras que limitam o exercício da competência tributária pelos entes federados que a detém.

• Essas limitações configuram um rol não exaustivo, já que no caput do art. 150 é previsto que essas limitações são definidas 'sem prejuízo de outras'.

• Art. 150. Sem prejuízo de outras garantias asseguradas ao contribuinte, é vedado à União, aos Estados, ao Distrito Federal e aos Municípios: (...)

• Dessa forma, o texto constitucional permite que outras regras limitadoras do poder de tributar sejam criadas, além das que se encontram na lista contida no artigo 150. Logo, essas hipóteses não são taxativamente previstas na CF e, por isso, a questão está errada.

SHANGYU YANG 1.in a society the member of a species cooperate with one another to achieve objectives collectively that they could not achieve as individuals. 2.The traditional social sciences focus their attention upon the behaviour of the species Homo sapiens, examining how people interact with one another and how they organise themselves for co-operative activities.but then devote a great deal of attention to dysfunctional behaviour, such as crime and war and malfunctional phenomena, such as unemployment and pollution. 3.i think the social science is reaserch the in a society the member of a species i nteract with one another same species or with one o t h e r s s p e c i e s ' interact.

Author Response

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

We thank the reviewers for their thoughtful assessment of our work and their valuable critiques which we will address in the “Recommendations for the authors” section below. In particular, we appreciate Reviewer #3 noting the value of the C. elegans model system and our efforts to bridge models with our study. We agree with the reviewer that there is a need to clarify the rationale, presentation and interpretation of our results. We have substantially revised the text in our manuscript and Figure legend to address this issue, and provided extensive new commentary and citations to lay out the logic behind our experiments. Indeed, it was our oversight not being more thorough about this initially. We have further adjusted our conclusions to be less unequivocal. Finally, we added an RPM-1 signaling diagram (Fig. 8A) to more clearly annotate the players in the RPM-1/MYCBP2 signaling network that were evaluated genetically in Fig. 8. Importantly, we provide clearer commentary on how genetic enhancer effects with known RPM-1 binding proteins and the absence of genetic suppression in vab-1/Eph receptor double mutants with components of the RPM-1/FSN-1 ubiquitin ligase complex are consistent with the biochemical finding that MYCBP2 stabilizes but does not degrade EphB2. Text edits reflecting these points are in the abstract, the C. elegans results section starting on line 411, and the discussion on lines 499, 502-504 and 541.

Following extensive discussions between the three reviewers, all three agree that the C. elegans data, as presented, does not add to, and in fact might harm, your bottom line. Our combined suggestion is to take this data out unless you plan to improve it substantially. All reviewers are perplexed by Figure 2F and the presumed interactions of cytosolic proteins with the extracellular domain of EPHB2. At the very least, please provide some suggestions/model/interpretation.

We have adjusted our manuscript substantially to address this. Please see detailed comments in the individual Reviewer sections below.

We would like to thank the reviewers for their thorough examination of our manuscript, constructive criticisms, and helpful suggestions.

Reviewer #1 (Recommendations For The Authors):

The work is extensive in my view, and mostly of high quality. See minor comments on some of the figures below.

Thank you very much.

Two more major comments :

• I don't think the C. elegans work adds to - in fact I think it hurts - the statement that this regulatory mechanism is specific to EphB2. I would advise the authors to take it out.

We agree that C. elegans has a sole Eph receptor called VAB-1 and is therefore not a specific model for EPH2B. However, testing MYCBP2 specificity for EPHB2 was not the goal or our perceived value for the C. elegans experiments. We now clarify this in the text of the Results section.

Rather, we are providing evidence that the C. elegans ephrin receptor interacts genetically with known MYCBP2/RPM-1 binding proteins. Moreover, we now provide an extensive array of citations to note that genetic enhancer interactions between different RPM-1/MYCBP2 binding proteins is well established. The reviewer has nicely highlighted for us that we handled the C. elegans genetics in too cursory a fashion in our original manuscript. We appreciate this being noted and have now aimed to make this substantially clearer. We hope the reviewer agrees that our revised C. elegans section accomplishes this goal.

Furthermore, we extensively revised the text of the Results to emphasize a key point: our observation that axon termination defects are not suppressed in vab-1; fsn-1 and vab-1; rpm-1 double mutants excludes the possibility that the VAB-1 Eph receptor is a substrate that is inhibited or degraded by the RPM-1/FSN-1 ubiquitin ligase complex. If the VAB-1 Eph receptor were ubiquitinated and degraded by the RPM-1/FSN-1 complex, we would have observed a suppression of phenotype in vab-1; rpm-1 double mutants. The precedent for this genetic relationship between the RPM-1 ubiquitin ligase and its substrates that are degraded has been established by several prior studies (PMID: 15707898; PMID: 31676756; PMID: 35421092). We now more clearly note that the absence of genetic suppression in vab-1; rpm-1 double mutants and vab-1; fsn-1 double mutants is consistent with the non-canonical stabilizing role of MYCBP2 on EPHB2 that was observed in our biochemical experiments with mammalian cells.

We also adjusted the text of the manuscript to stress that we are testing genetic interactions between the VAB-1 Eph receptor and known RPM-1 binding proteins. This is a key point, as genetic enhancer interactions are consistent with the Eph receptor functioning in the RPM-1 signaling network. This concept has been well established for RPM-1 binding proteins as now noted in our revised text with an extensive number of additional citations to published work.

Based on the above arguments, we respectfully disagree with the reviewer that our C. elegans data should be removed from the paper. To re-iterate, we are not trying to evaluate specificity for MYCBP2 and EPHB2 in C. elegans. Rather, our goals are twofold: 1) To ask whether there is an evolutionarily conserved functional genetic link between Eph receptors and known RPM-1 binding proteins. 2) To provide further in vivo genetic evidence invalidating the hypothesis that Ephrin receptors could be ubiquitination substrates that are inhibited/degraded by MYCBP2.

Text edits reflecting these points are in the abstract, the C. elegans results section starting on line 411, and the discussion on lines 499, 502-504 and 541.

• The cellular responses are not robust and the effects of MYCBP2 KO - although significant - are minor in most cases. But I don't think more experiments will help here.

We interpret the comment about the robustness to mean that the extent to which a given cellular response is affected by the loss of MYCBP2 is minor. First, the cellular responses themselves are typical of previous studies and depend on the cellular biology underlying them. For example, a growth collapse of ~50-60% over a background of 10% (Fig. 7) is typical for these sorts of assays (PMID: 37369692; PMID: 33972524; PMID: 17785182). A decrease of cell area by ~25% (Fig. 3) is quite substantial if one considers how much of a cell’s volume is taken up by the nucleus and organelles. Second, the phenotypes elicited by the loss of MYCBP2 are likely brought on by a decrease in EphB2 protein levels, but not its complete absence, as suggested by our biochemical experiment. Given that EphB2 complete loss only affects the cellular responses to a limited extent, the minor effects are not a surprise (e.g. for GC collapse: PMID: 23143520). Nevertheless, the subtle changes in cellular phenotypes, elicited by EPHB2 signaling are often sufficient to achieve proper cell positioning and cell response to guidance cues. For instance, regulation of the growth cone collapse of the outgrowing axons requires delicate changes that are dynamic and temporal.

Minor:

Fig 1C - EPHA3 and EPHB2 seem to run in different sizes, is this the case? In 2A they run at the same size.

We believe this size discrepancy is due to different percentages of SDS-PAGE gels used to resolve proteins. In Fig. 1C, we used a 6% gel for a Western blot analysis of both EPHA3/-B2-FLAG (~130 kDa) and MYCBP2 (~510 kDa). In Fig. 2A however, we performed Western blot analysis using 10% resolving gel to separate and detect EPHA3/-B2-FLAG along with MYC-FBXO45 (~30 kDa). We have reviewed the results obtained from additional biological replicates of this experiment, and observed a similar pattern in gel migration of EPHA3/-B2-FLAG across all replicates.

Fig1F - I can't trust the MYCBP2 blot.

Indeed, the MYCBP2-EPHB2 co-IP with endogenous proteins was not convincing. We now repeated this experiment using rat cortical neurons, and the results replace the previous Fig. 1F panel as mentioned on line 158.

In Fig2b the authors claim that there is enhancement in the binding of MYCBP2 and EPHB2 upon FBXO45 expression. For this type of statement quantification is required.

The quantification is now included in Fig. 2C and its significance is mentioned on line 180. Our conclusion about the enhancement stands.

Fig2G - it remained unclear to me where the binding site to MYCBP2 is, how long is the cytoplasmic tail in the DeltaICD protein?

Based on our experimental observations from Fig. 2E-H, we concluded that the fragment encompassing the extracellular domain(s) and/or transmembrane (TM) domain of EPHB2 is necessary for the protein complex formation with MYCBP2. We would like to accentuate that the EPHB2-MYCBP2 interaction might not be direct, and might involve other transmembrane protein(s) acting as a scaffold for EPHB2 and MYCBP2 binding. We did not pursue experiments to determine the exact region of the extracellular-TM portion of EPHB2 that is required for the interaction with MYCBP2.

The cytoplasmic tail in ΔICD protein consists of 25 aa of the N-terminal fragment of EPHB2 juxtamembrane (JM) region, which is adjacent to the TM helix, and followed by the 8 aa FLAG tag (EPHB2 ΔICD domain composition: extracellular domains – TM domain – 25 aa fragment of JM region – FLAG). We have determined the TM and JM sequences based on Hedger et al. (PMID: 25779975) and included the N-terminal portion of the JM region to facilitate proper ΔICD protein localization within the plasma membrane (PMID: 35793621). We modified the schematic in Fig. 2G to better visualise the EPHB2 truncations and now provide information on their size in the figure legend.

Always good to have a model of how all these proteins work together.

While we acknowledge that this would be helpful, we do not have a clear answer on how the EPHB2-MYCBP2 complex formation occurs. This requires further elucidation of the putative proteins involved in this ternary complex or testing the possibility that a MYCBP2 fragment is extruded extracellularly. Without these experiments there are too many possibilities to summarise into a clear model figure. We thus did not make any edits regarding these possibilities in the section starting on line 195.

Reviewer #2 (Recommendations For The Authors):

Overall, the experiments are classical experiments of co-immunoprecipitations, swapping experiments, collapse assays, and stripe assays which all are well carried out and are convincing.

Thank you for your encouraging comments.

Controls for the stripe assay may include Fc / Fc stripe assays.

We have performed these control experiments and now include their quantifications in the results sectioning concerning Fig. 3, starting on line 249, and those concerning Fig. 6 on line 381.

It is not clear to me why SD and not SEM has been used here for presentations.

Standard deviation (SD) measures the dispersion of a dataset relative to its mean. The standard error of the mean (SEM) measures how much discrepancy is likely in a sample’s mean compared with the population mean. Thus, SEM includes a statistical inference about the sampling distribution while SD is a less “processed” measurement that by definition is larger than SEM. SEM might make the data look less dispersed and many journals encourage the use of SD in bar graphs (PMID: 16223828).

Fig 7A: it is rather difficult to see 'branches' in Fig. 7A, better pictures and close-ups should be provided. How are branches defined? This piece of work needs more attention.

To remedy this shortcoming, we now provide inverted images with GFP signal in dark pixels overlaid on Fc (white) / eB2 (pink) stripes next to the original images.

Reviewer #3 (Recommendations For The Authors):

1) My most important suggestion to the authors would be to more carefully describe the results and their interpretation of the results. Sometimes, the distinction is not clear.

We modified the text throughout the manuscript to address this.

2) There are several cases, when the authors report on trends that are not statistically significant (1D, for example), or report no change, when it is clear that the addition of one more sample could have dramatically made a difference (4M - see point 12).

We agree that some of the nonsignificant differences could become significant if we added more Ns. But we prefer not to move our experimental design towards N-chasing and p-hacking (PMID: 25768323). The number of biological replicates is normally pre-determined before the onset of the experiment. Of course, some replicates can be discarded if there is a valid reason, such as a technical issue with the experiment or a positive control not working but this is not relevant for the dataset we have provided.

3) Data in 1F is very difficult to interpret.

As in response to Reviewer #1: Indeed, the MYCBP2-EPHB2 co-IP with endogenous proteins was not convincing. We now repeated this experiment using rat cortical neurons, and the improved results are in revised Fig. 1F.

4) Figure 2 puts Figure 1 in a strange perspective. If I understand correctly, fig 2 claims that EPHB2 interaction with MYCBP2 depends on FBXO45 - if that is the case then how does the binding in Figure 1 occur?

Indeed, we propose that the EPHB2-MYCBP2 interaction depends on FBXO45. In Fig. 2, we reveal that FBXO45 enhances the formation of the EPHB2-MYCBP2 complex. Thus, we suspect that the endogenous FBXO45 present in HeLa cells and neurons would mediate the interaction between EPHB2 and MYCBP2 in Fig. 1 experiments. We were unable to show this by Western blotting due to lack of reliable commercial antibodies against FBXO45, the complex containing endogenous FBXO45 and EPHB2 is also implied by our AP-MS data (Fig. 1B) and published databases.

5) I am still trying to wrap my mind around the results in 2G-H. So do MYCBP2 and FBXO45 bind the extracellular domain of EPHBP2? What does that mean?

(see also our response to Reviewer #1, end of their section) Based on our experimental observations from Fig. 2G-H, we conclude that the fragment encompassing the extracellular domain(s) and/or transmembrane domain of EPHB2 is necessary for the protein complex formation with MYCBP2 and FBXO45. Although there is a possibility that MYCBP2 directly binds the extracellular portion of EPHB2, we have not formally tested this hypothesis. MYCBP2 has been previously shown to interact with the extracellular portion of transmembrane N-cadherin (CDH2) via BioID proximity labeling and AP-MS proteomics approaches (PMID: 32341084).

Considering the results in Fig. 2A-B, we suspect that EPHB2-MYCBP2 interaction is indirect, as FBXO45 enhances this association. Secretion of FBXO45 and direct binding of FBXO45 to the extracellular cadherin (EC1-2) domains of N-cadherin has been documented (PMID: 25143387; PMID: 32341084). Although, not tested, this is also a possibility for EPHB2-FBXO45 mode of interaction. Nevertheless, we also cannot rule out the possibility that an unknown transmembrane protein binds EPHB2 extracellularly and the same unknown protein binds MYCBP2/FBXO45 intracellularly. Resolving this model is beyond the scope of this study and will require us to pursue extensive new lines of investigation.

6) I don't understand the stable Hela cell line CRISPR - is this a stable MYCBP2 deletion? In which case why is there only a reduction, not complete elimination of the protein? Or, is this a stable integration of a plasmid generating gRNA against MYCBP2? In which case, I would expect a homozygous null to emerge at some point. In any case, this is not well explained.

These lines are not derived from single cells infected with the CRISPR sgRNA-carrying viruses, therefore they are not clonal and probably contain some cells that express normal levels of MYCBP2, hence its detection on a Western. This is now clarified starting on line 221 and on line 608.

7) In 3C - is this the right statistical analysis?? I would say you want to claim the different effect of the control +/- eB2 compared to the effect in the mutant +/- eB2. Still should be significant but I think a more correct analysis.

We now include this comparison in Fig. 3C as well in the results section starting on line 234.

8) The robustness of the assay in Figure 3D is underwhelming – how was the area measured?

This is a live imaging experiment. Fig. 3D plots cell area at 60 minutes after ephrin-B2 addition as a fraction of the same cell’s area at 0 minutes (ephrin-B2 addition). For control cells that is a decrease of ~25%. If one considers that a cell’s nucleus and organelles like the Golgi Apparatus take up most of its volume, the magnitude is not that surprising.

9) Figure 3F – did you try to plot the relative area of overlap divided by the total cellular area? You might get a more striking phenotype. Also – claiming that this confirms that MYCBP2 is REQUIRED for EPHB2 function is a bit overstated, especially given that we don’t know (do you?) the EPHB2 mutant phenotype in this assay.

We preferred to stay with the original method of image quantification which we use for other assays. With respect to the requirement of MYCBP2 for EPHB2 function in the stripe assay, our logic is rooted in the observation that native HeLa cells do not respond to ephrin-B2 stripes (45.46 ± 7.62% of cells on eB2 stripes v. Fc; data not shown). When they are transfected with EPHB2 expression plasmids they do, therefore we assume that EPHB2 expression endows them with a sensitivity to eB2 stripes. A loss of MYCBP2 attenuates this sensitivity. We clarified this starting on line 246 and on line 251.

10) I didn't quite get the difference between 4A and 4B.

We apologize for the confusion. In Fig 4A, we used a stable HeLa cell line that has tetracycline-inducible expression of EPHB2-FLAG. Using these cells, we subsequently generated CTRLCRISPR or MYCBP2CRISPR cells. In these cells we then induced EPHB2 expression with tetracycline and observed that deletion of MYCBP2 resulted in the reduction of EPHB2 protein levels. To confirm this observation and to rule out the possibility that EPHB2 protein reduction is an effect of the CRISPR lines generation, we tested whereas MYCBP2 deletion reduces EPHB2, which has been transiently overexpressed (Fig. 4B). We hence conclude that loss of MYCBP2 decreases EPHB2 that was either expressed from a stable locus (Fig. 4A) or from transient transfection (Fig. 4B). We modified the Results section starting on line 262 to make this point clear.

11) The entire link to lysosomal degradation should be strengthened. Perhaps I am confused, but if the reduced EPHB2 levels in MYCBP2 mutant cells result from impaired lysosomal degradation then inhibiting the lys-deg should bring the protein levels back to normal (i.e. CRISPR control) - no? As currently presented, I do not understand nor do I think the claim is strongly supported by the data.

Before treatment with inhibitors, EPHB2 levels in MYCBP2CRISPR cells are already 40% lower than they are in CTRLCRISPR cells and in all our attempts, inhibitors can only rescue/restore EPHB2 in MYCBP2CRISPR cells to a level that is lower than in CTRLCRISPR cells. But this restoration is greater in MYCBP2CRISPR than in MYCBP2CTRL cells (BafA1: 19% increase in CTRL cells and 40% in MYCBP2CRISPR cells; CoQ: 10% comparing to 35%). This indicates that EPHB2 degradation through the lysosomal pathway in MYCBP2CRISPR cells is stronger, explaining why EPHB2 degradation is promoted in MYCBP2CRISPR cells, compatible with reduced EPHB2 levels and enhanced EPHB2 ubiquitination.

12) 4M, O - reporting ns based on these data seems a bit strange to me... Add one point and it will be strongly significant.

See our response to point (2), above. We prefer not to invoke potential p-hacking.

13) 7d - so what are you claiming? That the cellular response to eB1 but not eB2 is affected by the addition of FBD1? this is almost the opposite of what you wrote in the text...

We treated the cells with two different ephrin-B ligands to make a stronger conclusion. When using ephrin-B1, growth cone collapse in FBD1 WT is not significant comparing to Fc treatment. When using ephrin-B2, growth cone collapse in FBD1 WT is not as significant as it is in FBD1 mut group (* versus ). We interpret this as meaning that the EPHB2-mediated growth cone collapse to both ligands is dampened, when we disrupt the EPHB2-MYCBP2 association. The difference between these two ligands might be due to their different affinities for the receptor or signalling kinetics.

14) By far the weakest link in this paper is the worm part. I think it's a pity because strengthening this would affect the significance of the finding. First, the authors mention new genes without introducing their relationship to the signaling pathway tested. Second, the textual logics should be strengthened. Finally and most importantly, when the difference between the phenotypic severity is so strong (vab-1 and rpm-1) then I think it's impossible to say anything from the double mutant.

We appreciate the reviewer noting that they appreciate the value and importance of the C. elegans model. The goals of our C. elegans experiments were twofold:

1) To evaluate genetic interactions between the VAB-1 Eph receptor and known RPM-1 binding proteins. This was not clearly explained in the original manuscript nor was the published precedent for these types of genetic enhancer experiments provided. We have now rectified this by substantially revising the text of the Results C. elegans section starting on line 431 and by adding several citations.

2) Our C. elegans genetics confirmed that the VAB-1 Eph receptor is not inhibited/degraded by the RPM-1/MYCBP2 ubiquitin ligase complex. We have now revised the text to draw this point out more clearly.

To further address the reviewer’s concerns, we have added a new schematic (Fig. 8A) to show the relationship between the RPM-1 and the RPM-1 binding proteins (FSN-1/FBXO45 and GLO-4/SERGEF) we are testing. We chose FSN-1 because it is part of the RPM-1 ubiquitin ligase complex and we chose GLO-4 because it functions outside the context of RPM-1 ubiquitin ligase signaling via the GLO-1 Rab GTPase to influence late endosomal/lysosomal biogenesis.

Regarding the reviewer’s concern that different penetrance/frequency of defects between rpm-1 mutants and vab-1 mutants means outcomes with vab-1; rpm-1 double mutants cannot be interpreted. We respectfully disagree. An extensive number of published studies have demonstrated that RPM-1 binding proteins have milder phenotypes than rpm-1 mutants and display genetic enhancer effects as double mutants with one another (PMID:17698012, PMID: 22357847, PMID: 25010424, PMID: 24810406). We now make this point much more clearly. While the frequency of axon termination defects in rpm-1 mutants is high it is not completely saturated as the defect is not 100%. Moreover, a major point of the vab-1; rpm-1 double mutants is that they do not have a significant reduction in phenotypic penetrance/frequency. Thus, our system is fully capable of resolving genetic suppression, which did not occur. We now make this point much more carefully and clearly.

To further address the reviewer’s concern, we have softened language about the VAB-1/Eph receptor functioning in the same pathway as RPM-1 throughout the manuscript. While we think this is still the case, because the frequency of axon termination defects is not fully saturated in rpm-1 mutants and defects could potentially become more severe (i.e. the hook might occur closer to the head of the animal rather than in the midbody). Nonetheless, this is not a critical point and we think it is more important to be clear about the two major goals and objectives of our C. elegans experiments. We hope the reviewer agrees that our rationale, logic and conclusions are more clearly and accurately drawn in the revised paper.

The repetition of happy made me smile a bit because happy is an emotion i always see myself in. Happiness

Reviewer #3 (Public Review):

Summary:

This manuscript develops a new method termed MINT for decoding of behavior. The method is essentially a table-lookup rather than a model. Within a given stereotyped task, MINT tabulates averaged firing rate trajectories of neurons (neural states) and corresponding averaged behavioral trajectories as stereotypes to construct a library. For a test trial with a realized neural trajectory, it then finds the closest neural trajectory to it in the table and declares the associated behavior trajectory in the table as the decoded behavior. The method can also interpolate between these tabulated trajectories. The authors mention that the method is based on three key assumptions: (1) Neural states may not be embedded in a low-dimensional subspace, but rather in a high-dimensional space. (2) Neural trajectories are sparsely distributed under different behavioral conditions. (3) These neural states traverse trajectories in a stereotyped order.

The authors conducted multiple analyses to validate MINT, demonstrating its decoding of behavioral trajectories in simulations and datasets (Figures 3, 4). The main behavior decoding comparison is shown in Figure 4. In stereotyped tasks, decoding performance is comparable (M_Cycle, MC_Maze) or better (Area 2_Bump) than other linear/nonlinear algorithms (Figure 4). However, MINT underperforms for the MC_RTT task, which is less stereotyped (Figure 4).

This paper is well-structured and its main idea is clear. The fact that performance on stereotyped tasks is high is interesting and informative, showing that these stereotyped tasks create stereotyped neural trajectories. The task-specific comparisons include various measures and a variety of common decoding approaches, which is a strength. However, I have several major concerns. I believe several of the conclusions in the paper, which are also emphasized in the abstract, are not accurate or supported, especially about generalization, computational scalability, and utility for BCIs. MINT is essentially a table-lookup algorithm based on stereotyped task-dependent trajectories and involves the tabulation of extensive data to build a vast library without modeling. These aspects will limit MINT's utility for real-world BCIs and tasks. These properties will also limit MINT's generalizability from task to task, which is important for BCIs and thus is commonly demonstrated in BCI experiments with other decoders without any retraining. Furthermore, MINT's computational and memory requirements can be prohibitive it seems. Finally, as MINT is based on tabulating data without learning models of data, I am unclear how it will be useful in basic investigations of neural computations. I expand on these concerns below.

Main comments:

1. MINT does not generalize to different tasks, which is a main limitation for BCI utility compared with prior BCI decoders that have shown this generalizability as I review below. Specifically, given that MINT tabulates task-specific trajectories, it will not generalize to tasks that are not seen in the training data even when these tasks cover the exact same space (e.g., the same 2D computer screen and associated neural space).

First, the authors provide a section on generalization, which is inaccurate because it mixes up two fundamentally different concepts: 1) collecting informative training data and 2) generalizing from task to task. The former is critical for any algorithm, but it does not imply the latter. For example, removing one direction of cycling from the training set as the authors do here is an example of generating poor training data because the two behavioral (and neural) directions are non-overlapping and/or orthogonal while being in the same space. As such, it is fully expected that all methods will fail. For proper training, the training data should explore the whole movement space and the associated neural space, but this does not mean all kinds of tasks performed in that space must be included in the training set (something MINT likely needs while modeling-based approaches do not). Many BCI studies have indeed shown this generalization ability using a model. For example, in Weiss et al. 2019, center-out reaching tasks are used for training and then the same trained decoder is used for typing on a keyboard or drawing on the 2D screen. In Gilja et al. 2012, training is on a center-out task but the same trained decoder generalizes to a completely different pinball task (hit four consecutive targets) and tasks requiring the avoidance of obstacles and curved movements. There are many more BCI studies, such as Jarosiewicz et al. 2015 that also show generalization to complex real-world tasks not included in the training set. Unlike MINT, these works can achieve generalization because they model the neural subspace and its association to movement. On the contrary, MINT models task-dependent neural trajectories, so the trained decoder is very task-dependent and cannot generalize to other tasks. So, unlike these prior BCIs methods, MINT will likely actually need to include every task in its library, which is not practical.

I suggest the authors remove claims of generalization and modify their arguments throughout the text and abstract. The generalization section needs to be substantially edited to clarify the above points. Please also provide the BCI citations and discuss the above limitation of MINT for BCIs.

2. MINT is shown to achieve competitive/high performance in highly stereotyped datasets with structured trials, but worse performance on MC_RTT, which is not based on repeated trials and is less stereotyped. This shows that MINT is valuable for decoding in repetitive stereotyped use-cases. However, it also highlights a limitation of MINT for BCIs, which is that MINT may not work well for real-world and/or less-constrained setups such as typing, moving a robotic arm in 3D space, etc. This is again due to MINT being a lookup table with a library of stereotyped trajectories rather than a model. Indeed, the authors acknowledge that the lower performance on MC_RTT (Figure 4) may be caused by the lack of repeated trials of the same type. However, real-world BCI decoding scenarios will also not have such stereotyped trial structure and will be less/un-constrained, in which MINT underperforms. Thus, the claim in the abstract or lines 480-481 that MINT is an "excellent" candidate for clinical BCI applications is not accurate and needs to be qualified. The authors should revise their statements according and discuss this issue. They should also make the use-case of MINT on BCI decoding clearer and more convincing.

3. Related to 2, it may also be that MINT achieves competitive performance in offline and trial-based stereotyped decoding by overfitting to the trial structure in a given task, and thus may not generalize well to online performance due to overfitting. For example, a recent work showed that offline decoding performance may be overfitted to the task structure and may not represent online performance (Deo et al. 2023). Please discuss.

4. Related to 2, since MINT requires firing rates to generate the library and simple averaging does not work for this purpose in the MC_RTT dataset (that does not have repeated trials), the authors needed to use AutoLFADS to infer the underlying firing rates. The fact that MINT requires the usage of another model to be constructed first and that this model can be computationally complex, will also be a limiting factor and should be clarified.

5. I also find the statement in the abstract and paper that "computations are simple, scalable" to be inaccurate. The authors state that MINT's computational cost is O(NC) only, but it seems this is achieved at a high memory cost as well as computational cost in training. The process is described in section "Lookup table of log-likelihoods" on line [978-990]. The idea is to precompute the log-likelihoods for any combination of all neurons with discretization x all delay/history segments x all conditions and to build a large lookup table for decoding. Basically, the computational cost of precomputing this table is O(V^{Nτ} x TC) and the table requires a memory of O(V^{Nτ}), where V is the number of discretization points for the neural firing rates, N is the number of neurons, τ is the history length, T is the trial length, and C is the number of conditions. This is a very large burden, especially the V^{Nτ} term. This cost is currently not mentioned in the manuscript and should be clarified in the main text. Accordingly, computation claims should be modified including in the abstract.

6. In addition to the above technical concerns, I also believe the authors should clarify the logic behind developing MINT better. From a scientific standpoint, we seek to gain insights into neural computations by making various assumptions and building models that parsimoniously describe the vast amount of neural data rather than simply tabulating the data. For instance, low-dimensional assumptions have led to the development of numerous dimensionality reduction algorithms and these models have led to important interpretations about the underlying dynamics (e.g., fixed points/limit cycles). While it is of course valid and even insightful to propose different assumptions from existing models as the authors do here, they do not actually translate these assumptions into a new model. Without a model and by just tabulating the data, I don't believe we can provide interpretation or advance the understanding of the fundamentals behind neural computations. As such, I am not clear as to how this library building approach can advance neuroscience or how these assumptions are useful. I think the authors should clarify and discuss this point.

7. Related to 6, there seems to be a logical inconsistency between the operations of MINT and one of its three assumptions, namely, sparsity. The authors state that neural states are sparsely distributed in some neural dimensions (Figure 1a, bottom). If this is the case, then why does MINT extend its decoding scope by interpolating known neural states (and behavior) in the training library? This interpolation suggests that the neural states are dense on the manifold rather than sparse, thus being contradictory to the assumption made. If interpolation-based dense meshes/manifolds underlie the data, then why not model the neural states through the subspace or manifold representations? I think the authors should address this logical inconsistency in MINT, especially since this sparsity assumption also questions the low-dimensional subspace/manifold assumption that is commonly made.

References

Weiss, Jeffrey M., Robert A. Gaunt, Robert Franklin, Michael L. Boninger, and Jennifer L. Collinger. 2019. "Demonstration of a Portable Intracortical Brain-Computer Interface." Brain-Computer Interfaces 6 (4): 106-17. https://doi.org/10.1080/2326263X.2019.1709260.

Gilja, Vikash, Paul Nuyujukian, Cindy A. Chestek, John P. Cunningham, Byron M. Yu, Joline M. Fan, Mark M. Churchland, et al. 2012. "A High-Performance Neural Prosthesis Enabled by Control Algorithm Design." Nature Neuroscience 15 (12): 1752-1757. https://doi.org/10.1038/nn.3265.

Jarosiewicz, Beata, Anish A. Sarma, Daniel Bacher, Nicolas Y. Masse, John D. Simeral, Brittany Sorice, Erin M. Oakley, et al. 2015. "Virtual Typing by People with Tetraplegia Using a Self-Calibrating Intracortical Brain-Computer Interface." Science Translational Medicine 7 (313): 313ra179-313ra179. https://doi.org/10.1126/scitranslmed.aac7328.

Darrel R. Deo, Francis R. Willett, Donald T. Avansino, Leigh R. Hochberg, Jaimie M. Henderson, and Krishna V. Shenoy. 2023. "Translating Deep Learning to Neuroprosthetic Control." BioRxiv, 2023.04.21.537581. https://doi.org/10.1101/2023.04.21.537581.

This is interesting because statistically women are higher achievers within school, and there are more women than men at almost all universities.

Ataques ICMP los usan para descubrir redes o denegar servicio.

DOI: 10.1016/j.isci.2023.107943

Resource: SCENIC (RRID:SCR_017247)

Curator: @scibot

SciCrunch record: RRID:SCR_017247

What is this?

DOI: 10.1016/j.cell.2023.08.032

Resource: (DSMZ Cat# ACC-267, RRID:CVCL_0028)

Curator: @scibot

SciCrunch record: RRID:CVCL_0028

What is this?

DOI: 10.1038/s41467-023-41927-x

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @scibot

SciCrunch record: RRID:SCR_002798

What is this?

DOI: 10.3389/fimmu.2023.1237715

Resource: (IMSR Cat# JAX_000651,RRID:IMSR_JAX:000651)

Curator: @scibot

SciCrunch record: RRID:IMSR_JAX:000651

What is this?

• RE 354870 AgR
• Órgão julgador: Primeira Turma
• Relator(a): Min. ROBERTO BARROSO
• Julgamento: 02/12/2014
• Publicação: 02/02/2015

• EMENTA: AGRAVO REGIMENTAL EM RECURSO EXTRAORDINÁRIO. IMUNIDADE TRIBUTÁRIA. ANULAÇÃO DE ATO ADMINISTRATIVO. EFEITOS. DIREITO ADQUIRIDO. REGIME JURÍDICO. INEXISTÊNCIA. Diante da constatação de que o sujeito passivo, antes reputado imune, jamais deveria sê-lo, não há óbice que possa impedir a Administração tributária de proferir ato declaratório no sentido de afastar a desoneração. Este ato possui cunho, inequivocamente, declaratório, na medida em que reconhece situação de direito desde sempre consolidada. Não obstante, cumpre salientar que não existe um direito adquirido a regime tributário beneficiado (RMS 27382 ED, Rel. Min. Dias Toffoli). Agravo regimental a que se nega provimento.

Inteiro Teor - Diante da constatação de que o sujeito passivo, antes reputado imune, jamais deveria sê-lo, não há óbice que possa impedir a Administração tributária de proferir ato declaratório no sentido de afastar a desoneração. Este ato possui cunho, inequivocamente, declaratório, na medida em que reconhece situação de direito desde sempre consolidada. A parte agravante jamais foi uma entidade imune. Este direito não lhe poderia ser assegurado por um entendimento equivocado. - Não obstante, cumpre salientar que não existe um direito adquirido a regime tributário beneficiado (RMS 27382 ED, Rel. Min. Dias Toffoli). Constatado, a qualquer tempo, que não houve o preenchimento dos requisitos, cumpre ao Fisco fazer incidir o tributo. Ademais, não pode ser desconsiderado o motivo que dá ensejo ao afastamento da regra de desoneração. Se o contribuinte já fez jus a imunidade e perdeu este direito, a posteriori, então a incidência fiscal será ex nunc. Apurado que o contribuinte jamais atendeu aos pressupostos necessários, o equívoco se deu ab initio. O ato será declaratório, munido de força retroativa, nos termos do Código Tributário Nacional.

Questão correlata:

Ano: 2017 Banca: CESPE / CEBRASPE Órgão: Prefeitura de Fortaleza - CE Prova: CESPE - 2017 - Prefeitura de Fortaleza - CE - Procurador do Município - A respeito das limitações constitucionais ao poder de tributar, julgue o item que se segue, de acordo com a interpretação do STF.

• As imunidades tributárias incondicionadas são autoaplicáveis e independem de regulamentação, sendo admitida, entretanto, a suspensão de seus efeitos, por ato declaratório da administração tributária, quando ficar demonstrada a sua inaplicabilidade a fato jurígeno tributário.

Alternativas - Certo - Errado (Alternativa correta)

Justificativa: - Não se trata, portanto, de SUSPENSÃO dos efeitos da norma e da imunidade, mas sim de AFASTAMENTO DA DESONERAÇÃO. Isso porque se "demonstrada a sua inaplicabilidade a fato jurígeno tributário", significa que não há imunidade no caso, desde o princípio.

• Sendo assim, não há que se falar em suspensão dos efeitos de imunidade, mas de mero reconhecimento de ausência de subsunção à norma.

• A suspensão só ocorre com as imunidades condicionadas, sendo decorrência da inobservância dos requisitos legais, conforme prevê o art. 14, §1º do CTN c/c art. 32 e §§ da Lei 9430/96.

1. The bias surrounding art was male prestige.

Este fragmento en la lectura es un claro ejemplo de la realidad actual: ¿Cuántos de nuestros alumnos, no les ha tocado, que comienzan sus platicas con ejemplos vistos en los dispositivos tecnológicos (redes sociales) o aun mas drástico, sus temas de platico son únicamente lo visto en sus redes sociales?

¡Muy cierto! Ahora formamos parte del Ecosistema= Docente/Tecnología Y todos estos dispositivos siempre serán herramientas para obtener información, ideas y auxiliar en la adquisición de nuevos conocimientos o reafirmar los mismos.

conexion a la percepcion de correccion o agrado de otras variedades--grupos regionales

esto es interesante, por que seria diferente? permitimos mas flexibilidad a todos los hablantes nativos, o solamente a los que quedan dentro de nuestro grupo social? y por que??

esto!!!!! el peso social de algunas caracteristicas chilenas segun el grado de asociacion que tenga con el estandar o el subestandar

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

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

The manuscript investigates the role of PAT1 gene family in Arabidopsis thaliana. Though the PAT1 protein has been previously investigated and displayed immune-related and developmental phenotypes, the other two members of the family, PATH1 and PATH2, have not been well studied. The authors set out to understand the role of these proteins in relation to the role of PAT1. They thus generated single, double, and triple mutants of the possible combinations of PAT1 genes and examined their phenotypes. As the study focused on the developmental effects of PAT1, the mutants were generated on the background of the summ2 mutant to avoid phenotypes related to immune response. The authors notice a developmental difference between the pat1 mutant combinations, suggesting that PAT1 acts differently than PATH1 and PATH2 and that the PATH proteins serve a redundant function. They also performed RNA-seq analysis to identify differentially-regulated genes in the mutant combinations. The study is interesting and well-executed, yet I believe some questions should still be addressed:

__Our response: __We thank the reviewer for acknowledging the significance of our findings. Please see our detailed answers to the reviewer’s suggestions in the following.

1. The research mainly focuses on the developmental phenotype of pat mutants but also tests the interaction of PATH proteins with RNA decapping enzymes to check their function and localization during different treatments. I found it a bit confusing since Figure 1 also shows the developmental phenotype of the mutants. I think editing the order of the figures would make the overall story more coherent.

__Our response: __We agree with the reviewer thus we moved old Fig 1C to new Fig 3A, we believe the new figure orders make the overall story more coherent.

My main concern is the correlation between the developmental phenotype of the mutants and the gene expression. Leaf samples for RNA extraction were taken when the plants were 6 weeks old, and the developmental phenotype is very evident. It is thus not possible to tell whether the differences in gene expression are a cause or effect of the developmental phenotype. I think performing qPCR of selected candidates at earlier developmental times might help solve this issue, as well as the characterization of younger plants for the developmental phenotypes (such as leaf number).

__Our response: __We followed the reviewer’s suggestions and performed qRT-PCR on IAA19, IAA29, SAUR23 and PIL2 in pats mutants under different developmental stages (Line 162, 169; Fig S4), we also characterized leaf number of pats mutants from younger stages (Line 109; new Fig 3C).

Overall, the manuscript is missing data regarding replicate numbers in the IP and confocal microscopy experiments.

__Our response: __We thank the reviewer for pointing this it out, the replicate numbers are provided now in our new figure legends.

Minor comments:

1. Figure 1C - the authors should add a picture of Col0 plants as well as the mutants.

Our response: To be reader friendly, the picture of Col-0 plant is added in Fig S1A. For the reviewer’s information, plant pictures in FigS1A and old Fig1C (new Fig 3A) were taken at the same time. 2.

Figure 3 - Calculating the leaf-to-petiole ratio in the different mutants would be good.

Our response: We now calculate PBR (petiole blade ratio) of all pats mutants in Fig3F (Line 121).

Figure 4 - the details in the figure are very unclear, especially in the PCA. It would be good to display the data in 2D for PC1 and PC3 and change the colors a bit.

Our response: We agree with the reviewer; thus, we remade the PCA plot from RNA-seq reads data in a 2D style and also changed the colors for each mutant (Fig 4A). We need to point out that the PCs number also changed because the old PCA plot were made by mistake from expression data.

Reviewer #1 (Significance (Required)): Both PATH proteins have been less investigated than PAT1, and in that sense, the work is novel. However, it seems that most of the phenotype is attributed to PAT1 rather than the other family members, limiting the interest to the broad plant science community.

Our response: We appreciate the reviewer think our work is novel. We agree that PAT1 plays the main role during plant development (old Line 171), however the pat triple mutant exhibit the most severe dwarfism as well as the most mis-regulated genes compared to any single or double mutants, indicating all 3 PATs are essential for development.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

Zuo et al., characterize the role of three cytoplasmic mRNA-decay activator proteins PAT1, PATH1 and PATH2 in the context of plant development and leaf morphology in Arabidopsis thaliana and Nicotiana benthamiana. The authors show that the triple pat mutant displays the most severe dwarfism of all combinatorial mutants. Through treatment with different stimulants the authors found that only IAA treatment induces the three homologues to form condensates (possibly PBs), while PAT1 forms condensates upon every tested stimulus. An extensive RNA seq experiment revealed miss-regulation of several hundred genes in the higher order mutants, several of which were involved in auxin responsive and leaf morphology determinant genes.

__Our response: __We thank the reviewer for the peer review. Please see our detailed answers to the reviewer’s suggestions in the following.

Major points: 1.Title is not meaningful as is and, in my opinion, does not reflect the main findings in the manuscript.

Our response: We now changed our title into “PAT mRNA decapping factors are required for proper development in Arabidopsis”.

The results section could benefit from improved flow between the paragraphs and more reasoning for the next steps taken to help readers understand the aims of the authors.

Our response: We followed the reviewer’s suggestion and modified the wording in our result part(Line 79,81,94,146-151).

L46: "So far little is known about the functions of these three PATs in plant development.", The authors themselves have studied these proteins in the context of seed germination and ABA control, as well as apical hook formation and auxin responses. Should at least be mentioned and the results discussed in this context.

Our response: We thank the reviewer for noticing our other work and we now included this information in the new introduction and discussion part (Line56&237).

What are the expression levels and patterns of PATH1 and PATH2 compared to PAT1? Is anything known about spatial or temporal regulation of these proteins?

Our response: All three PATs are expressed in roots, stems, leaves, flowers, siliques, and seeds during the whole developmental stages, PAT1 has higher expression level in leaves but lower expression levels in petals. (Klepikova et al., 2016;

https://www.arabidopsis.org/servlets/TairObject?id=138009&type=locus for PAT1; https://www.arabidopsis.org/servlets/TairObject?id=38646&type=locus for PATH1 and https://www.arabidopsis.org/servlets/TairObject?id=128694&type=locus for PATH2).

Figure 1:

o I do not agree that the authors have shown that "PATH1 and PATH2 are also mRNA decapping factors", rather that these proteins can co-localize (and possibly interact) with LSM1. Decapping assays for example with the known PAT1 de-capping targets from their previous work and their extensive mutant collection could be used to test this.

Our response: We thank the reviewer for pointing it out and reminding us about the characterized mRNA decapping target from our previous work, we now include the decapping assays in new Fig5 (Line 197).

From the BiFC experiment (Figure 1B) it looks like PATs are mostly soluble in the cytoplasm (like LSM1) and might be stress-induced components of PBs (like LSM1). Do PATs co-localize with other canonical PB markers that are more prone to condensation, like DCPs or VCS? BiFC could be performed after IAA treatment to confirm that the cytoplasmic foci are indeed LSM1-positive PBs.

Our response: We agree with the reviewer that PATs behave more like LSM1. Given time limit of the project, we unfortunately are not able to check the colocalization of PATs with DCPs or VCS. However, we performed BIFC after IAA treatment, and the cytoplasmic foci are indeed LSM1-positive foci (new Fig1B).

A: please provide uncropped images of all Western blots in supplemental data.

Our response: To be reader friendly, we decide to show the original western blots here (see in the file named "RC-Full-revision"), instead of in supplemental data. However, we will leave the final decision to the editor.

I applaud the authors for establishing this great higher order mutant collection that will be very useful for researchers in the field. However, I am confused about the description of these mutants. If I understood it correctly, these mutants were already used in a previous study by the authors, namely “Zuo, Z., et al., Molecular Plant-Microbe Interactions, 35(2), 125-130.” & Zuo, Z., et al., (2021). FEBS letters, 595(2), 253-263.” In this study the authors refer to a BioRxiv “Zuo, Z., et al., (2019).” As the reference for these Arabidopsis lines. Is this current manuscript a continuation of the BioRxiv? Please elaborate whether these lines have been used and described In previous studies.

Our response: We truly appreciate the reviewer for acknowledging the significance of our work. These pats mutants have been used in the FEBS letters paper (2021), MPMI paper (2022), and the new published paper in Life Science Alliance (2023, but preprinted in BioRxiv 2019 and 2022). However, they have not been fully described or characterized in any of the mentioned published stories. Characterization of these pats mutants were originally only included in preprint 2019 which was cited in FEBS letters paper (2021) and MPMI paper (2022).

L72: Is the strong developmental phenotype of the higher order mutants persistent under long day conditions? Considering the strong developmental phenotypes of the mutants, the flowering transition and morphology could be an interesting trait to study. Why did you choose short day conditions for this study?

Our response: The pat triple mutant also has strong developmental phenotype under long day condition and exhibits early flowering phenotype. We are currently preparing a manuscript regarding mRNA decay and flowering. We did not “choose” short day condition, we just started with short day condition and observed phenotypical differences hence we kept this condition.

L78: This statement is hard to see in Figure 1C and best described for Figure 3A.

Our response: We now change this statement for Fig 3.

L82: Please include a reasoning for testing PATs localization after hormone treatment. Do you have any indication that other PB proteins behave similar to either PAT1 or the PATHs after hormone treatment to substantiate that these foci observed are indeed PBs? What is known about PBs after hormone treatment in planta?

Our response: We were interested in investigating if all three PAT proteins may also form PBs in Arabidopsis thus we tested PATs localization with/without hormone treatment (old Line 84, new line 81). For the reviewer’s interest we also observe LSM1 localization after hormone treatment (Fig 2). PBs have been published to respond to light, cold treatment, PAMPs, ABA, ACC and auxin (Line 39-42).

Figure 2:

o How does the localization of LSM1 change under the same treatments? Does ist behave like PAT1 or the homologues?

Our response: Please see our new Fig 2 for LSM1 localization, and it behaves more like PAT1.

Which part of the root was imaged for this experiment? Is it possible that the observed foci are ARF-condensates as reported by Jing et al., 2022? Do you observe a gradual change in numbers or morphology along the root?

Our response: We use root elongation zone for this experiment. We don’t know if the foci are ARF-condensates, but it’s possible to study in the future. If the reviewer is interested, we are happy to share our materials. We do observe more foci in the cell division zone and less in the mature zone.

How did the authors decide on the concentrations for the stimulant treatments? Have you tried different doses, and could the responses be dose-dependent?

Our response: We did not try different doses; we searched for and applied the commonly used concentrations for different hormones.

A representative image is not sufficient for quantitative responses, like RNA granule condensation. Please provide a quantification of stimulant-induced foci after the different treatments.

Our response: Please see the quantification in our new Fig 2.

L91: Does that mean that most co-precipitated signal comes from the soluble fraction and not PB-localized? Would an RNAse treatment step eliminate the co-precipitation (optional)?

Our response: We believe it means LSM1 and PATs are in the same complex regardless of PB localization.

L92/93: Or alternatively that PAT1 localizes to PBs independent of the stress, while PATHs are signal-specific PB components?

Our response: We think PAT1 aggregates upon broad stimuli/stress, while PATHs respond to specific/limited stimuli, for example, auxin.

Figure 3:

o I wonder if these results fit better in conjunction with Figure 1, either as a combined figure or move before Figure 2.

Our response: We agree with the reviewer thus we moved old Fig 1C into Fig 3.

It is interesting that path2/pat1, while being dwarfed, is less serrated compared to pat1 or path1/pat1. Can you find any indications in your RNAseq set which genes might be involved?

Our response: ANAC016 might be involved, but more research needs to be done to confirm it and this is not the focus of the current project.

Indicate statistical test used to determine p-value

Our response: We now indicate the statistic test in Materials and Methods part (Line 369).

L116/L117: Doesn't the result in Figure 3E indicate that PATH1 and PATH2 are not fully redundant, but that PATs have specific and narrow roles in leaf development? L116 goes against your statement in L150 & L160. What is known about the expression patterns of PAT1, PATH1 and PHATH2?

Our response: We agree and thus modified our statement (Line 137). All three PATs are expressed in roots, stems, leaves, flowers, siliques, and seeds during the whole developmental stages. Please also see our answer to major comment #4.

L123: PC3 only explains 0.55% of the variance, so differences along this axis will be overinflated. In my interpretation the pat1/path2 mutant is clustering apart from the other higher order mutants, which is also reflected in the leaf phenotypes. A 2D PCA would be sufficient to describe most of the variation.

Our response: We agree and thus we changed the PCA plot into a 2D style, please also see our response to reviewer 1 minor comment #3.

Figure 4: o A: The 3D-PCA inflates the differences between higher order mutants along PC3, even though this axis explains only 0.55% of the variance, maybe a 2D-PCA would more intuitively cluster the samples together?

Our response: Please see our new PCA plot in Fig4A.

B: Please explain the scale in the figure legend and which genes were included? Only DEGs between triple mutant and summ2-8 or DEGs that were different in at least one higher order mutant?

Our response: We now explained more details in the figure legends. The genes which were included in Fig4B were DEGs that were differently expressed in at least one of the pat mutants.

C: several comparisons are missing from the upset-plot. Please show the complete plot, also is there a white box laid over the second bar in the upper graph? It would help the reader, if the results section would explain the plots and the comparisons took. Which differences are the authors interested in?

Our response: We covered all the comparisons we wanted to show, but we thank the reviewer for suggesting a more detailed explanation and we therefore explain Fig4C more in detail in Line 146. There is no white box over the second bar, it’s only 1 gene mis-regulated specifically by PATH1 (mis-regulated in plants with path1 mutation).

From Figure 4B, the triple mutant has an almost inverted expression of mis-regulated genes. High expression genes are now lowly expressed and vice-versa. Has this been reported for other RNA decay mutants before?

Our response: Our RNA-seq data indicate the pat tripe mutant has more than 1000 mis-regulated genes and based on microarray data on 2-week-old lsm1alsm1b plants (Perea-Resa et al, 2012), more than 600 genes are misregulated in lsm1alsm1b mutant.

How do you explain that mutants in RNA decay have a large group of repressed transcripts and a large group of enriched transcripts? Wouldn't you suspect a general higher expression in RNA decay mutants or which kind of feedback loop would you propose is happening here? Also, since both kinds of expression changes are recorded in your RNA seq can you speculate on the specificity? Why are some genes up- and others downregulated? Would you suspect that transcription factors are under PATs control?

Our response: We assume that the mRNA decapping machinery target genes should accumulate in mRNA decapping mutants, pat mutants in our case. On the other hand, the down-regulated genes could be target genes of other mRNA degradation pathways such as exosome pathway (Line 257); We agree with the reviewer that the down regulated genes in pat triple could also be negatively regulated by the mRNA decapping targets which could be transcription factor genes. For example, our previous research indicates the transcription factor gene ASL9/LBD3 is mRNA decapping targets under PATs control.

Where is the sequencing data deposited? This dataset can be of great value for researchers in the field, but the raw data needs to be made commonly available.

Our response: We thank the reviewer for acknowledging the significance of our work. The raw data has been submitted to NCBI, accession number is PRJNA1006171(Line 307)

Minor points:

1. Check order and nomenclature for protein / gene names in Abstract and Introduction

Our response: We now carefully double check the order and nomenclature for protein / gene names in abstract and introduction (Line 8,11,14,18,19,24)

L26 / L83 "aggregate" implies non-functionality, I would use "concentrate", "condensate" or "accumulate".

Our response: We thank the reviewer for pointing it out, we now use “concentrate” (Line 29&80)

L35, L45 & L54 all state the same. Maybe remove at least one mention to reduce redundancy?

Our response: We modified these statements hopefully in a satisfactory way. (Line 56)

L211: Did you use the same imaging settings for all lines?

Our response: We used the same settings for all the lines and treatment (Line 284)

L217: RNA quality "control" word missing?

Our response: The word “control” is added in Line 296

L477: Authors should cite the newest version of their BioRxiv: Zuo, Z., Roux, M. E., Chevalier, J. R., Dagdas, Y. F., Yamashino, T., H�jgaard, S. D., ... & Petersen, M. (2022). The mRNA decapping machinery targets LBD3/ASL9 to mediate apical hook and lateral root development in Arabidopsis. bioRxiv, 2022-07.

Our response: The latest version is cited in our new manuscript (Line 42)

Figure 3B-F, Figure 4C: check spelling on the axis titles.

Our response: We carefully checked the spelling on the axis titles in our new manuscript.

Reviewer #2 (Significance (Required)):

This manuscript represents a continuation of the author's characterization of the 3 PAT1s in Arabidopsis development after Zuo et al., 2021; Zuo et al., 2022a; Zuo et al., 2022b. The mutants and the corresponding RNA sequencing experiments are of value to the community working on RNA regulation and degradation or plant development. While the initial findings are interesting, the authors do not explore the stimulus-induced condensation differences between the homologues or try to link the extreme differences in expression profiles mechanistically or functionally. I think the manuscript could greatly benefit from contextualizing their work within the frame of their previous studies and what is known about PBs in terms of plant development. While the RNA seq is a comprehensive data set, a closer examination and a better representation of the results would help readers to access the findings.

__Our response: __We thank the reviewer for the constructive criticism. We hope the reviewer is satisfied by our modified manuscript.

Reviewer expertise: RNA granule biology, Arabidopsis, molecular biology

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

Summary:

In the study "PAT mRNA decapping factors function specifically and redundantly during development in Arabidopsis" authors investigate potential specific functions of Arabidopsis PAT1 orthologs in plant development. Authors observe differences in rosette phenotypes (leaf size, serration and number) of single and multiple mutants of PAT1 gene family, show variation in translocation of the corresponding PAT1 proteins to processing bodies under a set of stress conditions and perform transcriptomics on the established mutants to elucidate the impact of individual PATs on posttranscriptional regulation of plant gene expression. Authors conclude that PAT1 orthologs have both overlapping and specific roles in plant development.

__Our response: __We thank the reviewer for the peer review. Please see our detailed answers to the reviewer’s suggestions in the following.

Major comments:

1. The study contains intersting transcriptomics data that will be of use for the scientific community. However, analysis of the transcriptomics results could be discussed a bit more in depth. Authors could express their opinion about what gene expression changes might be caused by direct degradation via PAT1-dependent decapping mechanism and what changes are more likely to have occurred indirectly via other factors.

__Our response: __We followed the reviewer’s suggestion and thus we analysed and discussed more in depth about the transcriptomic data (Line145, 220 &232)

The intersting phenotypic observations are currently poorly linked to the transcriptomics/qPCR data provided, resulting in a somewhat fragmented story flow.

__Our response: __We appreciate the reviewer thought the pat mutants’ phenotype are interesting, however we disagre with the reviewer on the statement of “poorly linked to the transcriptomics/ qPCR data”. For instance, downregulation of developmental and auxin responsive genes could explain the stunt growth phenotype in the pat triple mutant. Furthermore, the published petiole elongation regulator genes XTR7/XTH15 and PIL2/PIF6 exhibit decreased expression level only in mutants with shorter petioles. Nevertheless, we hope our new data and analysis will satisfy the reviewer.

The transcriptomics was performed on the 6-weeks old plants. It would be helpful to learn more about authors reasoning for choosing this developmental stage for sampling. Why did authors decide against sampling at the earlier stages, before the observed leaves phenotypes were established?

__Our response: __The pat mutants growth phenotypes showed bigger difference among each other at the late stage, therefore we performed RNA-seq on these samples. But we agree with the reviewer (also reviewer 1, major comment #2), transcriptomic shift at earlier stage could also be responsible for the observed phenotype, thus we performed qRT-PCR on the pat mutants at earlier stages for certain genes to examine this (Line 162 &169)

Authors obtained intriguing results on specific translocation of PAT1, PATH1 and PATH2 to processing bodies in the root cells upon various stresses. Perhaps root transcriptomics of single PAT1, PATH1 and PATH2 knockouts under control conditions, treatment that translocate all three proteins to PBs(IAA) and selectively translocate only PAT1 (e.g. cytokinin) could shed more light on the redundancy an specificity of these proteins as the mRNA decapping factors.

__Our response: __We appreciate the reviewer found our findings interesting. The specific translocation of PAT1, PATH1 and PATH2 to PBs in the root cells upon various stimuli indicates functional specificity and redundancy in cellular level which correlates with mutants’ growth phenotype. However, we agree with the reviewer that root transcriptomic data on pat mutants are very interesting, we are more than willing to share these mutants with peers who want to persue this in more detail.

Do authors consider PAT1, PATH1 and PATH2 to be localized to different PBs sub-populations? It could be intersting to check co-localization of PAT1, PATH1 and PATH2 under various stress conditions. Could authors elaborate on their view of PBs composition and fate to which different PAT1s are recruited?

__Our response: __We agree with the reviewer that it’s interesting to check co-localization of PAT1, PATH1 and PATH2. We observed partial localization of CFP-PATH2(in blue) and Venus-PAT1(in yellow) when transiently expressed in Benthmiana. But for permanent lines, we failed at observing separate CFP-PATH2(Blue) signal due to too much signal leakage from Venus-PAT1(Green). Given the fact that PATs function redundantly, we would assume they are partially co-localized in cellular level.

Could authors speculate what features in the PAT1 protein might cause it being recruited to PBs more efficiently (or better to say, under a broader range of stresses) in comparison to PATH1 and 2?

__Our response: __The release of ribosome-free mRNPs induces PB formation (Brengues et al., 2005). We suspect PAT1 could bind broader mRNAs compared to PATH1 and PATH2, therefor PAT1-mRNPs could form PBs more efficiently. Moreover, Sachdev et al found yeast PAT1 enhances the condensation of Dhh1 and RNA and PAT1-DHH1 interaction is essential for PB assembly (Sachdev et al., 2019), we assume PAT1 might have better interaction with DHH1 compared to PATH1 and PATH2 thus promote PB formation more efficiently. Please see our discussion part (Line 252)

Are all three Arabidopsis PAT paralogs co-expressed in the same tissues /developmental stages?

__Our response: __Please see our response to reviewer 2 major comment #4.

Could authors elaborate a bit more why the triple pat1 knockout has a much more severe phenotype in comparison to a single pat1 loss-of-function mutant or any of the double pat1 mutants. Do authors observe complementary changes in the PAT1 genes expression in the mutant lines, e.g. is PATH1 expressed at a higher level in the absence of PAT1 and PATH2?

__Our response: __We now elaborate more about the reason why triple pat1 knockout has the most severe phenotype in the multiple pat mutants (Line 210). We do see higher transcriptional level of PAT1 in path1-4path2-1summ2-8 and also higher transcriptional level of PATH1 in pat1-1path2-1summ2-8 but the same PATH2 transcriptional level in pat1-1path1-4summ2-8 compared to summ2-8 (Fig S1C, Line 104)

Please provide the name of the used statistical test in all figure legends.

__Our response: __We now provide the statistical test in “Material and Methods” part (Line 367).

Minor comments:

1. Authors might want to reconsider the title as it is somewhat too vague in its current form.

__Our response: __We now changed our title into “ PAT mRNA decapping factors are required for proper developmental in Arabidopsis”

Line 9: explanation of PAT1 and PATH1 and 2 abbreviations is best placed at the first mentioning of the name.

__Our response: __We carefully followed the reviewer’s suggestion (Line 10)

Line 10: mRNA degradation is rather a posttranscriptional regulation of gene expression.

__Our response: __We agree and changed our statement in the new ms (Line 9).

Lines 11 and 12: path1 and path2 abbreviation are not explained. Please note that on the Figure 1A the same proteins are labelled as PAT1H1 and PAT1H2

__Our response: __We thank the reviewer for pointing it out, we now have PATH1 and PATH2 abbreviations explained in Line 10 and also correct the labels in Fig 1A.

Lines 22-25: Would you be so kind to rephrase or elaborate on what yoPBu mean. LSM1-7/PAT1 complex are known to bind oligoadenylated transcripts indeed and even stabilize their 3' ends, it is not clear what "engage transcripts containing deadenylated tails" means in this context.

__Our response: __We hope we now rephrase the statement in a clear way (Line 25)

Line 29: for the sake of clarity, it might be beneficial to list the known activators of the decapping DCP2 enzyme, including the VCS. Generally the introduction could benefit from a bit more in depth review of the decapping mechanism.

__Our response: __We hope the more detailed introduction will satisfy the reviewer (Line 27).

Line 51:"other 2 PATs" => "other two PATs". Generally the text is quite well written, but might need a bit of polishing.

__Our response: __The text is corrected now (Line 64).

Authors are absolutely correct in their attempt to provide full information about mutant backgrounds. However, for the sake of comprehension, it would be great to grant the double and triple mutants in the summ2 background shorter and more legible names. For example, the pat1-1path1-4path2-1summ2-8 mutant could be named as pat1/h1/h2/s.

__Our response: __We originally used pat1/h1/h2/s for the triple but a colleague pointed out “h1” or “h2” are not proper gene names and suggested us to rename them. But we agree that the double and triple pat names are comprehensive, to compromise we change the triple pat mutants into pat triple.

Figure 1B:

• it would be intersting to have authors opinion on why PBs are formed in this case under non-stress(?) conditions.

__Our response: __Forming PBs is a dynamic process, and we assume that even under normal conditions, there is still ongoing mRNA decay and translational repression which should be seen as some background level of PBs (Line 85).

Please note that expressing only the N-terminal part of CFP is a weak negative control for BiFC. No restoration of CFP can occur in such case and thus it is a given that no fluorescence can be observed in these samples. For example, co-expression of nCFP-PAT1 with cCFP-GUS, would be a more rigorous negative control, better aligned with the coIP experiments.

__Our response: __We had nCFP-Gus with cCFP-LSM1 as real negative control in old Fig 1B (bottom lane). We also agree with the reviewer that only the N-terminal part of CFP is a weak negative control for BiFC, therefore we removed the weak control and only left the rigorous negative control (new Fig 1B).

Please note that some arrows point at a structure that seems to be not discernible a signal.

__Our response: __It’s due to the poor quality of the picture from the PDF file, arrows in the original high-resolution figure do point at discernible foci.

Figure 1C: It might be helpful to also include a Col-0 WT plant

__Our response: __Col-WT plant is now included in Fig S1A.

It is not clear how qPCR data and complementation lines help to characterize the established PATH1 and PATH2 loss-of-function mutants. There is no immunodetection of the corresponding proteins in the knockouts, qPCR shows no dramatic decrease in the transcript level of PATH1 and H2 and the phenotypes of complemented lines presented in the Fig S1E at a glance look quite similar to the phenotypes of the corresponding knockout mutants. Complementation lines are not used for any other experiments in this study and it is not clear why authors decided to include this material into the article.

__Our response: __To characterize the path1 and path2 mutants, we first did qRT-PCR to check the transcriptional level expression, but like the reviewer mentioned, there was no dramatic decrease indicating the mutations of path1-4 and path2-1 did not change PATH1 and PATH2 transcriptional level expression. We also tried to raise antibodies against PATH1 and PATH2, however the antibodies failed to recognize any PAT proteins. Therefore, we used the complementation lines to characterize the mutations in PATH1 and PATH2. Since path1 and path2 single mutants don’t have obvious growth phenotype and the dwarf pat triple is barely possible to transform, we had to complement the pat1path1 and pat1path2 double mutants. If the reviewer takes a closer look, the growth phenotype of the complementation lines Venus-PATH1/ pat1-1path1-4summ2-8 and Venus-PATH2/ pat1-1path2-1summ2-8 are similar to pat1-1summ2-8 but not the background pat double mutants. The complementation lines were also used to study PATH1 and PATH2 cellular localization.

Figure S1C misses labels indicating what detection of what gene is shown on what chart.

__Our response: __We thank the reviewer for pointing it out, the gene names are indicated now in new FigS1C.

Experiments to visualize PBs under various stress stimuli were conducted on roots for the Figure 2 while coIP was performed on the green tissue. Could authors elaborate on whether PB formation could be expected to be the same in different plant organs? Somewhat related to the same topic, Figure 2 contains micrographs obtained on meristematic, transition and elongation root zones, in which epidermal cells are present at various developmental stages. Since PAT proteins are suggested to impact plant development, it might be prudent to obtain observations for all samples at the same developmental stage. Could authors provide their opinion about how representative the provided micrographs are for all root zones? Furthermore, Venus-PATH2 under ACC treatment shows punctate localization only in a single cell out of the three-ish cells visible on the micrograph, potentially indicating differences in PAT2 recruitment to PBs in trichoblasts and atrichoblasts. This in itself could be an intersting observation helpful for elucidating the specific roles of PAT1 orthologs.

__Our response: __CoIP results from Benthamiana leaves indicate Arabidopsis PATs and LSM1 are in the same complex, and PB visualization in root area suggests PATs respond to different hormone treatments. flg22 treatment has been published to induce PB formation in Arabidopsis root but dissemble PBs in Arabidopsis protoplasts, indicating a tissue specific manner of PB formation. We randomly chose 1 picture/treatment from 9 (3 plants * bio-triplicates) which showed the same. However, we thank the reviewer for pointing out the confocal pictures we chose were not all from elongation zone, we now carefully checked all our confocal pictures and made sure they are from the same developmental stages. We also discuss more of PATH2 localization in response to ACC (Line 251).

Figure 4C would greatly benefit from a more detailed description in the main text and figure legend of what authors show/conclude.

__Our response: __We thank the reviewer for the suggestion hence we describe Fig 4C in more detail in our new manuscript (Line 146).

Figure 5, please avoid using the same color for the bars for the triple pat knockout and the control summ2-8 line

__Our response: __We changed the colour scheme for all the mutants (new Fig 4E).

Figure 5B legend should include the name of the statistical test.

__Our response: __We now include the name of the statistical test in “Material and Methods” (Line 367).

Figure S2: The coIP experiment is a bit difficult to interpret due to the extremely low protein quantities in some of the input samples. Perhaps a repetition with more balanced input quantities would be beneficial. The figure legend does not contain information on how normalized intensity values were obtained.

__Our response: __We used the same amount of total protein for each sample (3mg) for each IP, PATH1 and PATH2 don’t express as high as PAT1. The numbers indicate the comparative ratio between PAT-HA protein signal and LSM1-GFP signal, and PAT1-HA/LSM1-GFP under non-treatment condition is normalized as 1.

Line 130: Fig S2 is referenced but Fig S3 is meant

__Our response: __We thank the reviewer for pointing out our mistake, the correct figure is now referenced.

Reviewer #3 (Significance (Required)):

Strength:

Regulation of gene expression by mRNA decay is an extremely intersting topic and is highly relevant in plant stress and developmental biology. This study provides a more in depth view on the potential specific roles of the three PAT1 orthologs in Arabidopsis plants. Authors established loss-of-function mutants of the corresponding genes and performed transcriptomics analysis that will be a valuable source for future studies. Furthermore, microscopy analysis of PATH1 and PATH2 translocation to PBs indicates their potential specific roles in plant stress response.

Weakness: The current version of this study suffers from vague presentation of the results. Starting from the title and ending with discussion authors provide a "general" view on their results and do not go into detailed interpretations. Thus, no mechanistic insight has been derived or at least suggested from the wealth of the transcriptomics, phenotypic and microscopy data.

The introduction should provide more detailed information on what is known on the PAT1 role in the mRNA decapping pathway and its relevance for plant stress response and development.

Please note, that the above mentioned suggestion of different sampling for transcriptomics analysis is not meant as a request for this particular study, but rather as an illustration of an expectation a reader would built while following the current version of the text. A thorough description of the strategy for transcriptomics and a more in depth analysis might significantly strengthen the study's coherence and impact.

Advance:

At this stage, the study looks more like an incremental advance of the work from the same laboratory performed for the single PAT1 protein. However, as mentioned in the comments above, the study might be made significantly stronger by elaborating the results analysis and highlighting potential discoveries.

Audience:

The topic of this study is of a significant interest to a broad audience performing research in plant stress biology and also developmental plant biology.

__Our response: __We thank the reviewer for acknowledging the significance of our work and the structural criticism. We hope our detailed answers to the reviewer’s suggestions and the additional data we included in the manuscript will satisfy the reviewer.

Reviewer's and co-reviewer's fields of expertise:

Molecular Biology, Plant cell biology, Plants Stress response, Autophagy, Stress granules

__Reviewer #4 (Evidence, reproducibility and clarity (Required)): __

PAT1 (Protein Associated with Topoisomerase II) are RNA-binding proteins involved in the control of mRNA decay in the cytoplasm. Plants possess multiple PAT1 family members, three in Arabidopsis, PAT1, PATH1 and PATH2. According to the literature, the pat1 mutant shows dwarfism and de-repressed immunity. In this paper, Zou et al. describe the function of PATH1 and PATH2. Two pieces of evidence are consistent with their role in the control of mRNA decay. First, Co-IP and bimolecular Fluorescence Complementation assays in tobacco indicate physical interaction and co-localization of PAT1, PATH1 or PATH2 with LSM1 (Fig. 1), which is a protein present in decapping complexes that form the cytoplasmic foci involved in mRNA decay. Second, PAT1, PATH1 and PATH2 are present in these cytoplasmic Processing Bodies (Fig. 2). Zou et al. generated path1 and path2 mutants, double mutants with pat1 and the triple mutant using independent alleles and the summ2 background to avoid autoimmunity interference. The mutants show leaf growth (Fig. 3) and gene expression (Fig. 4) phenotypes that are not exactly similar among the different family members, but there is significant redundancy revealed by these phenotypes.

__Our response: __We thank the reviewer for the peer review. Please see our detailed answers to the reviewer’s suggestions in the following.

1. The conclusions are straight forward and, apparently, well supported by the data. However, the authors should confirm that when they provide the number of replicates (n) in the legends to the figures, this actually refers to the number of biological replicates. The statements should be based on true biological replicates (not technical replicates). The statistical tests should also be explicitly indicated (including that used to identify DEG in the RNAseq experiment).

__Our response: __We carefully went through our figures and made sure the number of replicates (n) were correctly stated in figure legends and the statistical tests were indicated (Line 367)

Reviewer #4 (Significance (Required)):

The results are useful but mainly descriptive. Personally, I am interested in the mechanisms involved in the control of growth and the manuscript does not mechanistically link the action of PAT1, PATH1 and PATH2 to the transcriptome and the latter to the growth patterns.

__Our response: __We thank the reviewer for acknowledging the significance of our work of characterizing PATs and we hope our new data could satisfy the reviewer in regarding to “mechanistical link”.

In dem bisher umfassendsten Klimaprozess klagen 6 portugiesische Kinder und junge Menschen vor dem Europäischen Menschenrechts-Gerichtshof gegen 32 Nationen, um sie zu einer schnellen Senkung ihrer Emissionen zu zwingen. Sie haben inzwischen erreicht, dass ein Hearing vor der Grand Jury des Gerichtshofs stattfand. https://www.theguardian.com/environment/2023/sep/14/young-people-to-take-32-european-countries-to-court-over-climate-policies

TreeSet如文所示如此借由TreeMap实现自己的行为特性。

is there a difference between the expected and some essential natural way ? besides breathing and organs functioning can anything really be separated form the psychological and sociological forces that 'police' and/or instruct a given technique of the body.

Na medida em que: locução causal À medida que: proporção

DOI: 10.1101/2023.09.15.557918

Resource: (BioLegend Cat# 311410, RRID:AB_314879)

Curator: @scibot

SciCrunch record: RRID:AB_314879

What is this?

04102023 133054 4-277 SSA. NPAER. M; CFTV<br /> o LIDO

I have seen this play out both negatively and positively according to the capacity of my administration. I have worked with a total of seven different administrative leadership teams and fourteen different administrators, eight of whom were my direct administrative supervisors. Capacity really does make a huge difference!

Building and sustaining a coherent approach seems necessary to effectively running a school district. When everyone is on the same page with the same goals, than districtwide student learning can take place.

• Informativo nº 710
• 27 de setembro de 2021.
• RECURSOS REPETITIVOS
• Processo: REsp 1.892.589-MG, Rel. Min. Paulo de Tarso Sanseverino, Rel. Acd. Min. Ricardo Villas Bôas Cueva, Segunda Seção, por maioria, julgado em 16/09/2021. (Tema 1040)

Ramo do Direito DIREITO PROCESSUAL CIVIL

Paz, Justiça e Instituições EficazesTema <br /> Alienação fiduciária. Ação de busca e apreensão. Decreto-Lei n. 911/1969. Apreciação da contestação antes da execução da medida liminar. Impossibilidade. Tema 1040.

DESTAQUE - Na ação de busca e apreensão de que trata o Decreto-Lei n. 911/1969, a análise da contestação somente deve ocorrer após a execução da medida liminar.

INFORMAÇÕES DO INTEIRO TEOR - Pontua-se, de início, que não se discute a possibilidade de apresentação da contestação antes da execução da medida liminar, não havendo espaço para se falar em extemporaneidade, prematuridade ou necessidade de desentranhamento da peça.

• A controvérsia se restringe ao momento em que a contestação deve ser apreciada pelo órgão julgador.

• Observa-se que no artigo 3º do Decreto-Lei n. 911/1969 o legislador elegeu a execução da liminar como termo inicial de contagem do prazo para: 1) a consolidação da propriedade do bem no patrimônio do credor fiduciário; 2) o pagamento da integralidade da dívida pendente e a consequente restituição do bem ao devedor livre de ônus e 3) a apresentação de resposta pelo réu.

• Ou seja, a eleição da execução da medida liminar como termo inicial da contagem do prazo para contestação revela uma opção legislativa clara de assegurar ao credor fiduciário com garantia real uma resposta satisfativa rápida em caso de mora ou inadimplemento por parte do devedor fiduciante, incompatível com o procedimento comum.

• É essa agilidade inerente ao procedimento especial do Decreto-Lei n. 911/1969 que fomenta o instituto da alienação fiduciária tornando a sua adoção vantajosa tanto para o consumidor, que conta com melhores condições de concessão de crédito (taxas e encargos), quanto para o agente financeiro, por meio da facilitação dos mecanismos de recuperação do bem em caso de inadimplemento.

• É cediço que a mora e o inadimplemento, aliados à morosidade no deferimento de tutela satisfativa voltada à entrega do bem alienado ao credor fiduciário, são fatores determinantes para o encarecimento do crédito, de modo que o aparente rigorismo na norma é o que garante a utilidade do instituto, impedindo que ele caia em desuso.

• Não foi outro o norte seguido pela Segunda Seção, quando do julgamento do REsp 1.622.555/MG, ao afastar a aplicação da teoria do adimplemento substancial no regime da lei especial (Decreto n. 911/1969), sob pena de desvirtuamento do instituto da propriedade fiduciária, concebido pelo legislador justamente para conferir segurança jurídica às concessões de crédito, essencial ao desenvolvimento da economia nacional.

• Não há dúvidas, portanto, de que a legislação especial foi estruturada com um procedimento especial que prevê, em um primeiro momento, a recuperação do bem e, em uma segunda etapa, a possibilidade de purgação da mora e a análise da defesa.

• Vale anotar que o próprio sistema dispõe de mecanismos para remediar eventual abuso ou negligência do credor fiduciário ao prever o pagamento de multa em favor do devedor fiduciante, equivalente a 50% (cinquenta por cento) do valor originalmente financiado, devidamente atualizado, caso o bem já tenha sido alienado, na hipótese de improcedência da ação de busca e apreensão, além da responsabilidade do credor fiduciário por perdas e danos (artigo 3º, §§ 6º e 7º).

• Além disso, está absolutamente sedimentada a jurisprudência desta Corte no sentido de que, estando demonstrada a mora/inadimplemento, o deferimento na medida liminar de busca e apreensão é impositivo.

• Nesse contexto, condicionar o cumprimento da medida liminar de busca e apreensão à apreciação da contestação, ainda que limitada a eventuais matérias cognoscíveis de ofício e que não demandem dilação probatória (considerada ainda a subjetividade na delimitação dessas matérias), causaria enorme insegurança jurídica e ameaça à efetividade do procedimento.

• Informativo nº 769
• 4 de abril de 2023.
• QUARTA TURMA
• Processo: REsp 1.742.102-MG, Rel. Ministro Marco Buzzi, Quarta Turma, por unanimidade, julgado em 23/3/2023.

Ramo do Direito DIREITO CIVIL

Busca e apreensão. Decreto n. 911/1969. Consolidação da propriedade fiduciária. Venda do bem. Eventual saldo. Credor fiduciário. Ônus de comprovar a alienação e o preço de venda.

DESTAQUE - No procedimento da consolidação da propriedade fiduciária pelo Decreto n. 911/1969, compete ao credor fiduciário, após a consolidação da propriedade decorrente da mora do devedor, o ônus de comprovar a venda do bem e o valor auferido com a alienação.

INFORMAÇÕES DO INTEIRO TEOR - Após a retomada do bem pelo credor fiduciário, a venda (judicial ou extrajudicialmente) é premissa básica, constituindo essa uma obrigação estabelecida por lei, razão pela qual a mercancia do bem e a aplicação do preço auferido no pagamento do crédito e nas despesas de cobrança, é algo certo.

• Com a entrada em vigor da Lei n. 13.043/2014, o dispositivo específico do Decreto-Lei n. 911/1969 (artigo 2º) foi acrescido da obrigatoriedade do credor fiduciário promover a devida prestação de contas: "No caso de inadimplemento ou mora nas obrigações contratuais garantidas mediante alienação fiduciária, o proprietário fiduciário ou credor poderá vender a coisa a terceiros, independentemente de leilão, hasta pública, avaliação prévia ou qualquer outra medida judicial ou extrajudicial, salvo disposição expressa em contrário prevista no contrato, devendo aplicar o preço da venda no pagamento de seu crédito e das despesas decorrentes e entregar ao devedor o saldo apurado, se houver, com a devida prestação de contas".

• Como se vê, a administração de interesse de terceiro decorre do comando normativo que exige destinação específica do quantum e a entrega de eventual saldo ao devedor, principalmente após a entrada em vigor da Lei n. 13.043/2014, que alterou o art. 2° do Decreto-Lei n. 911/1969, a qual estabeleceu, expressamente, ser do proprietário fiduciário o dever de prestar contas.

• Obviamente, não é o devedor fiduciário quem deve promover a devida prestação de contas - não se presta contas de débito, notadamente quando não mais está na administração de interesses alheios - sendo tal obrigação do credor fiduciário, afinal foi esse quem retomou o bem e consolidou a propriedade fiduciária e tem o encargo de promover a sua venda e o devido abatimento dos custos da operação e da dívida, para, em havendo saldo, realizar a entrega ao devedor.

• Também não constitui ônus da devedora fiduciária apurar qual o valor/o preço obtido com a alienação, bem como das despesas relativas à cobrança do crédito, isso porque não foi ela quem promoveu a mercancia do produto (judicial ou extrajudicialmente), não tendo como lhe ser transferida uma obrigação alheia.

• Informativo nº 782
• 15 de agosto de 2023.
• RECURSOS REPETITIVOS
• Processo: REsp 1.951.662-RS, Rel. Ministro Marco Buzzi, Rel. para acórdão Ministro João Otávio de Noronha, Segunda Seção, por maioria, julgado em 9/8/2023. (Tema 1132).

REsp 1.951.888-RS, Rel. Ministro Marco Buzzi, Rel. para acórdão Ministro João Otávio de Noronha, Segunda Seção, por maioria, julgado em 9/8/2023 (Tema 1132).

Ramo do Direito DIREITO CIVIL, DIREITO PROCESSUAL CIVIL

Ação de busca e apreensão. Alienação fiduciária em garantia. Comprovação da mora. Notificação extrajudicial com Aviso de Recebimento (AR). Envio no endereço do devedor indicado no instrumento contratual. Suficiência. Tema 1132.

DESTAQUE - Para a comprovação da mora nos contratos garantidos por alienação fiduciária, é suficiente o envio de notificação extrajudicial ao devedor no endereço indicado no instrumento contratual, dispensando-se a prova do recebimento, quer seja pelo próprio destinatário, quer por terceiros.

INFORMAÇÕES DO INTEIRO TEOR - A controvérsia cinge-se a definir se, para a comprovação da mora nos contratos garantidos por alienação fiduciária, é suficiente, ou não, o envio de notificação extrajudicial ao endereço do devedor indicado no instrumento contratual e se é dispensável, por conseguinte, a prova de que a assinatura do Aviso de Recebimento (AR) seja do próprio destinatário.

• O art. 2º, § 2º, do Decreto-Lei n. 911/1969 é expresso ao prever que a mora nos contratos de alienação fiduciária decorrerá do simples vencimento do prazo para pagamento e poderá ser comprovada por carta registrada com Aviso de Recebimento, não exigindo que a assinatura constante do referido aviso seja a do próprio destinatário.

• Consequentemente, uma interpretação literal do dispositivo enseja a conclusão de que, para a constituição do devedor em mora, exige-se tão somente o vencimento do prazo para pagamento, não havendo dúvida sobre isso, porquanto o texto da lei utiliza a expressão "simples vencimento", que, nesse caso, quer literalmente dizer tão somente ou nada mais que o vencimento do prazo para pagamento.

• Com efeito, ao dispensar a interpelação do devedor para sua constituição em mora, o legislador estabelece regra que a doutrina denomina de dies interpellat pro homine, ou seja, a chegada do dia do vencimento da obrigação corresponde a uma interpelação, de modo que, não pagando a prestação no momento ajustado, encontra-se em mora o devedor. Assim, se a mora decorre do mero inadimplemento, prescinde de qualquer atitude do credor, já que advém automaticamente do atraso.

• Após dispor que a mora decorre do simples vencimento do prazo, o legislador estabeleceu, ainda, que a mora poderá ser comprovada por "carta registrada com Aviso de Recebimento", dispondo expressamente que não se exige "que a assinatura constante do referido aviso seja a do próprio destinatário". Nesse contexto, a literalidade da lei, que escolheu o vocábulo "poderá" em vez de "deverá", e os conceitos jurídicos que ela exprime, por si sós, já são elementos suficientes para dirimir a controvérsia.

• Verifica-se, portanto, que a lei estabeleceu que a comprovação é mera formalidade, pois primeiro usa o termo "poderá" e, na sequência, dispensa que a assinatura seja do próprio destinatário. Se é a própria lei que torna não exigível a demonstração cabal de ciência do próprio devedor, não pode ser outra a interpretação do Tribunal de origem e, menos ainda, a do STJ, cuja responsabilidade não se limita à análise do caso concreto, mas vincula, de forma transcendental, as relações contratuais à sua decisão.

• Além dessa interpretação literal do dispositivo, da análise sistemática ressai a conclusão de que pretendeu a lei tão somente estabelecer a forma do processo nas hipóteses em que a garantia do crédito deu-se por alienação fiduciária, na medida em que não se pode ignorar que a cláusula de alienação fiduciária nos contratos caracteriza-se por uma via de mão dupla, ou seja, é uma garantia bilateral, uma vez que a vantagem econômica do contrato é buscada por ambas as partes, não somente pelo credor.

• Assim, se, na origem, o contrato é um negócio jurídico bilateral, em que se estabelece a alienação fiduciária em garantia e cujo objetivo é a vantagem econômica e o equilíbrio das relações entre as partes, não se pode permitir que, na conclusão desse mesmo negócio, ocorra um desequilíbrio, ou seja, as regras sejam tendenciosas e, portanto, tragam mais ônus ao credor em benefício exclusivo do devedor.

• Também, uma análise teleológica do dispositivo legal enseja inafastável a conclusão de que a lei, ao assim dispor, pretendeu trazer elementos de estabilidade, equilíbrio, segurança e facilidade para os negócios jurídicos, de modo que é incompatível com o espírito da lei interpretação diversa, que enseja maior ônus ao credor, em benefício exclusivo do devedor fiduciante.

• Observa-se, ainda, que o entendimento pacífico da Segunda Seção já é no sentido de que, na alienação fiduciária, a mora constitui-se ex re, isto é, decorre automaticamente do vencimento do prazo para pagamento. Ou seja, a mora decorre do simples vencimento do prazo. Naturalmente, tal particularidade significa que o devedor estará em mora quando deixar de efetuar o pagamento no tempo, lugar e forma contratados (arts. 394 e 396 do Código Civil).

• Então, se o objetivo da lei é meramente formal, deve ser igualmente formal o raciocínio sobre as exigências e, portanto, sobre a própria sistemática da lei, concluindo-se que, para ajuizar a ação de busca e apreensão, basta que o credor comprove o envio de notificação por via postal ao endereço indicado no contrato, não sendo imprescindível seu recebimento pessoal pelo devedor.

• Por fim, frisa-se que essa conclusão abarca como consectário lógico situações outras igualmente submetidas à apreciação deste Tribunal, tais como quando a notificação enviada ao endereço do devedor retorna com aviso de "ausente", de "mudou-se", de "insuficiência do endereço do devedor" ou de "extravio do aviso de recebimento", reconhecendo-se que cumpre ao credor demonstrar tão somente o comprovante do envio da notificação com Aviso de Recebimento ao endereço do devedor indicado no contrato.

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Reply to the reviewers

First and foremost, we would like to extend our thanks to the two referees and managing editor of Review Commons for their constructive feedback and careful consideration of this manuscript. We have taken into consideration all of the suggestions from the reviewers and address all points below and in the following sections.

Minor comments from Reviewer #1:

“It is not clear why the authors used cell number as a measure of viability compared to the MTS assay used in figure 2.”

Response:

In Fig. 2, both MTS assay and CellTiter-Blue assay are used to assess cell viability at 24h and 72h, respectively, whereas counterstaining with DAPI is used to quantify cell number in viral infection assays. Quantification of viral infection using immunofluorescence requires fixation and permeabilization of the cells which is not compatible with assessment of metabolic activity through either the MTS assay or CellTiter-Blue assay post-imaging. One can perform these assays (e.g., MTS and CellTiter-Blue) prior to imaging, however there were concerns regarding viral assay image quantification after running these assays due to the metabolic demand of dye processing which may influence susceptibility to viral infection/propagation, and fluorescence of the metabolic sensor interfering with subsequent IF staining.

DAPI staining is not meant to show viability per se, however, one can gate for fragmented cell nuclei (indicative of lysed cell) to remove dead cells or nuclear debris from the measurement. However, pre-apoptotic or dying cells cannot be accounted for in this measurement.

In our case, due to the rapid doubling time of the immortalized cell lines used (e.g., Vero E6 ~24h, Calu-3 ~35-48h, L929 ~24h), we are able to see large differences in cell number as infected or lysed cells fail to replicate over the 48h experiment. Thus, DAPI staining can be used as a proxy to determine the overall health of the culture but is not directly a measure of cell viability.

Minor comments from Reviewer #1:

“Why did the authors adopt a different pre-treatment infection protocol for SARS-CoV-2 compared to MHV and OC43?”

Response:

The pre-treatment protocol was developed by collaborators in the BSL3 facility as standard practice for rapid treatment/screening of multiple compounds in a BSL3 lab where hands-on time was limited due to immense research focus on infectious disease (e.g., SARS-CoV-2) at the time. We screened many different nanoparticle formulations with this protocol before assessing nanoparticle effect on MHV and OC43 where we also used standardized protocols.

In reference to comments from Reviewer #2:

“The substance of the work is very interesting, but experience shows that the idea that by testing one isolate you can generalize about all other viruses is not accurate. Viruses mutate. SARS-CoV-2 has shown an exceptional capacity for mutation, and the mechanism by which viruses enter cells by endocytosis or membrane fusion plays a role in their exposure to products concentrated in the lysosome. Viruses that enter by membrane fusion (including certain isolates of SARS-CoV-2) should not a priori be as sensitive because they are not subject to phagolysosome fusion.”

“In this sense, it is important to evaluate efficacy on several viral strains and not on a single strain, as has long been the case for acute viral infections. As with HIV, viruses can present natural or acquired resistances linked to evolution under selection pressure or not.<br /> In practice, we cannot rely on testing two viruses, one that has disappeared (Wuhan) and the first virus of the Omicron generation (B1), to assess the therapeutic capacity of a new strategy.”

Response:

We would like to highlight that MFQ-NP efficacy was evaluated in several different coronavirus models (MHV, HCoV-OC43 and SARS-CoV-2) in addition to two distinct viral strains (SARS-CoV-2 WT-WA1 and Omicron BA.1) in this study. Not only this, but we have selected viruses from distinct Betacoronavirus lineages which infect different species (homo sapiens and mus musculus). At the time, we had chosen Omicron BA.1 as our model strain as it was the dominant strain in circulation and was temporally separated and genetically distinct from the original WT-WA1 strain.

We can appreciate that viruses mutate, and SARS-CoV-2 has exemplified this through its life cycle. Due to this rapid mutation rate, it is challenging to assess the current dominant variant (e.g., XBB.1.16 as of writing this) and complete manuscript preparation in addition to full peer-review prior to the emergence of a new, potentially more relevant, dominant variant. Additionally, it remains challenging to accurately predict the emergence and genotypic/phenotypic changes of new strains before they arise, necessitating investigation on ancestral strains or, at the least, the current dominant strain.

Lastly, we do not wish to speculate/generalize that MFQ-NPs or a similar approach works for “all other viruses”. As exemplified by efficacy studies in three Betacoronavirus lineages, and one of which using two distinct strains, we do argue that this approach is an effective means to inhibit Betacoronavirus infection. We speculate in the discussion that MFQ-NPs may be used as either a prophylactic or treatment for an array of other respiratory coronaviruses, however we neither show data or speculate efficacy against viruses outside of the coronavirus family.

In reference to comments from Reviewer #1:

“The claim that MFQ may impact cell entry is not supported by the data in the paper. At minimum, the impact of treatment on expression of viral entry receptors for all 3 viruses should be performed, viral attachment assays (see PMID 35176124) and viral pseudoparticle assays. Further the conclusion that MFQ inhibits replication as well as entry is not fully supported by the data presented. This could be improved using a single cycle infection experiment using a synchronised infection protocol. The gold standard to determine impacts on replication would be the use of a viral replicon however I appreciate the technical difficulties in performing these experiments.”

Response:

We agree that the mechanism by which MFQ-NPs inhibit coronavirus infection has not been fully interrogated through this work. We do have preliminary evidence addressed in Fig. 4 which suggests that mechanistically MFQ-NPs may work through targeting pH-dependent protease activity and lysosomal function downstream of viral uptake. However, we have not yet investigated the effect that MFQ-NPs may have on viral entry receptors or viral attachment.

To that end, we are proposing to perform RT-qPCR to measure changes in expression level of key membrane bound proteins responsible for viral uptake. For these assays, we plan to treat Calu-3 cells with MFQ-NPs, unloaded PGC-NPs, equivalent concentration of molecular MFQ, or DMSO as control to gauge expression level of ACE2 (Hs01085333_m1), TMPRSS2 (Hs01122322_m1), sialate O-acetyltransferase gene (CasD1, Hs01082700_m1), and sialic acid acetylesterase gene (SIAE, Hs00405149_m1) relative to expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs02786624_g1) using TaqMan probes (ThermoFisher, USA). Additionally, we will also measure the expression level of Cathepsin L (Hs00964650_m1). Cathepsin L is not a transmembrane protein, however strong evidence suggests that this lysosomal protease is essential for S protein processing and viral membrane – endolysosomal membrane fusion in the SARS-CoV-2 endocytic infection route.

These proteins are chosen as ACE2 and TMPRSS2 are known mediators of SARS-CoV-2 uptake and fusion, and HCoV-OC43 relies on uptake via sialoglycan-based receptors with 9-O-acetylated sialic acid (9-O-Ac-Sia) as a key component. Although CasD1 and SIAE themselves are not transmembrane receptors for HCoV-OC43, they regulate the addition or removal of O-acetyl ester groups from sialic acids, respectively.

Similarly, we plan to treat L929 cells with MFQ-NPs, unloaded PGC-NPs, equivalent concentration of molecular MFQ, or DMSO as control to gauge expression level of CEACAM1 (Mm04204476_m1) relative to expression of GAPDH (Mm99999915_g1). MHV spike protein binds to murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a) facilitating infection. NP treatment duration will be consistent with viral infection assays (e.g., 48 h) prior to RNA isolation and qPCR.

Results from PCR measurements may warrant further investigation into protein level expression measured by Western Blotting. However, we plan to begin with PCR as blotting for multiple membrane bound proteins is considerably challenging and higher cost than qPCR.

In addition to expression of viral entry receptors, we will also perform a viral attachment assay and internalization assay to further interrogate MFQ’s mechanism of action. To determine whether mefloquine inhibits SARS-CoV-2 binding/attachment, we will perform viral binding assays in Calu-3/ Vero E6-TMPRSS2-T2A-ACE2 cells that express ACE2 at high levels, and HEK293T cells that express ACE2 at lower levels. Assays will be performed using SARS-CoV-2 Spike pseudo-typed lentivirus which expresses a fluorescent reporter upon mammalian cell infection. SARS-CoV-2 Spike pseudo-virus is advantageous as it mimics SARS-CoV-2 entry mechanisms; however, it can be handled at BSL2, and it can be used to accurately quantify viral uptake since this virus is not replication competent.

SARS-CoV-2 can be internalized primarily via receptor-mediated endocytosis in cells which do not express TMPRSS2 (e.g., Vero E6) or via direct plasma membrane fusion in cells which do express TMPRSS2 (e.g., Calu-3 and Vero E6-TMPRSS2-T2A-ACE2). To test whether mefloquine inhibits the endocytosis of SARS-CoV-2, Vero E6 cells will be pre-treated with effective concentrations of MFQ-NPs, equivalent concentration of molecular MFQ, or unloaded PGC-NPs/DMSO as controls. Next, cells will be inoculated with SARS-CoV-2 Spike pseudo-virus at MOI 0.5 at 37 oC for 1 h. Cells will then be washed with PBS to remove unbound virus, media containing treatments will be reintroduced, and the pseudoviral reporter fluorescence will be measured 18-24 h after inoculation.

To test whether mefloquine inhibits TMPRSS2-mediated fusion during SARS-CoV-2 infection we will use TMPRS2 expressing Vero E6-TMPRSS2-T2A-ACE2 and Calu-3 cells. Cells will be pre-treated with Leupeptin/Pepstatin (inhibitors of endolysosomal proteases), camostat mesylate (serine protease inhibitor), MFQ-NPs, molecular MFQ, unloaded PCG-NPs, or DMSO as control for 1 h, and then inoculated with SARS-CoV-2 Spike pseudo-virus (MOI = 0.5). Cells will then be washed with PBS to remove unbound virus, media containing treatments will be reintroduced, and the pseudoviral reporter fluorescence will be measured 18-24 h after inoculation.

Minor comments from Reviewer #1

_<br /> “Further discussion in the introduction as to the potential mechanism of action of MFQ should be included. I would also suggest the authors read the work by Elizabeth Campbell and Bruno Canard concerning the potential difficulties in designing direct acting antivirals for coronaviruses.”

“The figure legends lack detail concerning the number of replicates and statistical comparisons. For viral infections MOIs used are also absent.”_

Response:

We have included a brief summary describing what the field knows of the mechanism of action of MFQ. Namely that MFQ does not directly inhibit the virus/cell membrane attachment process, rather MFQ somehow inhibits viral entry after attachment. We speculate a few mechanisms by which this may occur, which include: (1) inhibiting viral membrane fusion with the cell membrane or endolysosomal membrane, (2) inhibiting proteases responsible for processing SARS-CoV-2 S protein and exposing the fusion peptide, (3) modulating expression levels of ACE2, TMPRSS2, and/or cathepsin, or (4) promoting exocytosis of SARS-CoV-2 particles after uptake.

While it is not the primary goal of the manuscript to determine this mechanism of action, we have evidence currently that MFQ inhibits endolysosomal proteolysis (e.g., mechanism 2 above). Through viral attachment assays and evaluation of receptor expression levels we will also probe mechanism 3 in the revision process.

We have updated the figure legends and methods section to include further details concerning replicates and statistical comparisons. For viral infections we had previously listed the MOIs used in the Materials & Methods section but have also included them in the figure legends.

In reference to comments from Reviewer #2:

“On the one hand, both the introduction and the abstract contain too many elements that give a biased view of the extremely controversial literature. For example, the activity of Hydroxychloroquine and its toxicity have been explored most extensively on the "C19Early" website, which reports on trials carried out in a multitude of countries, including more than 300 trials with Hydroxychloroquine, and it is unreasonable in a scientific paper, designed to last, to report on the major beliefs at a given moment in order to develop a work that has nothing to do with this debate. The same applies to the efficacy of the vaccine. It is difficult to say that the vaccine was poorly distributed, with 20 billion doses, making it the most widely distributed vaccine in the history of mankind in such a short space of time, with results that were not as spectacular as the studies predicted, since the epidemic continued at a comparable level. I suggest that the authors concentrate on their work rather than getting involved in the controversies that are developing around treatments and vaccination.”

Response:

When possible, we have tried to highlight the mixed/controversial results, both positive and negative, of pre-existing therapeutics targeting SARS-CoV-2 and COVID-19 and cited them accordingly. Conjectural phrases such as “selective pressure may lead to 3CLpro mutations conferring nirmatrelvir resistance to new viral mutants” or “there is a growing concern that Molnupiravir, especially when administered at sub therapeutic doses, may result in the creation of more virulent SARS-CoV-2 mutants” are supported by observations in the literature and have been proposed by other experts in the field. Otherwise, we have revised the text to remove any unsupported speculative phrases.

We believe it is worth noting the existing therapeutic strategies in the field to provide context and rationale for our differentiated approach. We have extensively explored the C19Early site as well, and although it does a fantastic job of compiling relevant literature, this site also appears to have a biased view. Throughout preparation of this manuscript, we have considered FDA recommendations and clinical practice in prophylactic protection/treatment of COVID-19 paramount in guiding our introduction and discussion.

We have removed phrasing regarding the limited distribution of vaccines.

In reference to comments from Reviewer #1:

_“The authors claim that MFQ loaded nanoparticles have reduced cytotoxicity compared to 'free MFQ' dissolved in DMSO, however with the NP data and free MFQ data not plotted with the same units this conclusion is hard to reach (Fig.2). While I appreciate the molar units are presented in the text - the reduction in cytotoxicity with MFQ-NP appears to be relatively minor in the both the Calu3 and Vero cell models (doubling in IC50 in both instances). This may indicate quite a narrow therapeutic window for antiviral efficacy without unwanted cytotoxicity. Can the authors replot the data on scales using either molar or ug/ml and use the same dose range for all treatments to enable statistical determination as to whether MFQ-NP significantly reduce cytotoxicity.

To test the antiviral efficacy of the MFQ-NP the authors adopt 2 infection systems, either treating cells pre or post infection. The authors either use fluorescently tagged reporter viruses (OC-43/MHV) or immunofluorescence to visualise and quantify viral infection in cell-line models. Given a key aim of this paper is to determine whether MFQ-NP rather than free MFQ is a superior treatment option, it is challenging to assess this with the data presented in figures 5-6. The units of treatment between NP, MFQ-NP and free MFQ again differ, and the molar dose range of MFQ-NP and free NP is not the same. This makes it very hard to conclude whether MFQ-NPs are more effective then free MFQ. Formal dose response curves with the same dose of empty-NPs, MFQ-NPs and free MFQ are needed here, preferably with match cell viability data using the same assay as figure 2.”_

Response:

We will include additional plots with axis scaling for MFQ-NPs as concentration of MFQ in µM rather than concentration of NPs in µg/mL for further comparison to the free MFQ group. We chose concentration of NPs to match with the equivalent unloaded NP controls. Unfortunately, it would not be possible to create a formal dose response curve with the same dose of empty-NPs and free MFQ as those entities only co-exist in the MFQ-NPs treatment group.

We agree with the observation that the therapeutic window is likely narrow in vitro. This is observed in the viral inhibition and cytotoxicity experiments, where the most efficacious dosing of NPs (e.g., 50 – 100 µg/mL) in inhibiting viral infection is similar to the IC50 value (e.g., 54 µg/mL in Calu-3) at 24 h. Similarly, we see a biphasic dose response in our protease activity assay, suggesting that low doses actually increase endolysosomal protease activity which may promote viral infection.

We speculate this dose limiting toxicity and narrow therapeutic window will likely be improved more drastically in vivo (ongoing continuation of this study), however in vitro we still see at least a minor improvement in MFQ tolerability.

In reference to comments from Reviewer #1:

“Finally while animal experiments are likely beyond the scope of this study, use of air-liquid interface cultures of lung epithelial cells would be a significant improvement to the work and provide further support to their conclusions in a physiologically relevant system.”

Response:

While we appreciate the suggestion of ALI models and animal models to further assess MFQ-NPs in a more physiologically relevant system, we agree that these studies would be beyond the scope of this study. This investigation is planned as a continuation of the current study.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

Major comments:

• 1/ The model system used in this work is referred to as "organoids", with the premise that organoids, as representatives of the original tissue, can be used to study tissue development. However, the organoids presented in the study are spherical structures, and the paper does not provide any information about how and to what extent these organoids represent the original tissue. Furthermore, it would be difficult to expect these organoids to accurately represent human breast tissue, as they are derived, to the best of the reviewer's understanding (it is not explicitly noted, but rather the text refers the reader to several previous works), from primary human breast epithelial cells that had been cultured for 6 passages in 2D culture. The CD24/CD44 flow cytometry profile of these 2D cultures, as well as the organoids derived from them, shows a unimodal distribution of CD24 and CD44 expression, and does not show distinct cell populations, as typical in primary breast epithelial cells that have not been cultured in 2D

• Response 1:

  We understand the concern raised by the reviewer. Before initiating the study we have carefully characterized our model (See Revision Section part 2.2, for the revisions that have already been carried out).

  In parallel, to further address this point, we plan to perform an extensive characterization of our mammary primary cells as well as our 3D-matrigel embedded culture. This will be achieved through flow cytometry using other multiple lineage-specific surface markers for luminal progenitor cells (such as CD49f−/low/EpCAM+), and basal/myoepithelial cells (such as CD49f+/EpCAM−/low).

  Thus, overall our experiments will confirm that our growth conditions (which we intend to describe in greater details in the methods section) for multipotent mammary stem cells can generate multi-lineage organoids.

2/ There appears to be confusion between concepts: primarily confusing a basal phenotype with a stem cell phenotype, and progenitor activity with stem cell activity. Because the 3D organoid model does not display a replication of luminal differentiation, it cannot be used as a proxy for stem cell function. The results from the miRNA screen and the subsequent experiments support two conclusions: 1. miR-160b-3p leads to an enhanced mesenchymal phenotype, manifested by reduced CD24 and enhanced CD44 expression. 2. miR-160b-3p leads to increased organoid formation. The latter may be interpreted, at best, as higher basal progenitor activity, but is not a measure of stem cell activity, as that would require the cells to give rise to more than one lineage, which is not shown here.

• Response 2:

We agree with the reviewer on the confusions between several concepts that we did not discriminate enough. The planned characterization of our cultures will allow us to better define the nature of our primary cell population and avoid any confusion (See Response 1). Indeed, an organoid is a self-organized 3D tissue that typically originates from stem cells (pluripotent, fetal or adult). Therefore, we will replace the term “stem cell activity” through the article by “stem/progenitor activity”.

3/ Overall, the information from figures 1-4 indicate that miR-160b-3p is driving and is associated with mesenchymal differentiation, possibly with EMT.

Response 3:

We agree with the reviewer that our data suggest that miR-160b-3p is driving and is associated with mesenchymal differentiation. As suggested by reviewer, to further evaluate the possible involvement of EMT, we will analysed using RT-qPCR, as we described in Fessart et al, (May 30, 2016 https://doi.org/10.7554/eLife.13887), the expression of the main EMT genes in miR-106a-3p cells. These results will be also confirmed at the protein level.

4/ In contrast to the work in the breast 3D culture model, the experiments with hESCs are interesting and do support a role of this miR in stemness.

Response 4:

We would like to extend our gratitude to the reviewer for their valuable comments on this aspect of our work.

There are several relatively minor comments that cumulatively somewhat undermine the strength of the work:

5/ Abstract: the sentence "organoids can be directly generated from human epithelial cells by only one miRNA, miR-106a-sp" needs better clarification.

• We agree with the reviewer, this sentence will be modified accordingly.

6/ Page 5 line 103: HMECs is a term that generally refers to human mammary epithelial cells, not a specific derivation or subpopulation thereof.

• We will replace HMEC by human primary mammary epithelial cells.

7/ The graph in Fig. 1A is unnecessary, it shows only one bar.

• We will remove this panel in the revised version of the manuscript.

8/ It is unclear if all the HMECs were derived from the same donor, or several donors. There is no information about the donor and how the tissue and cells were derived. In general, it is not entirely clear how the cells were collected, processed, stored, and cultured from the time they were obtained from the donor until their use in the current study.

• We will include in the material section the details information on our primary cells and our culture conditions

9/ The key for Fig. 2D is unclear. The axes read "density" but the text refers to "intensity". Fluorescence intensity in flow cytometry is usually measured on a log scale. Differences on a linear scale are not usually considered meaningful. The authors should clarify why they chose a linear scale for this screen.

• The screening was conducted using a high throughput microscope that measures the relative signal intensity, thus the scale is based on linear signal intensity.

10/ In the miRNA screen, how long were the cells cultured after transfection, and was it enough time for them to shift phenotype?

• The timeline of the screening process is detailed in the Material section. The cells were cultured for 6 days following transfection for the CD44/CD24 staining and 8 days following transfection for the 3D culture, which provides sufficient time for shifting the phenotype.

11/ Page 6 line 154: The authors likely mean z-score, not z factor (two different things).

• We thank the reviewer for pointing this, this will be corrected.

12/ Page 7 line 161: "mir-106a-3p directly promotes the "transdifferentiation" of CD44low/CD24high cells phenotype into CD44high/CD24low cell phenotype" - is an unsupported statement, given that there could be several alternative explanations for the observed change in population ratios, including effects on survival or growth of cells of a certain population.

• Transdifferentiation (also known as lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves the ectopic expression of transcription factors and/or other stimuli. We agree with the reviewer's observation that we did not fully demonstrate the transdifferentiation process in terms of lineage. Therefore, we will use flow cytometry to analyse multiple lineage-specific surface markers for luminal progenitor cells (such as CD49f−/low/EpCAM+), and basal/myoepithelial cells (such as CD49f+/EpCAM−/low) following miR-106a-3p expression.
• Additionally, we acknowledge that there may be other alternative explanations; so we have already assessed the impact of mir-106a-3p on the population doubling time in culture to determine whether it affects the cells' survival or growth of the cells (See Section Part 2.2 for a description of experiments already carried out).

13/ There is need for quantification of the phenotypes described in Fig. 3C

• We thank the reviewer for this valuable suggestion and we will indeed characterize the 3D structure using flow cytometry.

14/ Figures 3 D-F it is not clear if the graphs display percentage or mean number (there is discrepancy between figure text and figure legend text), and when percentage, not clear for figure 3F out of what.

• We appreciate the reviewer's suggestion, and we have replaced the term 'axis' with 'Organoid Formation Capacity (OFC),' which is the commonly used nomenclature for this measure. OFC corresponds to the percentage of organoids per cell seeded, as explained in the legend.

15/ Fig. 5B, what is the statistical significance of the enrichment?

• In Figure 5B, the statistical significance of the enrichment is an FDR value of 0.024 and it is based on the multiple rotation gene-set testing (mroast) from the Limma R package (https://doi.org/10.1093/bioinformatics/btq401). This information will be included in the figure legend.

Reviewer #2 (Evidence, reproducibility and clarity):

In this work, Robert et al. utilize mammary organoid culture as an in vitro model of stem cell renewal and maintenance. Authors show that a putative mammary stem cell population, characterized as CD44high CD24low, is enriched in organoid culture relative to 2D monolayer culture. They conduct a microRNA (miR) screen and identify miR-106a-3p as a miRNA that enriches for stem cell (CD44high CD24low) and organoid formation capacity, confirming these findings using miR-106a-3p overexpressing cells. Authors also show that CBX7 overexpression achieves a similar enrichment in CD44high CD24low and organoid formation capacity in an miR-106a-3p dependent manner, though the rationale behind the connection between CBX7 and miR-106a-3p is not well defined. Finally, the manuscript conducts a transcriptomic analysis on miR-106a-3p-OE cells and aims to functionally validate its role in embryonic stem cells (ESCs) as a model that is versatile for renewal and differentiation studies. How this relates to mammary adult stem cells (ASCs) is not entirely clear. Overall, the manuscript contains significant technical and conceptual limitations, and the data presented do not support the major conclusions of the study. There are two overarching issues that question the validity of most of the findings in this study. Firstly, there is no clear demonstration that the structures reported as 3D organoids are indeed driven by multipotent stem cells (in all data and experimental approaches associated with this). Secondly, there is a lack of evidence to support the claim that CD44high CD24low cells are stem cells with an exclusive or enhanced capacity to generate multi-lineage organoids.

In addition to these general points, there are several specific major issues summarized below:

1/ Authors base their findings about mammary ASC renewal using a poorly defined organoid culture system that they claim is driven by ASC renewal and differentiation. These organoid growth conditions were originally developed to support growth of bronchial epithelial cells, and the authors have not presented data to objectively assess the validity of this extrapolation to expansion of mammary organoids. The authors did not present data to support the notion that these growth conditions generate multi-lineage organoids that expand from multipotent mammary stem cells.

• Response 1:
• This concern has been also raised by the 1st reviewer (See Response 12 from Reviewer 1).
• Our experiments will confirm that our growth conditions (which will be more comprehensively described in the methods section) can generate multi-lineage organoids from multipotent mammary stem cells. We will include these characterizations in the new version of the manuscript.

2/ Authors claim that miR-106a-3p downregulates stem cell differentiation and utilize 'organoid' culture to track the temporal expression of OCT4, SOX2 and NANOG during phases of organoid renewal and differentiation. However, mammary stem cell differentiation is associated with the emergence of luminal progenitor, mature luminal and myoepithelial lineages that are characterized by the expression of a well-defined set of markers. The authors did not investigate the expression of these markers in their 'differentiation' settings. Furthermore, modulators of major pathways reported to be critical for mammary ASC maintenance are lacking, including modulators of WNT, TGF/BMP, Notch and other pathways. As such, it is difficult to ascertain that organoids emerging under such culture conditions are the result of stem cell renewal and differentiation, as opposed to lineage-restricted proliferative non-ASCs, thus questioning the validity of many of the findings in this work.

• Response 2:

• As suggested by the reviewer, we will investigate the expression of emergence of luminal progenitor, mature luminal and myoepithelial lineages in our 'differentiation' settings using flow cytometry. This analysis will involve the examination of multiple lineage-specific surface markers, including CD49f−/low/EpCAM+ for luminal progenitor cells and CD49f+/EpCAM−/low for basal/myoepithelial cells in the 3D context.

• Secondly, we agree with the reviewer that major pathways such as Wnt, TGFb/BMP and Notch have been reported to be critical for mammary ASC maintenance. As suggested by the reviewer, to further evaluate the possible involvement of these pathways, we have analyzed our transcriptomic data, using PROGENy pathway, to investigate which pathways are regulated. The major pathways are depicted in a new figure and will be included in the manuscript (See Revision Section part 2.2, for the revisions that have already been carried out).

• There was significant enrichment indicating down-regulation of TGFβ, MAPK, WNT, PI3K genes along with gene sets representing other oncogenic pathways, are up-regulated such as the hypoxia response, JAK/STAT pathway, and p53 pathway activity (Figure A and B). Stem cells possess self-renewal activities and multipotency, characteristics that tend to be maintained under hypoxic microenvironments [1], thus this is not surprising to observe an up-regulation of Hypoxia pathway and activation of HIF1α transcription factor. In mammary stem cells, it has also been shown that p53 is critical to control the maintenance of a constant number of stem cells pool [2, 3]. Remarkably, it has been shown that cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) express a constitutive activation of Jak-STAT pathway [4], which we observed as up-regulated along with STAT1 and STAT2 transcription factors. In parallel, we observe a down-regulation of Wnt signaling as well as PI3K pathways. Wnt is known to play important role in the maintenance of stem cells; its inhibition has been shown to lead to the inactivation of PI3 kinase signaling pathways to ensures a balance control of stem cell renewal [5]. Moreover, we observe a down-regulation of SMAD4 and SMAD3 transcription factors correlated with a down-regulation of TGFβ pathway. Signals mediated by TGF-β family members have been implicated in the maintenance and differentiation of various types of somatic stem cells [6].

• References

• Semenza GL. Dynamic regulation of stem cell specification and maintenance by hypoxia-inducible factors. Molecular aspects of medicine2016 Feb-Mar;47-48:15-23.

• Solozobova V, Blattner C. p53 in stem cells. World journal of biological chemistry2011 Sep 26;2(9):202-14.
• Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell2009 Sep 18;138(6):1083-95.
• Hernandez-Vargas H, Ouzounova M, Le Calvez-Kelm F, Lambert MP, McKay-Chopin S, Tavtigian SV et al. Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells. Epigenetics2011 Apr;6(4):428-39.
• He XC, Zhang J, Tong WG, Tawfik O, Ross J, Scoville DH et al. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nature genetics2004 Oct;36(10):1117-21.
• Watabe T, Miyazono K. Roles of TGF-beta family signaling in stem cell renewal and differentiation. Cell research2009 Jan;19(1):103-15.

3/ The authors base their work on the claim that CD44high CD24low cells represent bona fide mammary ASCs. This claim is not support by functional work to show that organoid generation is exclusive to or enhanced in CD44high CD24low cells relative to other cells. The claim of differentiation of this stem cell population in vitro is not supported by data to show emergence of luminal progenitor, mature luminal and myoepithelial lineages that are characterized by the expression of a well-defined set of reported markers as abovementioned. Although miR-106a-3p is claimed to downregulate stem cell differentiation based on a gene set enrichment analysis, authors did not investigate the expression of mammary differentiation markers or the association of miR-106a-3p-transfected HMECs with gene set pathways involved in mammary gland differentiation.

• Response 3:
• This concern has been also raised by the 1st reviewer (See Response 1 from Reviewer 1). As explained in response 1, to address this point, we will perform an extensive characterization of our mammary primary cells as well as our 3D-matrigel embedded cultures using flow cytometry This analysis will involve multiple lineage-specific surface markers, including luminal alveolar progenitor (such as CD49f−/low/EpCAM+) and basal/myoepithelial cells (such as CD49f+/EpCAM−/low).
• Secondly, as suggested by the reviewer, we will investigate the expression of mammary differentiation markers or the association of miR-106a-3p-transfected human mammary epithelial cells with gene set pathways involved in mammary gland differentiation by bioinformatics using our transcriptomic data.

4/ Line 129: "Together, these results indicate that cells grown as organoids acquired a CD44high / CD24low expression pattern similar to that of stem/progenitor cells, which suggests that 3D organoids can be used to enrich breast stem cell markers for further screening". This conclusion is not supported by the data presented. It is not clear if the expression of these markers was acquired upon 3D culture. It could be that 3D culture better maintained and expanded already existing CD44high CD24low cells in 2D culture. It is also unclear if these cells are indeed organoid-forming. Authors have not isolated and tested the organoid-forming capacity of CD44high CD24low cells relative to other cells. Along the same line, the conclusion that " mir-106a-3p directly promotes the "transdifferentiation" of CD44low/CD24high cell phenotype into CD44high/CD24low cell phenotype" isn't justified. Experiments required to conclude that 'transdifferentiation' is involved are lacking. miR-106a-3p overexpression could be creating conditions that are permissive for expansion of already existing CD44high CD24low cells as opposed to 'transdifferentiation' of other cell types into this phenotype. The authors could FACS-isolate CD44low CD24low cells and treat these with control or miR-106a-3p to conclusively establish 'transdifferentiation'.

• Response 4:
• This concern has been also raised by the 1st reviewer (See Response 12 from Reviewer 1). Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves the ectopic expression of transcription factors and/or other stimuli. We agree with the reviewer that we did not demonstrate the transdifferentiation process in terms of lineage. Therefore, we will use flow cytometry to analyze multiple lineage-specific surface markers for luminal progenitor (CD49f−/low/EpCAM+), and basal/myoepithelial cells (CD49f+/EpCAM−/low) following miR-106a-3p expression.
• Additionally, we cannot exclude the possibility that the 3D culture better maintained and expanded the pre-existing CD44high CD24low cells, as suggested by the reviewer. The reviewer recommends FACS-isolating CD44low CD24low cells. We apologise for any lack of clarity in our description. Technically, our primary cells have a low percentage of colony-forming efficiency (CFE) in 3D and not enough cells for FACS isolation of CD44low and CD24low cells. Therefore, we leveraged our knowledge that the expression of CBX7 potentiates the growth of 3D structures. We decided to FACS-isolate the different subpopulations of CD44 and CD24 cells to further elucidate the expression of miR-106a-3p in the different CD44/CD24 cell subpopulations. CBX7-transfected human mammary epithelial cells showed enrichment in CD44high/CD24low cells as compared to empty vector-transfected human mammary epithelial cells (Figure 4A-B). Subsequently, we separated the CD44high/CD24low (green) population from the CD44high/CD24high cell populations (blue) using flow cytometry to analyze the role of the endogenous expression of miR-106a-3p. The CD44high/CD24low population was the only one to exhibit endogenous expression of miR-106a-3p (Figure 4C). Blocking the endogenous expression of miR-106a-3p with LNA-anti-miR-106a-3p or LNA-control (Figure 4D) impacted organoid generation (Figure 4E-F). We will modify the text accordingly to include this point and this figure will be moved in the supplemental figure.

5/ Line 242: "These data demonstrate that miR-106a-3p is involved in the early cell differentiation process into the three germ layers ". The authors have not conducted functional or mechanistic work to show that miR-106a-3p is involved in morphological or transcriptomic differentiation changes in ESCs. This and other data would be necessary to substantiate this conclusion.

• Response 5:
• Indeed, we agree that we cannot conclude that the miR-106a-3p is involved in the early cell differentiation process into the three germ layers without demonstrating the differentiation changes in ESCs through transcriptomic analysis. To clarify the take-home message, we did not include the transcriptomic characterization of the differentiation changes in ESCs. Instead, we focused on assessing the impact on Oct4, Sox2, and Nanog expression. However, to further understand the impact of miR-106a-3p depletion on hESCs differentiation, we have also monitored the expression of specific genes upon induction of the three embryonic germ layers (See Section Part 2.2 for a description of the experiments already carried out).

Selected technical points:

6) Figure 1A: The Y-axis label is misleading and suggests multiple organoids seeded per cell? Organoid Formation Efficiency (OFE) or Organoid Formation Capacity (OFC) are the commonly used nomenclature for this.

• Response 6:
• Since reviewer 1 found that the graph in Fig. 1A to be unnecessary, we have decided to remove this panel in the revised version of the manuscript. Furthermore, as suggested, we will replace the axis “Number of organoids per cell seeded” with “Organoid Formation Capacity” (OFC), which is the commonly used nomenclature for this measure in the article.

7) Figure 2G: X-axis unclear. Is this meant to investigate the percentage of cells expressing CD44/CD24 as double high, double low, high/low and low/high?

• Response 7:
• We thank the reviewer for this careful examination of the manuscript. We apologize for this error, and will make the corresponding adjustment in Figure 2G.

8) Figure 4C: In HMEC-CBX7 cells, it is unclear whether the high miR-106a-3p levels in CD44high CD24low cells are due to CBX7 expression. An important control, HMEC-Control Vector, is missing.

• Response 8:
• This control has been done (see Section 3, for details on experiments that have been carried out).

Reviewer #3 (Evidence, reproducibility and clarity):

In this study the authors identify miR-106a-3p as a potent inducer of organoid formation from HMECs. Overexpression of miR-106a-3p induced formation of more organoids, increased the number of stem/progenitor cells and overall positively affected the stemness properties of the organoids, likely by affecting SOx2, Oct4 and Nanog expression.

Major comments:

1/ the flow of the paper is confusing, it appears that the authors are trying to combine several non-completed studies into one paper. It is not immediately evident how is the rationale or the conclusion supported by published data. For example, is there published evidence that Sox2/Oct4/Nanog are expressed in healthy mammary gland stem cells in vivo, or whether they have a role in establishment of stem cell population?

• Response 1:
• There is still a controversy about the existence of unipotent, bipotent or multipotent stem cells in mammary gland tissue [7-9]. Notably, OCT4, SOX2, and NANOG collectively form the core transcriptional network responsible for maintaining pluripotency in embryonic stem cells [10]. Evidence has accumulated over the past few years, accumulating evidence has supported the presence of stem cells in both mouse and human mammary [11]. Various strategies have been used to identify and isolate human breast stem/progenitor cells, including FACS sorting based on cell surface antigen expression. In addition, an in vitro cell culture system has been described allowing the propagation of human mammary epithelial cells in an undifferentiated state through their ability to proliferate in suspension as non-adherent mammospheres [12].. Simoe et al have demonstrated that stem cells isolated from both normal human breast and breast tumor cells display an increased expression of the embryonic stem cell genes NANOG, OCT4 and SOX2 [13]. Moreover, they have shown that the ectopic expression of any one of these factors, but in particular NANOG and SOX2, in breast cancer cells increases the pool of stem cells and enhances the cells' ability to form mammospheres. They observed higher expression of NANOG, OCT4, and SOX2 in the stem cell populations CD44+CD24−/low and EMA+CALLA+ compared to the rest of the sample population. Cells overexpressing these factors displayed an increase in the stem cell populations, thereby confirming the role of Nanog, Oct4, and Sox2 in the maintenance of human mammary stem cells. We will include this rationale in the manuscript.

• References

• Van Keymeulen A, Rocha AS, Ousset M, Beck B, Bouvencourt G, Rock J et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature2011 Oct 9;479(7372):189-93.

• Deome KB, Faulkin LJ, Jr., Bern HA, Blair PB. Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer research1959 Jun;19(5):515-20.
• Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML et al. Generation of a functional mammary gland from a single stem cell. Nature2006 Jan 5;439(7072):84-8.
• Boyer LA, Lee TI, Cole MF, Johnstone SE, Levine SS, Zucker JP et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell2005 Sep 23;122(6):947-56.
• LaMarca HL, Rosen JM. Minireview: hormones and mammary cell fate--what will I become when I grow up? Endocrinology2008 Sep;149(9):4317-21.
• Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes & development2003 May 15;17(10):1253-70.
• Simoes BM, Piva M, Iriondo O, Comaills V, Lopez-Ruiz JA, Zabalza I et al. Effects of estrogen on the proportion of stem cells in the breast. Breast cancer research and treatment2011 Aug;129(1):23-35.

2/ The organoids are all spherical, while mammary gland is characterized by branching. Therefore, the organoids are not recapitulating the gland morphology and the validation should include wider range of molecular markers.

• Response 2:
• This concern has been also raised by both reviewers 1 and 2. As explained in response 1 to reviewer 1, we will perform a comprehensive characterization of our mammary primary cells as well as our 3D-matrigel embedded culture culture using flow cytometry to examine multiple lineage-specific surface markers, including luminal alveolar progenitor (such as CD49f−/low/EpCAM+), and basal/myoepithelial cells (such as CD49f+/EpCAM−/low). As a result, our experiments will collectively confirm that our growth conditions (which will be more thoroughly described in the methods section) for multipotent mammary stem cells can indeed generate multi-lineage organoids.

3/ The choice of control is not clear. For example, why was miR-106a-5p chosen as a control? And why choose miR-106a-5p when a better candidate would be miR-106b-3p, that produces very high number of organoids as well? And how did the other miRNAs affected the CD44/24 profile?

• Response 3:
• We apologize for any lack of clarity in our description. It's important to note that miRNAs consist of two strands, -5p and -3p, and both strands can coexist and play distinct roles. For example, paired species of members in the let-7 and mir-126 families coexist and have different regulatory functions in reprogramming and differentiation of embryonic stem cells [1]. Several deep sequencing studies have demonstrated the coexistence of 5p/3p pairs in approximately half of the miRNA populations analyzed [2, 3]. Therefore, to determine whether the biological effect specifically resulted from the -3p strand, we also assessed the -5p strand.

• References

• Koh W, Sheng CT, Tan B, Lee QY, Kuznetsov V, Kiang LS et al. Analysis of deep sequencing microRNA expression profile from human embryonic stem cells derived mesenchymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of hepatic nuclear factor 4 alpha. BMC genomics2010 Feb 10;11 Suppl 1(Suppl 1):S6.

• Jagadeeswaran G, Zheng Y, Sumathipala N, Jiang H, Arrese EL, Soulages JL et al. Deep sequencing of small RNA libraries reveals dynamic regulation of conserved and novel microRNAs and microRNA-stars during silkworm development. BMC genomics2010 Jan 20;11:52.
• Kuchenbauer F, Mah SM, Heuser M, McPherson A, Ruschmann J, Rouhi A et al. Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells. Blood2011 Sep 22;118(12):3350-8.

4/ What is the CD44/CD24 profile in the organoid cultures from Figure 7?

• Response 4:
• The CD44/CD24 profile from the organoid cultures from Figure 7 will be assessed in the revised manuscript.

Part 2.2 Following the experiment that have been already carried out that will be included in the manuscript after revisions.

Reviewer #1 (Evidence, reproducibility and clarity):

Major comments:

The model system used in this work is referred to as "organoids", with the premise that organoids, as representatives of the original tissue, can be used to study tissue development. However, the organoids presented in the study are spherical structures, and the paper does not provide any information about how and to what extent these organoids represent the original tissue. Furthermore, it would be difficult to expect these organoids to accurately represent human breast tissue, as they are derived, to the best of the reviewer's understanding (it is not explicitly noted, but rather the text refers the reader to several previous works), from primary human breast epithelial cells that had been cultured for 6 passages in 2D culture. The CD24/CD44 flow cytometry profile of these 2D cultures, as well as the organoids derived from them, shows a unimodal distribution of CD24 and CD44 expression, and does not show distinct cell populations, as typical in primary breast epithelial cells that have not been cultured in 2D.

• Response:
• First, we assessed the aldehyde dehydrogenase activity (ALDH) [14] in our primary cells to demonstrate that these culture conditions preserve stem/progenitor properties (See Figure A, below). Additionally, we conducted immuno-staining of primary cells in 2D culture for lineage-specific luminal markers (CK18, MUC1) and basal markers (CK14, CK5), which revealed the heterogeneity of our population (See Figure B, below).

FIGURE FOR REVIEWERS

• Reference

• Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M et al. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell stem cell2007 Nov;1(5):555-67.

• For the characterization of the 3D culture, we have presented early time points of 3D cell growth, which are mainly spherical (until Day 8). However, differentiation occurs in a stepwise manner, where single stem cells proliferate to form small spheroids before undergoing multilineage differentiation into organoids that initiate branching (Day 10). Subsequently, ducts undergo budding and lobule formation (Day 20). We have included a time-course representation of this 3D growth to illustrate the differentiation process (See Figure 1C).

• Under these culture conditions, the 3D structure retains its self-renewal activity and the ability to reseed and regenerate secondary tissues. We have also assessed the self-renewal capacity of our 3D structure (See Figure 1D).

FIGURE FOR REVIEWERS<br />

Page 7 line 161: "mir-106a-3p directly promotes the "transdifferentiation" of CD44low/CD24high cells phenotype into CD44high/CD24low cell phenotype" - is an unsupported statement, given that there could be several alternative explanations for the observed change in population ratios, including effects on survival or growth of cells of a certain population.

• Indeed, we cannot exclude the possibility of other alternative explanations. Therefore, we have already assessed the impact of miR-106a-3p on population doubling in culture to determine whether it has an effect on the survival or growth of the cells. We observed that miR-V cells stopped growing after approximately 15 population doublings, indicating their limited proliferative potential. In contrast, we found that miR-106a extended the lifespan of the cells, suggesting an effect on cell survival (Figure below). We will include this information in the manuscript.

FIGURE FOR REVIEWERS

• Why was GATA3 not included in the last analysis depicted in Fig. 7?

• We apologize for any lack of clarity in our description. It's important to note that GATA3 was not included in Figure 7. As explained in the text, GATA3 plays a role in regulating Nanog expression. However, it's worth noting that the cells did not form organoids when GATA3 was depleted, as illustrated in the results below. We will include these results in the revised version of the manuscript.

FIGURE FOR REVIEWERS

Reviewer #2:

2/ Authors claim that miR-106a-3p downregulates stem cell differentiation and utilize 'organoid' culture to track the temporal expression of OCT4, SOX2 and NANOG during phases of organoid renewal and differentiation. However, mammary stem cell differentiation is associated with the emergence of luminal progenitor, mature luminal and myoepithelial lineages that are characterized by the expression of a well-defined set of markers. The authors did not investigate the expression of these markers in their 'differentiation' settings. Furthermore, modulators of major pathways reported to be critical for mammary ASC maintenance are lacking, including modulators of WNT, TGF/BMP, Notch and other pathways. As such, it is difficult to ascertain that organoids emerging under such culture conditions are the result of stem cell renewal and differentiation, as opposed to lineage-restricted proliferative non-ASCs, thus questioning the validity of many of the findings in this work.

• We agree with the reviewer that major pathways such as Wnt, TGFβ/BMP and Notch have been reported to be critical for mammary ASC maintenance. As suggested by the reviewer, to further evaluate the possible involvement of these pathways, we have analyzed our transcriptomic data, using the PROGENy R package (version 1.16.0) (https://doi.org/10.1038/s41467-017-02391-6) and DoRothEA (version 1.6.0)-decoupleR (version 2.1.6) computational pipeline (https://doi.org/10.1101/gr.240663.118, https://doi.org/10.1093/bioadv/vbac016), to investigate the differential activation of major signaling pathways and transcriptional factors. The major pathways are depicted in the figure below and will be included in the manuscript.

FIGURE FOR REVIEWERS

• There was significant enrichment indicating down-regulation of TGFβ, MAPK, WNT, PI3K genes along with gene sets representing other oncogenic pathways, are up-regulated such as the hypoxia response, JAK/STAT pathway, and p53 pathway activity (Figure A and B). Stem cells possess self-renewal activities and multipotency, characteristics that tend to be maintained under hypoxic microenvironments [15], thus this is not surprising to observe an up-regulation of Hypoxia pathway and activation of HIF1α transcription factor. In mammary stem cells, it has also been shown that p53 is critical to control the maintenance of a constant number of stem cells pool [16,17]. Remarkably, it has been shown that cell sorting of the cells with a putative cancer stem cell phenotype (CD44+/CD24 low) express a constitutive activation of Jak-STAT pathway [18], which we observed as up-regulated along with STAT1 and STAT2 transcription factors. In parallel, we observe a down-regulation of Wnt signaling as well as PI3K pathways. Wnt is known to play important role in the maintenance of stem cells; its inhibition has been shown to lead to the inactivation of PI3 kinase signaling pathways to ensure a balance control of stem cell renewal [19]. Moreover, we observe a down-regulation of SMAD4 and SMAD3 transcription factors correlated with a down-regulation of TGFβ pathway. Signals mediated by TGF-β family members have been implicated in the maintenance and differentiation of various types of somatic stem cells [20].

• References

• Semenza GL. Dynamic regulation of stem cell specification and maintenance by hypoxia-inducible factors. Molecular aspects of medicine2016 Feb-Mar;47-48:15-23.

• Solozobova V, Blattner C. p53 in stem cells. World journal of biological chemistry2011 Sep 26;2(9):202-14.
• Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B et al. The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell2009 Sep 18;138(6):1083-95.

• Hernandez-Vargas H, Ouzounova M, Le Calvez-Kelm F, Lambert MP, McKay-Chopin S, Tavtigian SV et al. Methylome analysis reveals Jak-STAT pathway deregulation in putative breast cancer stem cells. Epigenetics2011 Apr;6(4):428-39.

• He XC, Zhang J, Tong WG, Tawfik O, Ross J, Scoville DH et al. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling. Nature genetics2004 Oct;36(10):1117-21.

• Watabe T, Miyazono K. Roles of TGF-beta family signaling in stem cell renewal and differentiation. Cell research2009 Jan;19(1):103-15.

5) Line 242: "These data demonstrate that miR-106a-3p is involved in the early cell differentiation process into the three germ layers ". The authors have not conducted functional or mechanistic work to show that miR-106a-3p is involved in morphological or transcriptomic differentiation changes in ESCs. This and other data would be necessary to substantiate this conclusion.

• Response:
• Indeed, we cannot conclude that miR-106a-3p is directly involved in the early cell differentiation process into the three germ layers without demonstrating the differentiation changes in ESCs through transcriptomic analysis. To clarify the take-home message, it's important to note that we did not include the transcriptomic characterization of differentiation changes in ESCs. Instead, we focused on assessing the impact of miR-106a-3p depletion on the expression of Oct4, Sox2, and Nanog. However, to gain a deeper understanding of the effects of miR-106a-3p depletion on hESCs differentiation, we also monitored the expression of specific genes upon the induction of the three embryonic germ layers (see Figure A, B, and C below). We observed that the expression of endodermal genes was not or only weakly affected by the level of miR-106a-3p expression (Figure A), whereas the expression of mesoderm- and ectoderm-specific genes increased upon miR-106a-3p down-regulation (Figure B and C). We will include these findings in the manuscript.

FIGURE FOR REVIEWERS

Reviewer 2 (Minor points)

3) Figure 4C: In HMEC-CBX7 cells, it is unclear whether the high miR-106a-3p levels in CD44high CD24low cells are due to CBX7 expression. An important control, HMEC-Control Vector, is missing.

• Response
• The control HMEC-Vector is shown in panel Figure 4A for the FACS analysis. In Figure 4C, we did not include the control since there is no expression of miR-106a-3p, but it's important to note that this control was included in the experiment. As illustrated, there is no miR-106a-3p expression in the HMEC control cells. We intend to include this panel in Figure 4 of the manuscript.

FIGURE FOR REVIEWERS

OBRIGAÇÕES PRINCIPAIS: envolve dinheiro, pagar tributos ou multas

OBRIGAÇÕES ACESSÓRIAS: entrega de declaração, nf etc

Isenção: existe a competência e o fato gerador ocorre.

Imunidade tributária: limitação do poder de tributar

• A plan is a process that controls the order of operations
• fundamental plan in Humans = TOTE
• T (test): matches possible solutions
• O (operate): proceeds if solution is sensible
• T (post operation test): compares solution with goal
• E (exit): if goals are reached

because of Spaniards' whiteness, they are not subject to the same kind of stigma that latinos do when speaking Mock Spanish since when latinos do it, it play into the long held stereotypes against them.

Añadieron alguna nota o comentario a sus notas de campo?

Su profesor Manuel Saavedra es su informante clave o algún otro docente colaborador/a.

Investigadores: estudiantes de 8vo básico Informantes: estudiantes de 1ero básico (o primer ciclo)

whiskey was seen as barbaric and opposite to the values of the protestant work ethic and an orderly homelife.

servants are so respectful and traditionally British

focus on development and work instead of meaningless endeavours of fashion or nobility

documents how the early settlers are learning from the Indigenous population

leading on adventure

S:The speaker is Leon Botstein, he is the president of Bard College and the author of the book called "Jefferson's Children: Education and the Promise of American Culture". O: The Occasion is to bring awareness to how broken the American school system is and how it needs to be fixed. A: The audience for this article is school systems as well as students and teachers. P: The purpose is to have schools and the United States realize that our school system is failing kids. S: The subject is the American school system, in particular the high school system. Tone: The tone of this is informative and also argumentative towards the school systems.

S: Leon Botstein O: published in 1999 (2 years after the author published a different book on education); also school shootings may have prompted this piece A: high school students, perhaps educators, opinion-based, easy language, strong arguments makes this piece accessible to a broad audience P: to get a reaction (very strong, controversial claims) and expose the flaws of the modern high school experience Tone: worked-up, borderline upset, emotionally-driven while still trying to be serious/professional, a charged piece

consider how your connection might specifically deal with "emotions" in Voices

no se entiende, sacar

mal uso de tiempos verbales: presente o pasado?

Estas preguntas sobre temas de aceleración van al final, habíamos dicho que pueden contaminar al resto de las preguntas si van al principio

redacción

"... pertenece a la comunidad de academic_s de ciencias sociales en Chile" Creo que ampliaría esta definición ... pero habría que revisar el consentimiento informado del comité de ética

It is important to make a research study reliable, meaning that the results can be reproduced when the research is repeated under the same conditions. Along with valid, meaning the results really measure what they are supposed to measure. AS & ML

Havendo urgência e depósito do valor ofertado, poderá haver imissão na posse com contraditório postergado.

• Informativo nº 767
• 21 de março de 2023.
• PRIMEIRA TURMA
• Processo: REsp 1.930.735-TO, Rel. Ministra Regina Helena Costa, Primeira Turma, por unanimidade, julgado em 28/2/2023, DJe 2/3/2023.

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL

Desapropriação de imóvel por utilidade pública. Ausência do depósito previsto no art. 15 do Decreto-Lei n. 3.365/1941. Exigência legal para o deferimento de pedido de imissão provisória na posse que não impede a continuidade da demanda.

DESTAQUE - A ausência do depósito previsto no art. 15 do Decreto-Lei n. 3.365/1941 para o deferimento de pedido de imissão provisória na posse veiculado em ação de desapropriação por utilidade pública não implica a extinção do processo sem resolução do mérito, mas, tão somente, o indeferimento da tutela provisória.

INFORMAÇÕES DO INTEIRO TEOR - A disciplina normativa acerca da desapropriação por utilidade pública está radicada, no plano infraconstitucional, no Decreto-Lei n. 3.365/1941, cuja normatividade estabelece incumbir ao expropriante, após expedição do ato declaratório e superada a fase administrativa de composição amigável, provocar o poder judiciário mediante ação de desapropriação com o objetivo de fixar o montante devido a título de justa indenização.

• O art. 13 do Decreto-Lei n. 3.365/1941 estabelece os seguintes requisitos para a petição inicial da ação expropriatória: "Art. 13. A petição inicial, além dos requisitos previstos no Código de Processo Civil, conterá a oferta do preço e será instruída com um exemplar do contrato, ou do jornal oficial que houver publicado o decreto de desapropriação, ou cópia autenticada dos mesmos, e a planta ou descrição dos bens e suas confrontações".

• Assim, a par de instruir a petição inicial com exemplar do jornal no qual publicado o decreto de utilidade pública e de indicar a oferta do preço, incumbe ao expropriante atender aos requisitos genéricos previstos nos arts. 319 e 320 do CPC/2015, notadamente a juntada dos documentos indispensáveis à propositura da ação.

• Por sua vez, como a incorporação do bem ao patrimônio público exige, nos termos do art. 5º, XXIV, da Constituição Federal, prévia e justa indenização em dinheiro - cujo adimplemento somente ocorre quando definitivamente fixado o valor da compensação financeira e quitado o respectivo precatório -, a legislação autoriza o expropriante, em casos urgentes, a pleitear sua imissão provisória na posse da área objeto de desapropriação, de modo a permitir a tempestiva satisfação do interesse público que fundamenta tal forma de intervenção estatal na propriedade privada.

• Nesses casos, o art. 15 do Decreto-Lei n. 3.365/1941 estabelece dois requisitos para possibilitar a imissão provisória na posse, quais sejam, a alegação de urgência e o depósito de quantia ofertada pelo expropriante.

• Ademais, em ações dessa natureza, o instituto da imissão provisória na posse não detém autonomia, porquanto possui natureza jurídica de pedido de tutela antecipada voltado a permitir, antes da transferência definitiva da propriedade ao patrimônio estatal ao final da demanda, a realização das obras e serviços inadiáveis.

• Assim, embora o depósito da quantia estimada pelo ente público para o pagamento de indenização constitua pressuposto legal para o deferimento de pedido de imissão provisória na posse, sua ausência não implica a extinção da ação expropriatória sem resolução do mérito, mas, tão somente, o indeferimento da tutela antecipada, cuja rejeição não obsta a continuidade do processo para viabilizar a incorporação do bem ao patrimônio estatal.

Examen fisico

Implores God, expressing their desperation to hold onto the sand more tightly.

Em abril de 1987, a Comissão Brundtland publicou um relatório denominado “Nosso Futuro Comum” – que trouxe o conceito de desenvolvimento sustentável como sendo o desenvolvimento que encontra as necessidades atuais sem comprometer a habilidade das futuras gerações de atender suas próprias necessidades.

"Este proceso a veces se traduce en agregar el conocimiento de culturas locales a la educación sin cuestionar cómo se presentan o representan esas culturas. Esto significa que a menudo se mantienen las formas de pensar y de mostrar estas culturas de la misma manera que las culturas dominantes, lo que lleva a que la interculturalidad siga centrándose en aspectos étnicos."

• ADI 4757
• Procedência parcial da ação direta para conferir interpretação conforme à Constituição Federal: (i) ao § 4º do art. 14 da Lei Complementar nº 140/2011 para estabelecer que a omissão ou mora administrativa imotivada e desproporcional na manifestação definitiva sobre os pedidos de renovação de licenças ambientais instaura a competência supletiva dos demais entes federados nas ações administrativas de licenciamento e na autorização ambiental, como previsto no art. 15 e (ii) ao § 3º do art. 17 da Lei Complementar nº 140/2011, esclarecendo que a prevalência do auto de infração lavrado pelo órgão originalmente competente para o licenciamento ou autorização ambiental não exclui a atuação supletiva de outro ente federado, desde que comprovada omissão ou insuficiência na tutela fiscalizatória.

O autor está usando a teleologia como uma "baliza" para orientar seu percurso de análise dos filmes dos anos 60. Isso significa que ele está considerando a ideia de um propósito ou fim como um guia para compreender a estrutura e o significado das obras cinematográficas que está examinando.

A invenção formal se refere à capacidade de Glauber Rocha de inovar na linguagem cinematográfica, criando um novo horizonte para o cinema brasileiro. A alegoria se refere ao uso simbólico e metafórico da narrativa do filme para expressar questões relacionadas ao destino e à identidade nacional.

São aquelas que, enquanto não se verificarem, não trazem qualquer consequência para o negócio jurídico, vigorando o ajustado, cabendo inclusive o exercício dele decorrentes. Sobrevindo a condição resolutiva, extingue-se, para todos os efeitos, os direitos que a ela se opõem.

São aquelas que, enquanto não se verificarem, impedem que o negócio jurídico gere efeitos.

culture is essential in clinical diagnostic laboratories, and etiologic diagnosis of common bacterial and fungal pathogens.

Infection control include asepis technique in operating rooms, hospital ward includes handwashing, isolation procedures, thus prevent diseases and reducing cost of medical care

halfway: o giua 2 diem voi khoang cach bang nhau

Reside on the family farm: song o canh dong cua gia dinh

READ THIS ONE

Read This One

The sand grains might be a metaphor for something else, like passing time. Nevertheless, the speaker is concerned about being hit by the waves.

normal flora is beneficial and can be harmful o the human body

cuando existe una combinación de los dos modelos de enseñanza , se emplean los recursos tecnológicos y se toma de ellos lo mas eficaz u factible para el desarrollo del trabajo colaborativo

Según el autor (Diaz) se considera el proceso de enseñanza-aprendizaje donde existe la interacción entre el docente y el alumnado, por medio del desarrollo de las actividades y el empleo de los recursos y empleo educativo

El aprendizaje Ubicuo es la modalidad de adquirir conocimientos desde diversas percepciones y espacios contextuales

The stanzas that ensue are indented, each line shorter than normal, mimicking a block quote. Perhaps Eliot is citing the lyrics of a song that "crept by me upon the waters" in the stanza above. Now, in a sense, the readers aren't simply reading TWL anymore --- they are listening to it. The importance of sound over sight is seen in many sources, particularly Augustine's Confession, the Sermon on the Mount from the Bible, Fire Sermon Discourse from the Buddha, and Purgatorio. Reflecting on his sins, Augustine writes in Book 10, "But they who know how to praise Thee for it, 'O all-creating Lord,' take it up in Thy hymns, and are not taken up with it in their sleep. Such would I be. These seductions of the eyes I resist..." Here, songs and hymns, perceived using the sense of hearing, can serve as a pathway to conversion. And the necessity to "resist" the "seductions of the eyes" can be elucidated by the Sermon on the Mount: "Blessed are the pure in heart: for they shall see God." Evidently, Augustine is still in the process of demonstrating sorrow for his sins and not yet "pure in heart," just like how the sinners have to ascend from Inferno to Purgatory to the Heaves, where a "vision of God [is] represented by the yellow triangle at the very top." In order to be forgiven for one's sins, then, one needs to first listen and be stripped of sight.

As we've discussed in class, Eliot's belief in the possibility of a universal religion has allowed for a commonality to be observed between Buddhism and Christianity. In Fire Sermon Discourse, the Buddha preaches also for an "aversion for the eye [and] an aversion for forms," which to me possesses two meanings. The first being an aversion to the perception --- implying sight --- of "forms," or, in other words, using eyes to see objects that make up the tangible world around us. The second being an aversion to "formalities" --- one formality being the act of gift-giving, which is also mentioned in the Bible: "That thine alms may be in secret."

Thinking about the broader meaning of these song-like stanzas, it's important to note how the Rhine daughters' words --- "Weialala leia" --- repeated in "The Fire Sermon," evokes the tune of an implied song that they dance to while persuading Siegfried to return the ring. Yet, as Siegfried chooses to keep the ring for himself, perhaps the state of "The Fire Sermon" remains in a state of "Burning burning burning burning," never reaching a final state of salvation.

This quote comes from the final line of Parsifal, an ancient legend and opera about a man who completes the Grail Quest due to his ignorance. However, Parsifal is categorized as a "pure fool." This could be in correspondence with my previous comment about the Fool tarot card. Earlier in the text, Lil was called a "proper fool." The difference between "pure" and "proper" here shows the difference of the word "fool" when applied to men and women. As a woman, you are a fool for not following the world of the man. It has nothing to do with knowledge and everything to do with power. However, when applied to men, the word "fool" is synonymous with protagonist. Their ignorance is their power. This concept also has some backing within the story of Adam and Eve. For Parsifal, his power comes from ignorance that becomes complicity. He does not understand sin. But within that lack of understanding, he cannot save those who are victims. Rather, he becomes a mindless puppet accomplishing these tasks. The king he saved, Amfortas, was a man who had fallen to sin and seduction.

A symbiotic commentary on the self-destruction of human trivial desires, both Eliot and John Day—in Parliament of the Bees—utilize the irony of pleasuring the self at the cost of the betterment of society to push their respective narratives. Stemming from a story about goddess Diana and Acteon, who is punished for seeing Diana in the nude while she bathes. Yet, Eliot reverses the narrative of female empowerment to immortalize the ever-haunting barbarism of London in a post-Great War world. Seeking momentary happiness that will result in long-term personal and societal harm, the main speaker attends a brothel to satisfy his shallow desires for sexual gratification. At the unfortunate cost of respect for the feminine body and sexuality as a whole, his physical act goes unpunished, but his psychological choice of attending a brothel is returned in the form of a forever dismal life, surrounded by what does not foretell a benevolent future.

Unlike the line "O wedding day-afters, oh lacy bits of things!" in Laforgue's "Complaint of a Certain Sunday," the trash/leftovers mentioned here are interestingly absent from the river. Furthermore, the source of these obviously human-produced waste remains nebulous - "other testimony of summer nights" - as opposed to the clear setting of the aftermath of a "wedding," a large celebration creating a great deal of waste. In effect, something that seems positive - the absence of waste - is also rendered boring and uncelebrated, affirming Laforgue's outlook on life ("And until nature shows a nice and kind concern / Attempt to live the humdrum turn"). Interestingly, the line that Sophie and Grace have both mentioned above - "The nymphs are departed" - also exhibits the motif of an unnatural absence, only this time, it's the absence of something that is considered objectively positive, the nymphs in Spenser's "Prothalamion" with baskets of flowers for the two brides. Both of these absences, one supposedly positive, one supposedly negative, all lead to an inexplicable feeling of melancholy. As advocator of spiritual democracy Edward Carpenter would contend, through a myriad of voices and landscapes, "I see a great land poised as in a dream," the beginning of "The Fire Sermon" can be best described as a dreamscape. In this "dream," perhaps for a waste-less world, the side-effect of purity becomes silence, established through the knowledge of the narrator that eventually, the song will "end," leaving the Sweet Thames to "run softly," an action void of much noise. Here, a paradox is introduced, whether it's better for to have "a great land waiting for its own people to come and take possession of it," thus creating human-made waste and destruction, or to leave a land barren, untouched but mundane.

muy importante

DOI: 10.1016/j.xcrm.2023.101206

Resource: (Abcam Cat# ab37152, RRID:AB_778939)

Curator: @scibot

SciCrunch record: RRID:AB_778939

What is this?

:o

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RECURSO ESPECIAL REPETITIVO. TEMA 1000/STJ. PROCESSUAL CIVIL. CPC/2015. COMINAÇÃO DE ASTREINTES NA EXIBIÇÃO DE DOCUMENTOS REQUERIDA CONTRA A PARTE 'EX ADVERSA'. CABIMENTO NA VIGÊNCIA DO CPC/2015. NECESSIDADE DE PRÉVIO JUÍZO DE PROBABILIDADE E DE PRÉVIA TENTATIVA DE BUSCA E APREENSÃO OU OUTRA MEDIDA COERCITIVA. CASO CONCRETO. INSCRIÇÃO NEGATIVA EM CADASTRO DE INADIMPLENTES. PEDIDO AUTÔNOMO DE EXIBIÇÃO DO CONTRATO PERTINENTE À INSCRIÇÃO NEGATIVA. INDEFERIMENTO DA INICIAL PELO JUÍZO DE ORIGEM. APLICAÇÃO DA TEORIA DA CAUSA MADURA PELO TRIBUNAL DE ORIGEM. PROCEDÊNCIA DO PEDIDO DE EXIBIÇÃO COM COMINAÇÃO DE ASTREINTES. DESCABIMENTO. NECESSIDADE DE PRÉVIO JUÍZO DE PROBABILIDADE E DE PRÉVIA TENTATIVA DE BUSCA E APREENSÃO OU OUTRA MEDIDA COERCITIVA. ANULAÇÃO DA SENTENÇA E DO ACÓRDÃO RECORRIDO. RETORNO DOS AUTOS AO JUÍZO DE ORIGEM. 1. Delimitação da controvérsia: exibição incidental ou autônoma de documentos requerida contra a parte 'ex adversa' em demanda de direito privado. 2. Tese para os fins do art. 1.040 do CPC/2015: "Desde que prováveis a existência da relação jurídica entre as partes e de documento ou coisa que se pretende seja exibido, apurada em contraditório prévio, poderá o juiz, após tentativa de busca e apreensão ou outra medida coercitiva, determinar sua exibição sob pena de multa com base no art. 400, parágrafo único, do CPC/2015" (Tema 1000/STJ). 3. Caso concreto: - 3.1. Controvérsia acerca da cominação de astreintes em ação autônoma de exibição ajuizada com o escopo de ter acesso ao contrato que teria dado origem a uma inscrição negativa em cadastro de inadimplentes. - 3.2. Indeferimento da petição inicial pelo juízo de origem, tendo o Tribunal de origem reformado a sentença e, aplicando a teoria da causa madura, julgado procedente o pedido de exibição, com cominação de astreintes. - 3.3. Descabimento da cominação de astreintes sem prévio juízo de probabilidade acerca da existência da relação jurídica e do documento, nos termos da tese firmada neste voto. - 3.4. Necessidade de prévia tentativa de busca e apreensão ou outra medida coercitiva, antes da cominação de astreintes. - 3.5. Desconstituição da sentença e do acórdão recorrido para que seja retomado o curso da ação de exibição de documentos para possibilitar a aplicação da tese consolidada neste voto, como se entender de direito. 4. RECURSO ESPECIAL PROVIDO, EM PARTE. (REsp n. 1.777.553/SP, relator Ministro Paulo de Tarso Sanseverino, Segunda Seção, julgado em 26/5/2021, DJe de 1/7/2021.)

Bacterial taxonomy has three interrelated areas but separate ,that is Identification, Classification and nomenclature Identification is is the use of practical scheme to distinguish and isolate specific organisms amongst complex microbial flora, to authenticate and verify special properties of a culture in clinical setting and isolating causative organism of a disease. at the end of it all , this will help in section of specific pharmacologic treatment directed to eradicate micro-organisms .,mitigation towards vaccine pathology and measure by public health that prevents further transmission like handwashing. Classification ; Is where organisms are categorized into taxonomic group. Nomenclature is where organisms are named by an established group

Taxonomy is classification of organisms in an ordered system that indicates a natural relationship Identification isolates ,distinguishes, microorganisms ,classification categorizes organisms in taxonomic groups. Nomenclature is naming of an organism by a established group.

Identification schemes are not classification schemes. For example, few E. coli strains have been identified as the causative agent of hemolytic uremic syndrome(HUS) in infants, though E. coli strains are broadly classified as a member of the Enterobacteriaceae.

• Informativo nº 748
• 12 de setembro de 2022.
• SEGUNDA TURMA
• Processo: AgInt no RMS 61.658-RS, Rel. Min. Mauro Campbell Marques, Segunda Turma, por unanimidade, julgado em 10/05/2022, DJe 27/05/2022.

Ramo do Direito DIREITO ADMINISTRATIVO

Concurso público. Nomeação em cargo público. Exigibilidade de habilitação em nível superior (bacharelado). Superveniência de lei estadual. Alteração da legislação aplicável para permissão de tecnólogo. Inaplicabilidade. Observância das exigências previstas no edital.

DESTAQUE - A exigência dos requisitos previstos em edital para nomeação em cargo público não pode ser afastada por legislação posterior mais benéfica ao candidato.

INFORMAÇÕES DO INTEIRO TEOR - Cinge-se a controvérsia em determinar se os requisitos exigidos no edital de concurso podem ser desconsiderados quando as exigências previstas em lei para ocupar o respectivo cargo são alteradas após a homologação do edital para beneficiar candidato que não cumpre as exigências previstas no edital.

• No caso, a secretaria estadual lançou edital para a abertura de concurso público destinado ao provimento de cargo que tinha os seus requisitos disciplinados por lei estadual, exigindo bacharelado superior, em qualquer curso de nível devidamente complementado com especialização em administração ou em gestão pública.

• Contudo, sobreveio lei estadual que reestruturou a carreira, modificando tanto a nomenclatura deste cargo, quanto os seus requisitos mínimos, passando a exigir meramente uma graduação em geral, suplementada por Curso de Especialização em Administração ou Gestão Pública, com duração mínima de 360 horas, realizado por instituição de educação superior devidamente credenciada pelo Ministério da Educação (MEC).

• De acordo com a Ministra Assusete Magalhães (voto-vista), a ratio essendi da Súmula 266/STJ é no sentido de que os requisitos, exigidos no edital do certame para o exercício de determinado cargo público, devem ser comprovados no momento da posse.

• Entretanto, o cerne da controvérsia é verificar se esses requisitos, exigidos no edital do concurso - e que eram consentâneos com a legislação vigente à época da publicação do edital -, podem ser alterados posteriormente, mesmo que por lei, seja para prejudicar ou para beneficiar os candidatos.

• A Administração Pública, ao publicar o edital do concurso, baseando-se na lei à época vigente, para seleção de candidatos, anuncia a existência de vagas disponíveis, expõe os requisitos que devem ser cumpridos pelos candidatos - podendo estipular critérios de diferenciação entre os participantes, desde que previstos em lei, e cláusulas de barreira, para classificação ou para eliminação de candidatos -, criando expectativas a serem satisfeitas, em caso de aprovação, e descrevem as regras e os procedimentos que serão adotados durante o processo de seleção.

• Assim, a entrada em vigor de nova legislação, em momento posterior ao edital do certame e à homologação do concurso, não pode ter aplicabilidade ao concurso público já realizado e homologado, seja para prejudicar, seja para beneficiar o candidato, em face da isonomia entre os participantes, só podendo a novel legislação ser aplicada aos concursos abertos após a sua vigência.

• Em face da observância do princípio da vinculação ao edital do concurso e da isonomia entre os candidatos, não há como considerar preenchido, no caso, no momento da posse, o requisito da escolaridade - com o diploma de tecnólogo, e não o de bacharel -, ao arrepio das normas editalícias e legais vigentes na data do edital do concurso, que, ademais, fora homologado antes da vigência da lei estadual que reestruturou a carreira.

É lei complementar que define a criação de outras contribuições sociais para o financiamento da seguridade social.

• RE 1016605
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. MARCO AURÉLIO
• Redator(a) do acórdão: Min. ALEXANDRE DE MORAES
• Julgamento: 16/09/2020

• Publicação: 16/12/2020

• RECURSO EXTRAORDINÁRIO. REPERCUSSÃO GERAL. TEMA 708. CONSTITUCIONAL. TRIBUTÁRIO. IMPOSTO SOBRE A PROPRIEDADE DE VEÍCULOS AUTOMOTORES (IPVA). RECOLHIMENTO EM ESTADO DIVERSO DAQUELE QUE O CONTRIBUINTE MANTÉM SUA SEDE OU DOMICÍLIO TRIBUTÁRIO. IMPOSSIBILIDADE.

• Cuida-se, na origem, de ação por meio da qual empresa proprietária de veículos automotores busca declaração judicial de que não está sujeita à cobrança do Imposto sobre a Propriedade de Veículos Automotores (IPVA) por parte do Estado em que se encontra domiciliada, mas sim pelo Estado em que licenciados os veículos.
• O Estado de Minas Gerais, no qual a empresa tem sua sede, defende a tributação com base na Lei Estadual 14.937/2003, cujo art. 1º, parágrafo único, dispõe que “o IPVA incide também sobre a propriedade de veículo automotor dispensado de registro, matrícula ou licenciamento no órgão próprio, desde que seu proprietário seja domiciliado no Estado”.
• Embora o IPVA esteja previsto em nosso ordenamento jurídico desde a Emenda 27/1985 à Constituição de 1967, ainda não foi editada a lei complementar estabelecendo suas normas gerais, conforme determina o art. 146, III, da CF/88. Assim, os Estados poderão editar as leis necessárias à aplicação do tributo, conforme estabelecido pelo art. 24, § 3º, da Carta, bem como pelo art. 34, § 3º, do Ato das Disposições Constitucionais Transitórias - ADCT.
• A presente lide retrata uma das hipóteses de “guerra fiscal” entre entes federativos, configurando-se a conhecida situação em que um Estado busca aumentar sua receita por meio da oferta de uma vantagem econômica para o contribuinte domiciliado ou sediado em outro.
• A imposição do IPVA supõe que o veículo automotor circule no Estado em que licenciado. Não por acaso, o inc. III do art. 158 da Constituição de 1988 atribui cinquenta por cento do produto da arrecadação do imposto do Estado sobre a propriedade de veículos automotores aos Municípios em que licenciados os automóveis.
• Portanto, o art. 1º, parágrafo único da Lei Mineira 14.937/2003 encontra-se em sintonia com a Constituição, sendo válida a cobrança do IPVA pelo Estado de Minas Gerais relativamente aos veículos cujos proprietários se encontram nele sediados.
• Tese para fins de repercussão geral: “A Constituição autoriza a cobrança do Imposto sobre a Propriedade de Veículos Automotores (IPVA) somente pelo Estado em que o contribuinte mantém sua sede ou domicílio tributário.”
• Recurso extraordinário a que se nega provimento.

Tema 708 - Possibilidade de recolhimento do Imposto sobre a Propriedade de Veículos Automotores (IPVA) em estado diverso daquele em que o contribuinte mantém sua sede ou domicílio tributário.

• Tese: A Constituição autoriza a cobrança do Imposto sobre a Propriedade de Veículos Automotores (IPVA) somente pelo Estado em que o contribuinte mantém sua sede ou domicílio tributário.

• ADI 524
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. SEPÚLVEDA PERTENCE
• Redator(a) do acórdão: Min. RICARDO LEWANDOWSKI
• Julgamento: 20/05/2015 Publicação: 03/08/2015

Ementa EMENTA: AÇÃO DIRETA DE INCONSTITUCIONALIDADE. INCISO VI DO ART. 32 DA CONSTITUIÇÃO DO ESTADO DO ESPÍRITO SANTO. SERVIDOR PÚBLICO. NEPOTISMO. VEDAÇÃO AO EXERCÍCIO DE FUNÇÕES SOB A DIREÇÃO IMEDIATA DE CÔNJUGE OU PARENTE ATÉ O SEGUNDO GRAU CIVIL. VIOLAÇÃO AO INCISO II DO ART. 37 DA CONSTITUIÇÃO FEDERAL E AO PRINCÍPIO DA ISONOMIA. INEXISTÊNCIA. PROIBIÇÃO QUE DECORRE DO CAPUT DO ART. 37 DA CF. PROCEDÊNCIA PARCIAL PARA EMPRESTAR INTERPRETAÇÃO CONFORME A CONSTITUIÇÃO. INCIDÊNCIA EXCLUSIVA SOBRE CARGOS DE PROVIMENTO EM COMISSÃO, FUNÇÃO GRATIFICADA E CARGOS E DIREÇÃO E ASSESSORAMENTO.

Tema 342: A imunidade tributária subjetiva aplica-se a seus beneficiários na posição de contribuinte de direito, mas não na de simples contribuinte de fato, sendo irrelevante para a verificação da existência do beneplácito constitucional a repercussão econômica do tributo envolvido.

• Desse modo, um município não pode requerer a imunidade com relação ao ICMS incidente sobre a energia elétrica, uma vez que o contribuinte de direito desse tributo não é a municipalidade, e sim a empresa que presta esse serviço.
• Por outro lado, o município que importar um equipamento de informática para ser utilizado em uma escola tem direito à imunidade com relação ao Imposto de Importação, uma vez que, nessa hipótese, será ele contribuinte de direito da exação

• É irrelevante para a definição da aplicabilidade da imunidade tributária recíproca a circunstância de a atividade desempenhada estar ou não sujeita a monopólio estatal. O alcance da salvaguarda constitucional pressupõe o exame (i) da caracterização econômica da atividade (lucrativa ou não), (ii) do risco à concorrência e à livre-iniciativa e (iii) de riscos ao pacto federativo pela pressão política ou econômica.

• A imunidade tributária recíproca não se aplica à Petrobras, pois: trata-se de sociedade de economia mista destinada à exploração econômica em benefício de seus acionistas, pessoas de direito público e privado, e a salvaguarda não se presta a proteger aumento patrimonial dissociado de interesse público primário; a Petrobras visa à distribuição de lucros, e, portanto, tem capacidade contributiva para participar do apoio econômico aos entes federados; a tributação de atividade econômica lucrativa não implica risco ao pacto federativo. [RE 285.716 AgR, rel. min. Joaquim Barbosa, j. 2-3-2010, 2ª T, DJE de 26-3-2010.]

Incide o IPTU considerado imóvel de pessoa jurídica de direito público cedido a pessoa jurídica de direito privado, devedora do tributo. [RE 601.720, voto do rel. p/ o ac. min. Marco Aurélio, j. 19-4-2017, P, DJE de 5-9-2017, Tema 437.]

Imunidade recíproca. Empresa Brasileira de Correios e Telégrafos. Peculiaridades do serviço postal. Exercício de atividades em regime de exclusividade e em concorrência com particulares. Irrelevância. ICMS. Transporte de encomendas. Indissociabilidade do serviço postal. Incidência da Imunidade do art. 150, VI, a, da Constituição. Condição de sujeito passivo de obrigação acessória. Legalidade. (...) O transporte de encomendas está inserido no rol das atividades desempenhadas pela ECT, que deve cumprir o encargo de alcançar todos os lugares do Brasil, não importa o quão pequenos ou subdesenvolvidos. Não há comprometimento do status de empresa pública prestadora de serviços essenciais por conta do exercício da atividade de transporte de encomendas, de modo que essa atividade constitui conditio sine qua non para a viabilidade de um serviço postal contínuo, universal e de preços módicos. [RE 627.051, rel. min. Dias Toffoli, j. 12-11-2014, P, DJE de 11-2-2015, Tema 402.] Vide RE 601.392, rel. p/ o ac. min. Gilmar Mendes, j. 28-2-2013, P, DJE de 5-6-2013, Tema 235

Princípio da legalidade tributária.

O princípio da legalidade é mitigado quanto a * Imposto de Importação (II) * Imposto de Exportação (IE) * IOF * IPI * CIDE-Combustível * ICMS-Combustível

STF (RE 838.284/SC, j. 06/10/2016):

Não viola a legalidade tributária a lei que, prescrevendo o teto, possibilita o ato normativo infralegal fixar o valor de taxa em proporção razoável com os custos da atuação estatal, valor esse que não pode ser atualizado por ato do próprio conselho de fiscalização em percentual superior aos índices de correção monetária legalmente previstos.

Importante destacar a explicação irretocável, realizada pelo juiz federal Márcio André Lopes Cavalcante, sobre o julgado:

A Lei nº 6.994/82 delegou ao Conselho Profissional a fixação dos valores das taxas correspondentes aos serviços relativos a atos indispensáveis ao exercício da profissão, observados os respectivos limites máximos. Para o STF o fato de a lei ter fixado valor máximo da taxa já é suficiente para que seja respeitado o princípio da legalidade tributária prevista no art. 150, I da CF/88:

Dessa forma o legislador tributário pode se valer de cláusulas gerais, e as taxas cobradas em razão do exercício do poder de polícia podem ter algum grau de indeterminação por força da ausência de minuciosa definição legal dos serviços compreendidos. E, diante de taxa ou contribuição parafiscal, é possível haver maior abertura dos tipos tributários. Afinal, nessas situações, sempre há atividade estatal subjacente o que acaba deixando ao regulamento uma carga maior de cognição da realidade, sobretudo em matéria técnica.

Deve-se permitir essa flexibilização em homenagem à praticidade e à eficiência da Administração Pública.

• Informativo nº 727
• 7 de março de 2022.
• SEGUNDA TURMA
• Processo
• RMS 66.794-AM, Rel. Min. Francisco Falcão, Segunda Turma, por unanimidade, julgado em 22/02/2022.
• Ramo do Direito DIREITO ADMINISTRATIVO

Serviço público. Contrato de concessão. Intervenção. Contraditório prévio. Desnecessidade.

DESTAQUE - Não se exige contraditório prévio à decretação de intervenção em contrato de concessão com concessionária de serviço público.

INFORMAÇÕES DO INTEIRO TEOR - Conforme se extrai do regime jurídico do art. 175 da Constituição Federal e da Lei de Concessões - Lei n. 8.987/1995 -, o Estado delega a prestação de alguns serviços públicos, resguardando a si, na qualidade de poder concedente, a prerrogativa de regulamentar, controlar e fiscalizar a atuação do delegatário.

• No âmbito desse controle e fiscalização, a intervenção no contrato de concessão constitui um dever e uma prerrogativa de que dispõe o poder concedente, visando assegurar a adequação na prestação do serviço público, bem como o fiel cumprimento das normas contratuais, regulamentares e legais pertinentes, segundo dispõe o art. 32 da Lei n. 8.987/1995.

• De um lado, o poder concedente deve "instaurar procedimento administrativo para comprovar as causas determinantes da medida e apurar responsabilidades, assegurado o direito de ampla defesa" (art. 33 da Lei n. 8.987/1995). De outro, não se pode desconsiderar que eventuais ilegalidades no curso do procedimento devem ser aferidas em consonância com a regra geral do ordenamento jurídico de que a decretação da nulidade depende de comprovação de prejuízo.

• Nos termos do art. 33 da Lei n. 8.987/1995: "Declarada a intervenção, o poder concedente deverá, no prazo de trinta dias, instaurar procedimento administrativo para comprovar as causas determinantes da medida e apurar responsabilidades, assegurado o direito de ampla defesa." Verifica-se que, em se tratando de intervenção, o direito de defesa do concessionário só é propiciado após a decretação da intervenção, a partir do momento em que for instaurado o procedimento administrativo para apuração das irregularidades. Isso porque a intervenção possui finalidades investigatória e fiscalizatória, e não punitiva.

• Assim, é dispensável estabelecer contraditório prévio à decretação da intervenção, ausente determinação na Lei n. 8.987/1995.

• Informativo nº 718
• 22 de novembro de 2021.
• PRIMEIRA SEÇÃO
• Processo: MS 22.606-DF, Rel. Min. Gurgel de Faria, Primeira Seção, por unanimidade, julgado em 10/11/2021.

Ramo do Direito DIREITO ADMINISTRATIVO

Servidor público. Processo disciplinar. Sanção. Dosimetria. Controle de legalidade. Possibilidade. Penalidade de suspensão. Prazo máximo. Tempo de experiência. Ilegalidade. Antecedentes funcionais. Valoração negativa. Condenação anterior. Necessidade.

DESTAQUE - É necessária condenação anterior na ficha funcional do servidor ou, no mínimo, anotação de fato que o desabone, para que seus antecedentes sejam valorados como negativos na dosimetria da sanção disciplinar.

INFORMAÇÕES DO INTEIRO TEOR - No caso, a comissão processante sugeriu a aplicação da pena de suspensão, por 15 dias, nos termos do art. 130, por infração ao disposto nos arts. 116, I, III e IX e 117, IV, todos da Lei n. 8.112/1990.

• Na espécie, embora constasse motivação objetiva para aplicar a sanção de suspensão, no lugar de advertência, a Administração não justificou de maneira técnica a razão pela qual fixou aquela penalidade no prazo máximo da lei.

• Portanto, o exame é sobre a legalidade dos critérios técnicos e objetivos apresentados pela União para a escolha da penalidade aplicada, sem se imiscuir no critério discricionário da escolha.

• A interpretação sistemática dada aos arts. 117, IV, 128, parágrafo único, e 129, todos da Lei n. 8.112/1990, é no sentido de que, em regra, a conduta do servidor seria punível com advertência, admitindo-se, porém, a aplicação de sanção de suspensão, se a natureza e a gravidade da infração cometida, os danos que dela provierem para o serviço público, as circunstâncias agravantes ou atenuantes e os antecedentes funcionais assim justificassem.

• Em relação aos antecedentes funcionais, a administração entendeu por qualifica-los como negativos, por compreender que sendo o servidor veterano, com larga experiência, deveria ter conduzido com mais zelo e mais cuidado o processo administrativo que estava sob sua responsabilidade.

• Verifica-se que os antecedentes do servidor foram inapropriadamente valorados como negativos. A Administração poderia considerar como desfavorável o fato de o servidor ter sido tão imprudente, mesmo tendo larga experiência, se a legislação autorizasse o exame da culpabilidade do agente, tal como o art. 59 do CP autoriza.

• No entanto, o Estatuto dos Servidores Públicos Federais só admite considerar, na "dosimetria" da sanção disciplinar, os antecedentes funcionais, que ostentam concepção técnica própria. Nesse passo, para que aqueles fossem considerados negativos, deveria constar na ficha funcional do impetrante alguma condenação anterior, ou, no mínimo, alguma anotação de fato que desabonasse seu histórico funcional.

• Informativo nº 714
• 25 de outubro de 2021.
• PRIMEIRA TURMA

• Processo: AREsp 1.402.806-TO, Rel. Min. Manoel Erhardt (Desembargador convocado do TRF da 5ª Região), Primeira Turma, por unanimidade, julgado em 19/10/2021.

• Ramo do Direito: DIREITO ADMINISTRATIVO

• Ação de improbidade administrativa. Responsabilização de particular que figura isoladamente no polo passivo da demanda. Jurisprudência pacífica do STJ pela impossibilidade. Agente público acionado pelos mesmos fatos em demanda conexa. Distinção detectada. Viabilidade.

DESTAQUE - É viável o prosseguimento de ação de improbidade administrativa exclusivamente contra particular quando há pretensão de responsabilizar agentes públicos pelos mesmos fatos em outra demanda conexa.

INFORMAÇÕES DO INTEIRO TEOR - Primeiramente, destaque-se que não se está a discutir a já conhecida e reverenciada compreensão desta Corte Superior de que é inviável o manejo da ação civil de improbidade exclusivamente contra o particular, sem a concomitante presença de agente público no polo passivo da demanda.

• No caso, houve o ajuizamento de duas ações de improbidade, uma pelo Ministério Público Federal, outra pelo DNIT. Os agentes públicos envolvidos na idêntica trama factual narrada nas duas demandas foram excluídos da ação ajuizada pelo Parquet, que é a ora analisada, restando nesta apenas o particular acionado.

• Em sua fundamentação, a Corte Regional aduziu que, "com o reconhecimento da litispendência e a extinção do feito originário contra os agentes públicos, a ação de improbidade foi mantida somente contra o particular, o que não pode ser admitido. Com efeito, inexistindo agentes públicos no polo passivo da ação de improbidade administrativa, destinatários do preceito legal que enumera os atos tidos como ímprobos, não há como prosperar a ação originária em que pretende o agravado a condenação do agravante pela prática de ato de improbidade administrativa".

• O Tribunal Regional asseverou, portanto, que, muito embora houvesse ação conexa promovida contra os Agentes Públicos, a demanda apreciada contaria apenas com o particular no polo passivo, o que não poderia ser admitido em ações de improbidade.

• Essa conclusão é dissonante de ilustrativos desta Corte Superior de que não é o caso de aplicar a jurisprudência do STJ, segundo a qual os particulares não podem ser responsabilizados com base na LIA sem que figure no polo passivo um agente público responsável pelo ato questionado, pois houve a devida pretensão de responsabilizar os agentes públicos em outra demanda conexa (REsp 1.732.762/MT, Rel. Min. Herman Benjamin, Segunda Turma, DJe 17.12.2018).

ADI 7042 - 7. (...) (b) declarar a inconstitucionalidade parcial, com interpretação conforme sem redução de texto, do § 20 do art. 17 da Lei 8.429/1992, incluído pela Lei 14.230/2021, no sentido de que não inexiste “obrigatoriedade de defesa judicial”; havendo, porém, a possibilidade de os órgãos da Advocacia Pública autorizarem a realização dessa representação judicial, por parte da assessoria jurídica que emitiu o parecer atestando a legalidade prévia dos atos administrativos praticados pelo administrador público, nos termos autorizados por lei específica

• ADI 7042
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ALEXANDRE DE MORAES
• Julgamento: 31/08/2022
• Publicação: 28/02/2023

Ementa: CONSTITUCIONAL E ADMINISTRATIVO. CONSTITUCIONALIZAÇÃO DE REGRAS RÍGIDAS DE REGÊNCIA DA ADMINISTRAÇÃO PÚBLICA, PROTEÇÃO AO PATRIMONIO PÚBLICO E RESPONSABILIZAÇÃO DOS AGENTES PÚBLICOS CORRUPTOS PREVISTAS NO ARTIGO 37 DA CF. VEDAÇÃO À EXCLUSIVIDADE DO MINISTÉRIO PÚBLICO PARA PROPOSITURA DA AÇÃO POR ATO DE IMPROBIDADE ADMINISTRATIVA E DO ACORDO DE NÃO PERSECUÇÃO CIVIL (CF, ARTIGO 129, §1º). LEGITIMIDADE CONCORRENTE E DISJUNTIVA ENTRE FAZENDA PÚBLICA E MINISTÉRIO PÚBLICO. VEDAÇÃO À OBRIGATORIEDADE DE ATUAÇÃO DA ASSESSORIA JURÍDICA NA DEFESA JUDICIAL DO ADMINISTRADOR PÚBLICO. AÇÃO PARCIALMENTE PROCEDENTE.

1. Reconhecida a legitimidade ativa da Associação Nacional dos Procuradores dos Estados e do Distrito Federal – ANAPE e da Associação Nacional dos Advogados Públicos Federais – ANAFE para o ajuizamento das presentes demandas, tendo em conta o caráter nacional e a existência de pertinência temática entre suas finalidades institucionais e o objeto de impugnação. Precedentes.

2. Vedação constitucional à previsão de legitimidade exclusiva do Ministério Público para a propositura da ação por ato de improbidade administrativa, nos termos do artigo 129, §1º da Constituição Federal e, consequentemente, para oferecimento do acordo de não persecução civil.

3. A legitimidade da Fazenda Pública para o ajuizamento de ações por improbidade administrativa é ordinária, já que ela atua na defesa de seu próprio patrimônio público, que abarca a reserva moral e ética da Administração Pública brasileira.

4. A supressão da legitimidade ativa das pessoas jurídicas interessadas para a propositura da ação por ato de improbidade representa uma inconstitucional limitação ao amplo acesso à jurisdição (CF, art. 5º, XXXV) e a defesa do patrimônio público, com ferimento ao princípio da eficiência (CF, art. 37, caput) e significativo retrocesso quanto ao imperativo constitucional de combate à improbidade administrativa.

5. A legitimidade para firmar acordo de não persecução civil no contexto do combate à improbidade administrativa exsurge como decorrência lógica da própria legitimidade para a ação, razão pela qual estende-se às pessoas jurídicas interessadas.

6. A previsão de obrigatoriedade de atuação da assessoria jurídica na defesa judicial do administrador público afronta a autonomia dos Estados-Membros e desvirtua a conformação constitucional da Advocacia Pública delineada pelo art. 131 e 132 da Constituição Federal, ressalvada a possibilidade de os órgãos da Advocacia Pública autorizarem a realização dessa representação judicial, nos termos de legislação específica.

7. Ação julgada parcialmente procedente para (a) declarar a inconstitucionalidade parcial, com interpretação conforme sem redução de texto, do caput e dos §§ 6º-A e 10-C do art. 17, assim como do caput e dos §§ 5º e 7º do art. 17-B, da Lei 8.429/1992, na redação dada pela Lei 14.230/2021, de modo a restabelecer a existência de legitimidade ativa concorrente e disjuntiva entre o Ministério Público e as pessoas jurídicas interessadas para a propositura da ação por ato de improbidade administrativa e para a celebração de acordos de não persecução civil; (b) declarar a inconstitucionalidade parcial, com interpretação conforme sem redução de texto, do § 20 do art. 17 da Lei 8.429/1992, incluído pela Lei 14.230/2021, no sentido de que não inexiste “obrigatoriedade de defesa judicial”; havendo, porém, a possibilidade de os órgãos da Advocacia Pública autorizarem a realização dessa representação judicial, por parte da assessoria jurídica que emitiu o parecer atestando a legalidade prévia dos atos administrativos praticados pelo administrador público, nos termos autorizados por lei específica;(c) declarar a inconstitucionalidade do art. 3º da Lei 14.230/2021. Em consequência, declara-se a constitucionalidade: (a) do § 14 do art. 17 da Lei 8.429/1992, incluído pela Lei 14.230/2021; e (b) do art. 4º, X, da Lei 14.230/2021.

• Informativo nº 686, 1º de março de 2021
• PRIMEIRA TURMA
• Processo
• AREsp 1.314.581/SP, Rel. Min. Benedito Gonçalves, Primeira Turma, por unanimidade, julgado em 23/02/2021.

Ramo do Direito DIREITO ADMINISTRATIVO

Tema:Improbidade administrativa. Homologação judicial de acordo. Art. 17, § 1º, da Lei n. 8.429/1992, com redação alterada pela Lei n. 13.964/2019. Possibilidade.

DESTAQUE - É possível acordo de não persecução cível no âmbito da ação de improbidade administrativa em fase recursal.

INFORMAÇÕES DO INTEIRO TEOR - A Lei n. 13.964/2019, denominada "Pacote Anticrime", alterou o § 1º do art. 17 da Lei n. 8.429/1992, o qual passou a dispor que: § 1º As ações de que trata este artigo admitem a celebração de acordo de não persecução cível, nos termos desta Lei.

• A referida Lei também introduziu o § 10-A ao art. 17 da LIA, com a seguinte redação: Havendo a possibilidade de solução consensual, poderão as partes requerer ao juiz a interrupção do prazo para a contestação, por prazo não superior a 90 (noventa) dias.

• Com efeito, a aludida alteração trouxe a possibilidade de acordo de não persecução cível no âmbito da ação de improbidade administrativa.

• A Segunda Turma desta Corte, ao se pronunciar a respeito da delação premiada e do acordo de leniência, em sede de ação de improbidade administrativa, conferiu interpretação restritiva aos referidos institutos à esfera penal, nos termos do sobredito art. 17, § 1º, da LIA.

• Na oportunidade, o eminente Relator, Ministro Mauro Campbell Marques, consignou que "a transação e o acordo são expressamente vedados no âmbito da ação de improbidade administrativa (art. 17, § 1º, da Lei n. 8.429/1992), ainda que entenda oportuno o debate pelo Congresso Nacional sobre o referido dispositivo legal, a fim de analisar sua atualidade, pertinência e compatibilidade com normas sancionatórias que preveem a possibilidade de acordo de não-persecução penal." (REsp 1.464.287/DF, Rel. Min. Mauro Campbell Marques, Segunda Turma, DJe 26/6/2020.)

• A Primeira Turma desta Corte, em julgamento realizado em 03/05/2016, antes, portanto, da alteração do art. 17, § 1º, da LIA, não conheceu do requerimento de homologação de acordo no âmbito de ação de improbidade administrativa.

• Na ocasião, o eminente Relator, Ministro Napoleão Nunes Maia Filho, ressalvou seu ponto de vista pela possibilidade de acordo, uma vez cumpridas pelas partes transigentes as obrigações do Termo de Ajustamento de Conduta, não se justificando a protelação da homologação do acordo.

• O Conselho Nacional do Ministério Público (CNMP) editou a Resolução n. 179, de 26 de julho de 2017, regulamentando o § 6º do art. 5º da Lei n. 7.347/1985, para disciplinar, no âmbito do Ministério Público, a tomada do compromisso de ajustamento de conduta.

• Nessa linha, o Conselho Superior do Ministério Público do Estado de São Paulo (CSMPSP) editou a Resolução n. 1.193, de 11 de março de 2020, a qual disciplina o acordo de não persecução cível no âmbito do MPSP, regulamentando o disposto no art. 17, § 1º, da LIA e no art. 7º, § 2º, da Resolução n. 179/2017 do Conselho Nacional do Ministério Público.

• No caso, o recorrente foi condenado por dano ao erário pela prática de conduta ímproba na modalidade culposa do art. 10 da LIA, decorrente da condenação por danos morais sofrida, nos autos de ação de indenização, em razão de conduta omissiva consubstanciada pelo não cumprimento de ordem judicial para que fornecesse medicamento a paciente, que acabou vindo a óbito.

• Dessa forma, tendo em vista a homologação do acordo pelo Conselho Superior do MPSP, a conduta culposa praticada pelo recorrente, bem como a reparação do dano ao Município, além da manifestação favorável do Ministério Público Federal ao acordo, tem-se que a transação deve ser homologada.

Author Response

Reviewer #1 (Public Review):

In this very strong and interesting paper the authors present a convincing series of experiments that reveal molecular mechanism of neuronal cell type diversification in the nervous system of Drosophila. The authors show that a homeodomain transcription factor, Bsh, fulfills several critical functions - repressing an alternative fate and inducing downstream homeodomain transcription factors with whom Bsh may collaborate to induce L4 and L5 fates (the author's accompanying paper reveals how Bsh can induce two distinct fates). The authors make elegant use of powerful genetic tools and an arsenal of satisfying cell identity markers.

Thanks!

I believe that this is an important study because it provides some fundamental insights into the conservation of neuronal diversification programs. It is very satisfying to see that similar organizational principles apply in different organisms to generate cell type diversity. The authors should also be commended for contextualizing their work very well, giving a broad, scholarly background to the problem of neuronal cell type diversification.

Thanks!

My one suggestion for the authors is to perhaps address in the Discussion (or experimentally address if they wish) how they reconcile that Bsh is on the one hand: (a) continuously expressed in L4/L4, (b) binding directly to a cohort of terminal effectors that are also continuously expressed but then, on the other hand, is not required for their maintaining L4 fate? A few questions: Is Bsh only NOT required for maintaining Ap expression or is it also NOT required for maintaining other terminal markers of L4? The former could be easily explained - Bsh simply kicks of Ap, Ap then autoregulates, but Bsh and Ap then continuously activate terminal effector genes. The second scenario would require a little more complex mechanism: Bsh binding of targets (with Notch) may open chromatin, but then once that's done, Bsh is no longer needed and Ap alone can continue to express genes. I feel that the authors should be at least discussing this. The postmitotic Bsh removal experiment in which they only checked Ap and depression of other markers is a little unsatisfying without further discussion (or experiments, such as testing terminal L4 markers). I hasten to add that this comment does not take away from my overall appreciation for the depth and quality of the data and the importance of their conclusions.

Great suggestions, we will discuss these two hypotheses as requested.

Bsh initiates Ap expression in L4 neurons which then maintain Ap expression independently of Bsh expression, likely through Ap autoregulation. During the synaptogenesis window, Ap expression becomes independent from Bsh expression, but Bsh and Ap are both still required to activate the synapse recognition molecule DIP-beta. Additionally, Bsh also shows putative binding to other L4 identity genes, e.g., those required for neurotransmitter choice, and electrophysiological properties, suggesting Bsh may initiates L4 identity genes as a suite of genes. The mechanism of maintaining identity features (e.g., morphology, synaptic connectivity and functional properties) in the adult remains poorly understood. It is a great question whether primary HDTF Bsh maintains the expression of L4 identity genes in the adult. To test this, in our next project, we will specifically knock out Bsh in L4 neurons of the adult fly and examine the effect on L4 morphology, connectivity and function properties.

Reviewer #2 (Public Review):

Summary:

In this paper, the authors explore the role of the Homeodomain Transcription Factor Bsh in the specification of Lamina neuronal types in the optic lobe of Drosophila. Using the framework of terminal selector genes and compelling data, they investigate whether the same factor that establishes early cell identity is responsible for the acquisition of terminal features of the neuron (i.e., cell connectivity and synaptogenesis).

Thanks for the positive words!

The authors convincingly describe the sequential expression and activity of Bsh, termed here as 'primary HDTF', and of Ap in L4 or Pdm3 in L5 as 'secondary HDTFs' during the specification of these two neurons. The study demonstrates the requirement of Bsh to activate either Ap and Pdm3, and therefore to generate the L4 and L5 fates. Moreover, the authors show that in the absence of Bsh, L4 and L5 fates are transformed into a L1 or L3-like fates.

Thanks!

Finally, the authors used DamID and Bsh:DamID to profile the open chromatin signature and the Bsh binding sites in L4 neurons at the synaptogenesis stage. This allows the identification of putative Bsh target genes in L4, many of which were also found to be upregulated in L4 in a previous single-cell transcriptomic analysis. Among these genes, the paper focuses on Dip-β, a known regulator of L4 connectivity. They demonstrate that both Bsh and Ap are required for Dip-β, forming a feed-forward loop. Indeed, the loss of Bsh causes abnormal L4 synaptogenesis and therefore defects in several visual behaviors. The authors also propose the intriguing hypothesis that the expression of Bsh expanded the diversity of Lamina neurons from a 3 cell-type state to the current 5 cell-type state in the optic lobe.

Thanks for the excellent summary of our findings!

Strengths:

Overall, this work presents a beautiful practical example of the framework of terminal selectors: Bsh acts hierarchically with Ap or Pdm3 to establish the L4 or L5 cell fates and, at least in L4, participates in the expression of terminal features of the neuron (i.e., synaptogenesis through Dip-β regulation).

Thanks!

The hierarchical interactions among Bsh and the activation of Ap and Pdm3 expression in L4 and L5, respectively, are well established experimentally. Using different genetic drivers, the authors show a window of competence during L4 neuron specification during which Bsh activates Ap expression. Later, as the neuron matures, Ap becomes independent of Bsh. This allows the authors to propose a coherent and well-supported model in which Bsh acts as a 'primary' selector that activates the expression of L4-specific (Ap) and L5-specific (Pdm3) 'secondary' selector genes, that together establish neuronal fate.

Thanks again!

Importantly, the authors describe a striking cell fate change when Bsh is knocked down from L4/L5 progenitor cells. In such cases, L1 and L3 neurons are generated at the expense of L4 and L5. The paper demonstrates that Bsh in L4/L5 represses Zfh1, which in turn acts as the primary selector for L1/L3 fates. These results point to a model where the acquisition of Bsh during evolution might have provided the grounds for the generation of new cell types, L4 and L5, expanding lamina neuronal diversity for a more refined visual behaviors in flies. This is an intriguing and novel hypothesis that should be tested from an evo-devo standpoint, for instance by identifying a species when L4 and L5 do not exist and/or Bsh is not expressed in L neurons.

Thanks for the appreciation of our findings!

To gain insight into how Bsh regulates neuronal fate and terminal features, the authors have profiled the open chromatin landscape and Bsh binding sites in L4 neurons at mid-pupation using the DamID technique. The paper describes a number of genes that have Bsh binding peaks in their regulatory regions and that are differentially expressed in L4 neurons, based on available scRNAseq data. Although the manuscript does not explore this candidate list in depth, many of these genes belong to classes that might explain terminal features of L4 neurons, such as neurotransmitter identity, neuropeptides or cytoskeletal regulators. Interestingly, one of these upregulated genes with a Bsh peak is Dip-β, an immunoglobulin superfamily protein that has been described by previous work from the author's lab to be relevant to establish L4 proper connectivity. This work proves that Bsh and Ap work in a feed-forward loop to regulate Dip-β expression, and therefore to establish normal L4 synapses. Furthermore, Bsh loss of function in L4 causes impairs visual behaviors.

Thanks for the excellent summary of our findings.

Weaknesses:

● The last paragraph of the introduction is written using rhetorical questions and does not read well. I suggest rewriting it in a more conventional direct style to improve readability.

We agree, and will update the text as suggested.

● A significant concern is the way in which information is conveyed in the Figures. Throughout the paper, understanding of the experimental results is hindered by the lack of information in the Figure headers. Specifically, the genetic driver used for each panel should be adequately noted, together with the age of the brain and the experimental condition. For example, R27G05-Gal4 drives early expression in LPCs and L4/L5, while the 31C06-AD, 34G07-DBD Split-Gal4 combination drives expression in older L4 neurons, and the use of one or the other to drive Bsh-KD has dramatic differences in Ap expression. The indication of the driver used in each panel will facilitate the reader's grasp of the experimental results.

We agree, and will update the figure annotation.

● Bsh role in L4/L5 cell fate:

o It is not clear whether Tll+/Bsh+ LPCs are the precursors of L4/L5. Morphologically, these cells sit very close to L5, but are much more distant from L4.

Our current data show L4 and L5 neurons are generated by different LPCs. However, currently we don’t have tools to demonstrate which subset of LPCs generate which lamina neuron type. We are currently working on a followup manuscript on LPC heterogeneity, but those experiments have just barely been started.

o Somatic CRISPR knockout of Bsh seems to have a weaker phenotype than the knockdown using RNAi. However, in several experiments down the line, the authors use CRISPR-KO rather than RNAi to knock down Bsh activity: it should be explained why the authors made this decision. Alternatively, a null mutant could be used to consolidate the loss of function phenotype, although this is not strictly necessary given that the RNAi is highly efficient and almost completely abolishes Bsh protein.

The reason we chose CRISPR-KO (L4-specific Gal4, uas-Cas9, and uas-Bsh-sgRNAs) is that it effectively removed Bsh expression from majority of L4 neurons. However, it failed to knock down Bsh in L4 neurons using L4-split Gal4 and Bsh-RNAi because L4-split Gal4 expression depends on Bsh. We will include this explanation in the text.

o Line 102: Rephrase "R27G05-Gal4 is expressed in all LPCs and turned off in lamina neurons" to "is turned off as lamina neurons mature", as it is kept on for a significant amount of time after the neurons have already been specified.

Thanks; we will make that change.

o Line 121: "(a) that all known lamina neuron markers become independent of Bsh regulation in neurons" is not an accurate statement, as the markers tested were not shown to be dependent on Bsh in the first place.

Good point. We will rephrase it as “that all known lamina neuron markers are independent of Bsh regulation in neurons”.

o Lines 129-134: Make explicit that the LPC-Gal4 was used in this experiment. This is especially important here, as these results are opposite to the Bsh Loss of Function in L4 neurons described in the previous section. This will help clarify the window of competence in which Bsh establishes L4/L5 neuronal identities through ap/pdm3 expression.

Thanks! We will include Gal4 information in the text for every manipulation.

● DamID and Bsh binding profile:

○ Figure 5 - figure supplement 1C-E: The genotype of the Control in (C) has to be described within the panel. As it is, it can be confused with a wild type brain, when it is in fact a Bsh-KO mutant.

Great point! Thank you for catching this and we will update it.

○ It Is not clear how L4-specific Differentially Expressed Genes were found. Are these genes DEG between Lamina neurons types, or are they upregulated genes with respect to all neuronal clusters? If the latter is the case, it could explain the discrepancy between scRNAseq DEGs and Bsh peaks in L4 neurons.

We did not use “L4-specific Differentially Expressed Genes”. Instead, we used all genes that are significantly transcribed in L4 neurons (line 209-210).

● Dip-β regulation:

○ Line 234: It is not clear why CRISPR KO is used in this case, when Bsh-RNAi presents a stronger phenotype.

As we explained it above, the reason we chose CRISPR-KO (L4-specific Gal4, uas-Cas9, and uas-Bsh-sgRNAs) is that it effectively removed Bsh expression from majority of L4 neurons. However, it failed to knock down Bsh in L4 neurons using L4-split Gal4 and Bsh-RNAi because L4-split Gal4 expression depends on Bsh. We’ll include this explanation in the text.

○ Figure 6N-R shows results using LPC-Gal4. It is not clear why this driver was used, as it makes a less accurate comparison with the other panels in the figure, which use L4-Split-Gal4. This discrepancy should be acknowledged and explained, or the experiment repeated with L4-Split-Gal4>Ap-RNAi.

I think you mean 6J-M shows results using LPC-Gal4. We first tried L4-Split-Gal4>Ap-RNAi but it failed to knock down Ap because L4-Split-Gal4 expression depends on Ap. We will add this to the text.

○ Line 271: It is also possible that L4 activity is dispensable for motion detection and only L5 is required.

Thanks! Work from Tuthill et al, 2013 showed that L5 is not required for any motion detection. We will include this citation in the text.

● Discussion: It is necessary to de-emphasize the relevance of HDTFs, or at least acknowledge that other, non-homeodomain TFs, can act as selector genes to determine neuronal identity. By restricting the discussion to HDTFs, it is not mentioned that other classes of TFs could follow the same Primary-Secondary selector activation logic.

That is a great point, thank you! We will include this in the discussion.

Reviewer #2 (Public Review):

Summary:<br /> In this paper, the authors explore the role of the Homeodomain Transcription Factor Bsh in the specification of Lamina neuronal types in the optic lobe of Drosophila. Using the framework of terminal selector genes and compelling data, they investigate whether the same factor that establishes early cell identity is responsible for the acquisition of terminal features of the neuron (i.e., cell connectivity and synaptogenesis).

The authors convincingly describe the sequential expression and activity of Bsh, termed here as 'primary HDTF', and of Ap in L4 or Pdm3 in L5 as 'secondary HDTFs' during the specification of these two neurons. The study demonstrates the requirement of Bsh to activate either Ap and Pdm3, and therefore to generate the L4 and L5 fates. Moreover, the authors show that in the absence of Bsh, L4 and L5 fates are transformed into a L1 or L3-like fates.

Finally, the authors used DamID and Bsh:DamID to profile the open chromatin signature and the Bsh binding sites in L4 neurons at the synaptogenesis stage. This allows the identification of putative Bsh target genes in L4, many of which were also found to be upregulated in L4 in a previous single-cell transcriptomic analysis. Among these genes, the paper focuses on Dip-β, a known regulator of L4 connectivity. They demonstrate that both Bsh and Ap are required for Dip-β, forming a feed-forward loop. Indeed, the loss of Bsh causes abnormal L4 synaptogenesis and therefore defects in several visual behaviors.

The authors also propose the intriguing hypothesis that the expression of Bsh expanded the diversity of Lamina neurons from a 3 cell-type state to the current 5 cell-type state in the optic lobe.

Strengths:

Overall, this work presents a beautiful practical example of the framework of terminal selectors: Bsh acts hierarchically with Ap or Pdm3 to establish the L4 or L5 cell fates and, at least in L4, participates in the expression of terminal features of the neuron (i.e., synaptogenesis through Dip-β regulation).

The hierarchical interactions among Bsh and the activation of Ap and Pdm3 expression in L4 and L5, respectively, are well established experimentally. Using different genetic drivers, the authors show a window of competence during L4 neuron specification during which Bsh activates Ap expression. Later, as the neuron matures, Ap becomes independent of Bsh. This allows the authors to propose a coherent and well-supported model in which Bsh acts as a 'primary' selector that activates the expression of L4-specific (Ap) and L5-specific (Pdm3) 'secondary' selector genes, that together establish neuronal fate.

Importantly, the authors describe a striking cell fate change when Bsh is knocked down from L4/L5 progenitor cells. In such cases, L1 and L3 neurons are generated at the expense of L4 and L5. The paper demonstrates that Bsh in L4/L5 represses Zfh1, which in turn acts as the primary selector for L1/L3 fates. These results point to a model where the acquisition of Bsh during evolution might have provided the grounds for the generation of new cell types, L4 and L5, expanding lamina neuronal diversity for a more refined visual behaviors in flies. This is an intriguing and novel hypothesis that should be tested from an evo-devo standpoint, for instance by identifying a species when L4 and L5 do not exist and/or Bsh is not expressed in L neurons.

To gain insight into how Bsh regulates neuronal fate and terminal features, the authors have profiled the open chromatin landscape and Bsh binding sites in L4 neurons at mid-pupation using the DamID technique. The paper describes a number of genes that have Bsh binding peaks in their regulatory regions and that are differentially expressed in L4 neurons, based on available scRNAseq data. Although the manuscript does not explore this candidate list in depth, many of these genes belong to classes that might explain terminal features of L4 neurons, such as neurotransmitter identity, neuropeptides or cytoskeletal regulators. Interestingly, one of these upregulated genes with a Bsh peak is Dip-β, an immunoglobulin superfamily protein that has been described by previous work from the author's lab to be relevant to establish L4 proper connectivity. This work proves that Bsh and Ap work in a feed-forward loop to regulate Dip-β expression, and therefore to establish normal L4 synapses. Furthermore, Bsh loss of function in L4 causes impairs visual behaviors.

Weaknesses:

● The last paragraph of the introduction is written using rhetorical questions and does not read well. I suggest rewriting it in a more conventional direct style to improve readability.

● A significant concern is the way in which information is conveyed in the Figures. Throughout the paper, understanding of the experimental results is hindered by the lack of information in the Figure headers. Specifically, the genetic driver used for each panel should be adequately noted, together with the age of the brain and the experimental condition. For example, R27G05-Gal4 drives early expression in LPCs and L4/L5, while the 31C06-AD, 34G07-DBD Split-Gal4 combination drives expression in older L4 neurons, and the use of one or the other to drive Bsh-KD has dramatic differences in Ap expression. The indication of the driver used in each panel will facilitate the reader's grasp of the experimental results.

● Bsh role in L4/L5 cell fate:<br /> o It is not clear whether Tll+/Bsh+ LPCs are the precursors of L4/L5. Morphologically, these cells sit very close to L5, but are much more distant from L4.<br /> o Somatic CRISPR knockout of Bsh seems to have a weaker phenotype than the knockdown using RNAi. However, in several experiments down the line, the authors use CRISPR-KO rather than RNAi to knock down Bsh activity: it should be explained why the authors made this decision. Alternatively, a null mutant could be used to consolidate the loss of function phenotype, although this is not strictly necessary given that the RNAi is highly efficient and almost completely abolishes Bsh protein.<br /> o Line 102: Rephrase "R27G05-Gal4 is expressed in all LPCs and turned off in lamina neurons" to "is turned off as lamina neurons mature", as it is kept on for a significant amount of time after the neurons have already been specified.<br /> o Line 121: "(a) that all known lamina neuron markers become independent of Bsh regulation in neurons" is not an accurate statement, as the markers tested were not shown to be dependent on Bsh in the first place.<br /> o Lines 129-134: Make explicit that the LPC-Gal4 was used in this experiment. This is especially important here, as these results are opposite to the Bsh Loss of Function in L4 neurons described in the previous section. This will help clarify the window of competence in which Bsh establishes L4/L5 neuronal identities through ap/pdm3 expression.

● DamID and Bsh binding profile:<br /> ○ Figure 5 - figure supplement 1C-E: The genotype of the Control in (C) has to be described within the panel. As it is, it can be confused with a wild type brain, when it is in fact a Bsh-KO mutant.<br /> ○ It Is not clear how L4-specific Differentially Expressed Genes were found. Are these genes DEG between Lamina neurons types, or are they upregulated genes with respect to all neuronal clusters? If the latter is the case, it could explain the discrepancy between scRNAseq DEGs and Bsh peaks in L4 neurons.

● Dip-β regulation:<br /> ○ Line 234: It is not clear why CRISPR KO is used in this case, when Bsh-RNAi presents a stronger phenotype.<br /> ○ Figure 6N-R shows results using LPC-Gal4. It is not clear why this driver was used, as it makes a less accurate comparison with the other panels in the figure, which use L4-Split-Gal4. This discrepancy should be acknowledged and explained, or the experiment repeated with L4-Split-Gal4>Ap-RNAi.<br /> ○ Line 271: It is also possible that L4 activity is dispensable for motion detection and only L5 is required.

● Discussion: It is necessary to de-emphasize the relevance of HDTFs, or at least acknowledge that other, non-homeodomain TFs, can act as selector genes to determine neuronal identity. By restricting the discussion to HDTFs, it is not mentioned that other classes of TFs could follow the same Primary-Secondary selector activation logic.

DOI: 10.1016/j.jchemneu.2023.102316

Resource: (Santa Cruz Biotechnology Cat# sc-376861, RRID:AB_2722522)

Curator: @scibot

SciCrunch record: RRID:AB_2722522

What is this?

Como docente de Ciencias sociales y como ciudadana, este proyecto es interesante porque busca dar soluciones a problemas reales, problemas que efectivamente desde las ciencias sociales se trabaja, aunque debería ser desde todas las áreas de conocimiento. Si bien el proyecto se desarrolla en la ciudad de Envigado, este ejercicio se puede aplicar a cualquier vereda, municipio, ciudad etc. y con lo anterior como docente me permite hacer un análisis reflexivo que me hace concluir que los procesos de investigación en el aula se puede lograr mientras los estudiantes despierten el interés y sean bien direccionados. Los problemas ambientales a ser universales se pueden buscar resolver, y que mejor forma que sean los estudiantes quienes generen procesos de formulación de preguntas, ejercicios colectivos, de exploración y reflexión y que con ello generen unos resultados que beneficien a la comunidad o a ellos mismos.<br /> Desde el Ministerio de ciencias, Instituciones y docentes se puede enseñar, guiar desde las ciencias y la tecnología.

Atenção: não há exigência de acordo sobre alimentos.

Yo siento que la gente simplemente se esta dando cuenta que ellos SI PUEDEN ser independientes.

En lugares como Latinoamerica y el Caribe, la familia es muy importante para la cultura. Me pregunto, qué en la sociedad pudo haber provocado que los mayores quisieran vivir solos? Fue una elección que ellos tomaron? O fue una elección que tomo su familia? En los Estados Unidos, no es raro que las personas mayores vivan en un asilo. Mi familia es de México y se considera una falta de respecto dejar que los mayores de tu familia vivan solos. Mi pregunto si esto cambiará con las futuras generaciones de mi familia.

VAP bundle consists of 1. HOB elevation 30-45 degrees 2. sedation vacation and weaning 3. PUD prophylaxix and DVT prophylaxis 4. oral care (chlorhexidine) KD

IHI Ventilator Bundle 1. Elevate HOB 30-45 degrees 2. Daily sedation vacation & reassessment for readiness to extubate 3. Peptic ulcer disease prophylaxis 4. DVT prophylaxis 5. Oral hygiene

-BS-J

I NEVER REACH THE RIGHT LOCATION whenever I click on your link. NUNCA ATINGI O LOCAL CERTO sempre que clico no seu link.

I must go the right way when attending the review classes.

Devo seguir o caminho certo ao frequentar as aulas de revisão

do lado do cliente, é necessário concatenar o número da mensagem com o texto da mensagem java msgEnviada = i + "," + "vou enviar esta ...."; do lado do servidor é necessário fazer o split da mensagem de forma a obter o número da mesma e o texto, separadamente java numero = msgRecebida.split(",")[0] msg = msgRecebida.split(",")[1] Código utilizado para validar a solução: ```java public void test() {

DatagramSocket aSocket = null;

try {

    aSocket = new DatagramSocket();
    aSocket.setSoTimeout(5000);
    int nMessage=1;

    String m= "vou enviar esta mensagem ao servidor";

    InetAddress aHost = InetAddress.getByName("localhost");
    int serverPort = 6789;
    while(nMessage < 10) {
        if(nMessage == 4 || nMessage == 7) nMessage++;
        byte[] mb= (String.valueOf(nMessage).concat(",").concat(m)).getBytes();

        DatagramPacket request = new DatagramPacket(mb,  mb.length, aHost, serverPort);

        aSocket.send(request);

        byte[] buffer = new byte[1000];

        DatagramPacket reply = new DatagramPacket(buffer, buffer.length);

        aSocket.receive(reply);
        System.out.println("Enviei/recebi a mensagem: " + new String(reply.getData()));
        if(nMessage >= 5)
            assertEquals("waitingfor,"+ 4, new String(reply.getData()).trim());
        else
            assertEquals(m, new String(reply.getData()).trim());
        nMessage++;
    }

}catch (SocketException e){System.out.println("Socket: " + e.getMessage()); fail("");

}catch (IOException e){System.out.println("IO: " + e.getMessage()); fail("");

}finally {if(aSocket != null) aSocket.close();}

} ```

Art. 182. A política de desenvolvimento urbano, executada pelo Poder Público municipal, conforme diretrizes gerais fixadas em lei, tem por objetivo ordenar o pleno desenvolvimento das funções sociais da cidade e garantir o bem- estar de seus habitantes. - § 4º É facultado ao Poder Público municipal, mediante lei específica para área incluída no plano diretor, exigir, nos termos da lei federal, do proprietário do solo urbano não edificado, subutilizado ou não utilizado, que promova seu adequado aproveitamento, sob pena, sucessivamente, de: - III - desapropriação com pagamento mediante títulos da dívida pública de emissão previamente aprovada pelo Senado Federal, com prazo de resgate de até dez anos, em parcelas anuais, iguais e sucessivas, assegurados o valor real da indenização e os juros legais.

Art. 184. Compete à União desapropriar por interesse social, para fins de reforma agrária, o imóvel rural que não esteja cumprindo sua função social, mediante prévia e justa indenização em títulos da dívida agrária, com cláusula de preservação do valor real, resgatáveis no prazo de até vinte anos, a partir do segundo ano de sua emissão, e cuja utilização será definida em lei.

• § 1º As benfeitorias úteis e necessárias serão indenizadas em dinheiro.

• § 2º O decreto que declarar o imóvel como de interesse social, para fins de reforma agrária, autoriza a União a propor a ação de desapropriação.

• § 3º Cabe à lei complementar estabelecer procedimento contraditório especial, de rito sumário, para o processo judicial de desapropriação.

• § 4º O orçamento fixará anualmente o volume total de títulos da dívida agrária, assim como o montante de recursos para atender ao programa de reforma agrária no exercício.

• § 5º São isentas de impostos federais, estaduais e municipais as operações de transferência de imóveis desapropriados para fins de reforma agrária.

PROCESSUAL CIVIL. ADMINISTRATIVO. DESAPROPRIAÇÃO. JUROS COMPENSATÓRIOS, MORATÓRIOS E HONORÁRIOS ADVOCATÍCIOS EM AÇÕES EXPROPRIATÓRIAS. DECRETO-LEI N. 3.365/1945, ARTS. 15-A E 15-B. ADI 2.332/STF. PROPOSTA DE REVISÃO DE TESES REPETITIVAS. COMPETÊNCIA. NATUREZA JURÍDICA DAS TESES ANTERIORES À EMENDA 26/2016. CARÁTER ADMINISTRATIVO E INDEXANTE. TESES 126, 184, 280, 281, 282, 283 E SÚMULAS 12, 70, 102, 141 E 408 TODAS DO STJ. REVISÃO EM PARTE. MANUTENÇÃO EM PARTE. CANCELAMENTO EM PARTE. EDIÇÃO DE NOVAS TESES. ACOLHIMENTO EM PARTE DA PROPOSTA. MODULAÇÃO. AFASTAMENTO. 1. Preliminares: i) a Corte instituidora dos precedentes qualificados possui competência para sua revisão, sendo afastada do ordenamento nacional a doutrina do stare decisis em sentido estrito (autovinculação absoluta aos próprios precedentes); e ii) não há que se falar em necessidade de sobrestamento da presente revisão à eventual modulação de efeitos no julgamento de controle de constitucionalidade, discussão que compete unicamente à Corte Suprema. 2. Há inafastável contradição entre parcela das teses repetitivas e enunciados de súmula submetidos à revisão e o julgado de mérito do STF na ADI 2332, sendo forçosa a conciliação dos entendimentos. 3. No período anterior à Emenda Regimental 26/2016 (DJe 15/12/2016), as teses repetitivas desta Corte configuravam providência de teor estritamente indexante do julgamento qualificado, porquanto elaboradas por unidade administrativa independente após o exaurimento da atividade jurisdicional. Faz-se necessário considerar o conteúdo efetivo dos julgados para seu manejo como precedente vinculante, prevalecendo a ratio decidendi extraída do inteiro teor em caso de contradição, incompletude ou qualquer forma de inconsistência com a tese então formulada. Hipótese incidente nas teses sob revisão, cuja redação pela unidade administrativa destoou em parte do teor dos julgamentos em recursos especiais repetitivos. 4. Descabe a esta Corte interpretar o teor de julgado do Supremo Tribunal Federal, seja em cautelar ou de mérito, sendo indevida a edição de tese repetitiva com pretensão de regular seus efeitos, principalmente com caráter condicional. 5. Cancelamento da Súmula 408/STJ ("Nas ações de desapropriação, os juros compensatórios incidentes após a Medida Provisória n. 1.577, de 11/06/1997, devem ser fixados em 6% ao ano até 13/09/2001 e, a partir de então, em 12% ao ano, na forma da Súmula n. 618 do Supremo Tribunal Federal."), por despicienda a convivência do enunciado com tese repetitiva dispondo sobre a mesma questão (Tese 126/STJ). Providência de simplificação da prestação jurisdicional. 6. Adequação da Tese 126/STJ ("Nas ações de desapropriação, os juros compensatórios incidentes após a Medida Provisória n. 1.577, de 11/06/1997, devem ser fixados em 6% ao ano até 13/09/2001 e, a partir de então, em 12% ao ano, na forma da Súmula n. 618 do Supremo Tribunal Federal.") para a seguinte redação: "O índice de juros compensatórios na desapropriação direta ou indireta é de 12% até 11.6.97, data anterior à publicação da MP 1577/97.". Falece competência a esta Corte para discutir acerca dos efeitos da cautelar na ADI 2.332, sem prejuízo da consolidação da jurisprudência preexistente sobre a matéria infraconstitucional. 7. Manutenção da Tese 184/STJ ("O valor dos honorários advocatícios em sede de desapropriação deve respeitar os limites impostos pelo artigo 27, § 1º, do Decreto-lei 3.365/41 ? qual seja: entre 0,5% e 5% da diferença entre o valor proposto inicialmente pelo imóvel e a indenização imposta judicialmente."). O debate fixado por esta Corte versa unicamente sobre interpretação infraconstitucional acerca da especialidade da norma expropriatória ante o Código de Processo Civil. 8. Adequação da Tese 280/STJ ("A eventual improdutividade do imóvel não afasta o direito aos juros compensatórios, pois esses restituem não só o que o expropriado deixou de ganhar com a perda antecipada, mas também a expectativa de renda, considerando a possibilidade do imóvel ser aproveitado a qualquer momento de forma racional e adequada, ou até ser vendido com o recebimento do seu valor à vista. ") à seguinte redação: "Até 26.9.99, data anterior à publicação da MP 1901-30/99, são devidos juros compensatórios nas desapropriações de imóveis improdutivos.". Também aqui afasta-se a discussão dos efeitos da cautelar da ADI 2332, mantendo-se a jurisprudência consagrada desta Corte ante a norma anteriormente existente. 9. Adequação da Tese 281/STJ ("São indevidos juros compensatórios quando a propriedade se mostrar impassível de qualquer espécie de exploração econômica seja atual ou futura, em decorrência de limitações legais ou da situação geográfica ou topográfica do local onde se situa a propriedade.") ao seguinte teor: "Mesmo antes da MP 1901-30/99, são indevidos juros compensatórios quando a propriedade se mostrar impassível de qualquer espécie de exploração econômica atual ou futura, em decorrência de limitações legais ou fáticas.". De igual modo, mantém-se a jurisprudência anterior sem avançar sobre os efeitos da cautelar ou do mérito da ADI 2.332. 10. Adequação da Tese 282/STJ ("Para aferir a incidência dos juros compensatórios em imóvel improdutivo, deve ser observado o princípio do tempus regit actum, assim como acontece na fixação do percentual desses juros. As restrições contidas nos §§ 1º e 2º do art. 15-A, inseridas pelas MP's n. 1.901-30/99 e 2.027-38/00 e reedições, as quais vedam a incidência de juros compensatórios em propriedade improdutiva, serão aplicáveis, tão somente, às situações ocorridas após a sua vigência.") à seguinte redação: "i) A partir de 27.9.99, data de publicação da MP 1901-30/99, exige-se a prova pelo expropriado da efetiva perda de renda para incidência de juros compensatórios (art. 15-A, § 1º, do Decreto-Lei 3365/41); e ii) Desde 5.5.2000, data de publicação da MP 2027-38/00, veda-se a incidência dos juros em imóveis com índice de produtividade zero (art. 15-A, § 2º, do Decreto-Lei 3365/41).". Dispõe-se sobre a validade das normas supervenientes a partir de sua edição. Ressalva-se que a discussão dos efeitos da ADI 2332 compete, unicamente, à Corte Suprema, nos termos da nova tese proposta adiante. 11. Cancelamento da Tese 283/STJ ("Para aferir a incidência dos juros compensatórios em imóvel improdutivo, deve ser observado o princípio do tempus regit actum, assim como acontece na fixação do percentual desses juros. Publicada a medida liminar concedida na ADI 2.332/DF (DJU de 13.09.2001), deve ser suspensa a aplicabilidade dos §§ 1º e 2º do artigo 15-A do Decreto-lei n. 3.365/41 até que haja o julgamento de mérito da demanda."), ante o caráter condicional do julgado e sua superação pelo juízo de mérito na ADI 2332, em sentido contrário ao da medida cautelar anteriormente deferida. 12. Edição de nova tese: "A discussão acerca da eficácia e efeitos da medida cautelar ou do julgamento de mérito da ADI 2332 não comporta revisão em recurso especial.". A providência esclarece o descabimento de provocação desta Corte para discutir efeitos de julgados de controle de constitucionalidade do Supremo Tribunal Federal. 13. Edição de nova tese: "Os juros compensatórios observam o percentual vigente no momento de sua incidência.". Evidencia-se a interpretação deste Tribunal sobre a matéria, já constante nos julgados repetitivos, mas não enunciada como tese vinculante própria. 14. Edição de nova tese: "As Súmulas 12/STJ (Em desapropriação, são cumuláveis juros compensatórios e moratórios), 70/STJ (Os juros moratórios, na desapropriação direta ou indireta, contam-se desde o trânsito em julgado da sentença) e 102/STJ (A incidência dos juros moratórios sobre compensatórios, nas ações expropriatórias, não constitui anatocismo vedado em lei) somente se aplicam às situações havidas até 12.01.2000, data anterior à vigência da MP 1.997-34.". Explicita-se simultaneamente a validade dos enunciados à luz das normas então vigentes e sua derrogação pelas supervenientes. Providência de simplificação normativa que, ademais, consolida em tese indexada teor de julgamento repetitivo já proferido por esta Corte. 15. Manutenção da Súmula 141/STJ ("Os honorários de advogado em desapropriação direta são calculados sobre a diferença entre a indenização e a oferta, corrigidas monetariamente."). 16. Cabe enfrentar, de imediato, a questão da modulação dos efeitos da presente decisão, na medida em que a controvérsia é bastante antiga, prolongando-se há mais de 17 (dezessete) anos pelos tribunais do país. Afasta-se a modulação de efeitos do presente julgado, tanto porque as revisões limitam-se a explicitar o teor dos julgamentos anteriores, quanto por ser descabido a esta Corte modular, a pretexto de controle de efeitos de seus julgados, disposições que, a rigor, são de competência exclusiva do Supremo Tribunal Federal, por versarem sobre consequências do julgamento de mérito de ADI em disparida de com cautelar anteriormente concedida. 17. Proposta de revisão de teses repetitivas acolhida em parte. (Pet n. 12.344/DF, relator Ministro Og Fernandes, Primeira Seção, julgado em 28/10/2020, DJe de 13/11/2020.)

Juros de 6% ao ano sobre a diferença apurada entra a oferta e o valor real do bem imóvel, observe que o termo inicial para os juros compensatórios é a data da imissão na posse.

Súmula 12/STJ: Em desapropriação, são cumuláveis juros compensatórios e moratórios.

• Nesse sentido, o termo inicial dos juros moratórios é a data do trânsito em julgado, conforme assenta a Súmula nº 70/STJ

• Poderá haver a incidência de juros moratórios sobre os juros compensatórios, conforme Súmula nº 102/STJ, não configurando anatocismo.

Súmula nº 113/STJ: Os juros compensatórios, na desapropriação direta, incidem a partir da imissão na posse, calculados sobre o valor da indenização, corrigido monetariamente.

• ADI 2332
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ROBERTO BARROSO
• Julgamento: 17/05/2018
• Publicação: 16/04/2019

Ementa: Administrativo. Ação Direta de Inconstitucionalidade. Regime Jurídico dos Juros Compensatórios e dos Honorários Advocatícios na Desapropriação. Procedência Parcial. 1. A jurisprudência desta Corte é firme no sentido de que o controle judicial dos pressupostos constitucionais para a edição das medidas provisórias tem caráter excepcional, justificando-se apenas quando restar evidente a inexistência de relevância e urgência ou a caracterização de abuso de poder do Presidente da República, o que não ocorre no presente caso. 2. É constitucional o percentual de juros compensatórios de 6% (seis por cento) ao ano para a remuneração do proprietário pela imissão provisória do ente público na posse do seu bem, na medida em que consiste em ponderação legislativa proporcional entre o direito constitucional do proprietário à justa indenização (art. 5º, XXIV, CF/88) e os princípios constitucionais da eficiência e da economicidade (art. 37, caput, CF/88). 3. Declaração da inconstitucionalidade do termo “até” e interpretação conforme a Constituição do caput do art. 15-A, de maneira a incidir juros compensatórios sobre a diferença entre 80% do preço ofertado pelo ente público e o valor fixado na sentença. 4. Constitucionalidade dos §§ 1º, 2º e 4º, do art. 15-A, do Decreto-lei nº 3.365/1941, ao determinarem a não incidência dos juros compensatórios nas hipóteses em que (i) não haja comprovação de efetiva perda de renda pelo proprietário com a imissão provisória na posse (§ 1º), (ii) o imóvel tenha “graus de utilização da terra e de eficiência na exploração iguais a zero” (§ 2º), e (iii) sobre o período anterior “à aquisição da propriedade ou posse titulada pelo autor da ação”. Voto reajustado para expressar o entendimento da maioria. 5. É constitucional a estipulação de parâmetros mínimo e máximo para a concessão de honorários advocatícios, previstos no § 1º, do art. 27, do Decreto-lei nº 3.365/1941. 6. Declaração da inconstitucionalidade da expressão “não podendo os honorários ultrapassar R$ 151.000,00 (cento e cinquenta e um mil reais)” por inobservância ao princípio da proporcionalidade e por possibilitar violação reflexa ao justo preço na indenização do expropriado (art. 5º, XXIV, CF/88).

1. Ação direta julgada parcialmente procedente. Fixação das seguintes teses: “(i) É constitucional o percentual de juros compensatórios de 6% (seis por cento) ao ano para a remuneração pela imissão provisória na posse de bem objeto de desapropriação; (ii) A base de cálculo dos juros compensatórios em desapropriações corresponde à diferença entre 80% do preço ofertado pelo ente público e o valor fixado na sentença; (iii) São constitucionais as normas que condicionam a incidência de juros compensatórios à produtividade da propriedade; (iv) É constitucional a estipulação de parâmetros mínimo e máximo para a concessão de honorários advocatícios em desapropriações, sendo, contudo, vedada a fixação de um valor nominal máximo de honorários.”

Tese: - I - É constitucional o percentual de juros compensatórios de 6% (seis por cento) ao ano para a remuneração pela imissão provisória na posse de bem objeto de desapropriação; - II - A base de cálculo dos juros compensatórios em desapropriações corresponde à diferença entre 80% do preço ofertado pelo ente público e o valor fixado na sentença; - III - São constitucionais as normas que condicionam a incidência de juros compensatórios à produtividade da propriedade; - IV - É constitucional a estipulação de parâmetros mínimo e máximo para a concessão de honorários advocatícios em desapropriações, sendo, contudo, vedada a fixação de um valor nominal máximo de honorários.

Fixação de honorários nas ações de desapropriação variarão entre 0,5% a 5% do valor apurado entre a diferença entre o valor ofertado e o avaliado.

Ainda sobre os honorários, deverá ser levado em consideração a Súmula nº 131/STJ:

• Nas ações de desapropriação incluem-se no cálculo da verba advocatícia as parcelas relativas aos juros compensatórios e moratórios, devidamente corrigidas.

Súmula nº 141/STJ:

• Os honorários de advogado em desapropriação direta são calculados sobre a diferença entre a indenização e a oferta, corrigidas monetariamente.

• ADI 2139
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CÁRMEN LÚCIA
• Julgamento: 01/08/2018

• Publicação: 19/02/2019

• EMENTA: AÇÃO DIRETA DE INCONSTITUCIONALIDADE. §§ 1º A 4º DO ART. 625-D DA CONSOLIDAÇÃO DAS LEIS DO TRABALHO - CLT, ACRESCIDO PELA LEI N. 9.958, DE 12.1.2000. COMISSÃO DE CONCILIAÇÃO PRÉVIA - CCP. SUPOSTA OBRIGATORIEDADE DE ANTECEDENTE SUBMISSÃO DO PLEITO TRABALHISTA À COMISSÃO PARA POSTERIOR AJUIZAMENTO DE RECLAMAÇÃO TRABALHISTA. INTERPRETAÇÃO PELA QUAL SE PERMITE A SUBMISSÃO FACULTATIVAMENTE. GARANTIA DO ACESSO À JUSTIÇA. INC. XXXV DO ART. 5º DA CONSTITUIÇÃO DA REPÚBLICA. AÇÃO JULGADA PARCIALMENTE PROCEDENTE PARA DAR INTERPRETAÇÃO CONFORME A CONSTITUIÇÃO AOS §§ 1º A 4º DO ART. 652-D DA CONSOLIDAÇÃO DAS LEIS DO TRABALHO - CLT.

• O Supremo Tribunal Federal tem reconhecido, em obediência ao inc. XXXV do art. 5º da Constituição da República, a desnecessidade de prévio cumprimento de requisitos desproporcionais ou inviabilizadores da submissão de pleito ao Poder Judiciário.
• Contraria a Constituição interpretação do previsto no art. 625-D e parágrafos da Consolidação das Leis do Trabalho pelo qual se reconhecesse a submissão da pretensão à Comissão de Conciliação Prévia como requisito para ajuizamento de reclamação trabalhista. Interpretação conforme a Constituição da norma.
• Art. 625-D e parágrafos da Consolidação das Leis do Trabalhos: a legitimidade desse meio alternativo de resolução de conflitos baseia-se na consensualidade, sendo importante instrumento para o acesso à ordem jurídica justa, devendo ser estimulada, não consubstanciando, todavia, requisito essencial para o ajuizamento de reclamações trabalhistas.
• Ação direta de inconstitucionalidade julgada parcialmente procedente para dar interpretação conforme a Constituição aos §§ 1º a 4º do art. 625-D da Consolidação das Leis do Trabalho, no sentido de assentar que a Comissão de Conciliação Prévia constitui meio legítimo, mas não obrigatório de solução de conflitos, permanecendo o acesso à Justiça resguardado para todos os que venham a ajuizar demanda diretamente ao órgão judiciário competente.

Duração intrajornada

• (MPT - Procurador do Trabalho/2020) Pedro foi contratado por uma universidade para lecionar (16) dezesseis horas por semana, às segundas e quintas-feiras, das 19h às 23h. Às terças e sextas-feiras, por sua vez, trabalhava das 07h às 11h. Não houve pactuação, nem coletiva nem individual, para estipular regra distinta acerca das horas fictas ou de qualquer um dos intervalos. Diante dessa narrativa, analise as seguintes assertivas:
• I – A Universidade poderá ser autuada pela fiscalização do trabalho por descumprimento de normas atinentes à duração do trabalho.
• II – Em reclamação individual, o empregado poderá cobrar apenas 15 minutos de horas extras por semana.
• III – Em reclamação individual, o empregado poderá cobrar 7 minutos e 30 segundos de horas extras por dia de trabalho.
• IV – Em reclamação individual, o empregado poderá cobrar 6 horas extras e 15 minutos por semana.
• Assinale a alternativa CORRETA:
• (A) Apenas a assertiva I está correta.
• (B) Apenas as assertivas I e IV estão corretas.
• (C) Apenas as assertivas I, III e IV estão corretas.
• (D) Todas as assertivas estão corretas.
• (E) Não respondida.

Correta é a alternativa B

• SUM-60. ADICIONAL NOTURNO. INTEGRAÇÃO NO SALÁRIO E PRORROGAÇÃO EM HORÁRIO DIURNO.(incorporada a Orientação Jurisprudencial nº 6 da SBDI-I) - Res. 129/2005, DJ 20, 22 e 25.04.2005
• I - O adicional noturno, pago com habitualidade, integra o salário do empregado para todos os efeitos. (ex-Súmula nº 60 - RA 105/1974, DJ 24.10.1974)
• II - Cumprida integralmente a jornada no período noturno e prorroga-da esta, devido é também o adicional quanto às horas prorrogadas. Exegese do art. 73, § 5º, da CLT. (ex-OJ nº 6 da SBDI-I - inserida em 25.11.1996)

• Precedente motivador da súmula:

ERR 113733/94/TST

• [...] A discussão dos presentes autos cinge-se à circunstância de que, tendo o trabalhador já cumprido toda a jornada em período noturno, e, prolongando tal jornada para além das 5 horas da manhã, ser-lhe-á devido o adicional noturno relativamente a estas horas além do marco das 5 horas.
• Ora, o adicional visa compensar o obreiro pelo sabido desgaste a que se sujeita quando labora em período noturno. Se assim é, com muito maior razão há de ser pago quando o laborista, já tendo cumprido uma jornada em período noturno, prorroga a prestação de serviços para além das cinco horas da manhã. Nesta hipótese, o desgaste do trabalhador é, sem dúvida, ainda maior. Não por outra razão que o art. 75, § 5º, da CLT dispõe: [...]

From this point, we can see that Dupin see this case from the other side. He did not take all the testimonies as the proof or observation but try to link them together to see the whole picture of the murderer.

this line can appear immediately after the previous sentence. Para break not needed.

this I don't quite understand

Router DR o BDR que es adyacente con algo diferente a DR o BDR

Art. 58. Nas incorporações em que a construção fôr contratada pelo regime de administração, também chamado "a preço de custo", será de responsabilidade dos proprietários ou adquirentes o pagamento do custo integral de obra, observadas as seguintes disposições:

• I - tôdas as faturas, duplicatas, recibos e quaisquer documentos referentes às transações ou aquisições para construção, serão emitidos em nome do condomínio dos contratantes da construção;

• II - tôdas as contribuições dos condôminos para qualquer fim relacionado com a construção serão depositadas em contas abertas em nome do condomínio dos contratantes em estabelecimentos bancários, as quais, serão movimentadas pela forma que fôr fixada no contrato.

Art. 55. Nas incorporações em que a construção seja feita pelo regime de empreitada, esta poderá ser a preço fixo, ou a preço reajustável por índices préviamente determinados.

• § 1º Na empreitada a preço fixo, o preço da construção será irreajustável, independentemente das variações que sofrer o custo efetivo das obras e qualquer que sejam suas causas.

• § 2º Na empreitada a preço reajustável, o preço fixado no contrato será reajustado na forma e nas épocas nêle expressamente previstas, em função da variação dos índices adotados, também previstos obrigatóriamente no contrato.

• § 3º Nos contratos de construção por empreitada, a Comissão de Representantes fiscalizará o andamento da obra e a obediência ao Projeto e às especificações exercendo as demais obrigações inerentes à sua função representativa dos contratantes e fiscalizadora da construção.

• § 4º Nos contratos de construção fixados sob regime de empreitada, reajustável, a Comissão de Representantes fiscalizará, também, o cálculo do reajustamento.

• § 5º No Contrato deverá ser mencionado o montante do orçamento atualizado da obra, calculado de acôrdo com as normas do inciso III, do art. 53, com base nos custos unitários referidos no art. 54, quando o preço estipulado fôr inferior ao mesmo.

• § 6º Na forma de expressa referência, os contratos de empreitada entendem-se como sendo a preço fixo.

Art. 44. Após a concessão do habite-se pela autoridade administrativa, incumbe ao incorporador a averbação da construção em correspondência às frações ideais discriminadas na matrícula do terreno, respondendo perante os adquirentes pelas perdas e danos que resultem da demora no cumprimento dessa obrigação. (Redação dada pela Lei nº 14.382, de 2022)

• § 1º Se o incorporador não requerer a averbação o construtor requerê-la-á sob pena de ficar solidàriamente responsável com o incorporador perante os adquirentes.

• § 2º Na omissão do incorporador e do construtor, a averbação poderá ser requerida por qualquer dos adquirentes de unidade.

• Art. 35. O incorporador terá o prazo máximo de 45 dias, a contar do têrmo final do prazo de carência, se houver, para promover a celebração do competente contrato relativo à fração ideal de terreno, e, bem assim, do contrato de construção e da Convenção do condomínio, de acôrdo com discriminação constante da alínea "i", do art. 32. (Vide Lei nº 4.864/65 que altera o prazo máximo concedido ao incorporador para 60 (sessenta) dias)

• § 4º Descumprida pelo incorporador e pelo mandante de que trata o § 1º do art. 31 a obrigação da outorga dos contratos referidos no caput dêste artigo, nos prazos ora fixados, a carta-proposta ou o documento de ajuste preliminar poderão ser averbados no Registro de Imóveis, averbação que conferirá direito real oponível a terceiros, com o conseqüente direito à obtenção compulsória do contrato correspondente.

• Art. 32. O incorporador somente poderá alienar ou onerar as frações ideais de terrenos e acessões que corresponderão às futuras unidades autônomas após o registro, no registro de imóveis competente, do memorial de incorporação composto pelos seguintes documentos:

• i) instrumento de divisão do terreno em frações ideais autônomas que contenham a sua discriminação e a descrição, a caracterização e a destinação das futuras unidades e partes comuns que a elas acederão;

• Art. 32. O incorporador somente poderá alienar ou onerar as frações ideais de terrenos e acessões que corresponderão às futuras unidades autônomas após o registro, no registro de imóveis competente, do memorial de incorporação composto pelos seguintes documentos: (Redação dada pela Lei nº 14.382, de 2022)

• a) título de propriedade de terreno, ou de promessa, irrevogável e irretratável, de compra e venda ou de cessão de direitos ou de permuta do qual conste cláusula de imissão na posse do imóvel, não haja estipulações impeditivas de sua alienação em frações ideais e inclua consentimento para demolição e construção, devidamente registrado;

Vide Código Civil:

• Art. 1.336.: São deveres do condômino: [...] § 1º O condômino que não pagar a sua contribuição ficará sujeito aos juros moratórios convencionados ou, não sendo previstos, os de um por cento ao mês e multa de até dois por cento sobre o débito.

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Reply to the reviewers

Point-by-point response to reviewers, including our plans for the revision:

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

* Summary: In this manuscript by the Sanson group, Lye and colleagues try to definitively answer the question of whether pulling forces from the ventral mesoderm have significant effects on convergent extension in the Drosophila germband (germband extension). While germband extension does occur in mutant embryos lacking mesoderm invagination, it has long been an open question in the field as to whether ventral pulling forces from the mesoderm have significant effects (positive or negative) on cell intercalation during germband extension. To definitely address this question, Lye and colleagues generated high-quality, directly comparable datasets from wild-type and twist mutant embryos, and then systematically assessed nearly all aspects of cell intercalation, myosin recruitment, and tissue elongation over time. They demonstrate that pulling forces from the ventral mesoderm have negligible impacts on the course of germband extension. While there are indeed some interesting differences between wild-type and twist embryos with respect to cell intercalation and myosin recruitment, such differences are relatively minor. They conclude that the events of germband extension neither require nor are strongly affected by external forces from the mesoderm. While this is largely a negative results paper, I believe that it should be published and that it will be an impactful paper within the field. Namely, it will settle once and for all the question of whether mesoderm invagination is required for optimal germband extension in the early Drosophila embryo, and it suggests that tissues are largely autonomous developmental units that are buffered from outside mechanical inputs.*

• * *Major comments: *

* It seems to me that the one obvious omission from this paper is a general measure of convergent extension over time. I think it would be useful to the reader to include some measure of change in tissue aspect ratio over time between wild-type and twist embryos. This could be included in Figure 5 or 6. *

• *

We are happy to include a graph with what we call “tissue strain rate”, which measures the deformation of the germ-band in the direction of extension (along AP) over time, and propose to add it as a panel in Supplementary Figure 6. Note that in our measures, the “tissue” strain rate is decomposed into contributions from two cell behaviors, the “cell intercalation” strain rate and the “cell shape” strain rate (Blanchard et al., 2009). “Tissue” and “cell shape” strain rate are directly measured, and “cell intercalation” strain rate is what remains when “cell shape” strain rate is removed from “tissue” strain rate. The “cell intercalation” strain rate calculated in that way is a “continuous” measure of cell intercalation, measuring the progressive shearing of cells during convergent extension. We also use a “discrete” measure of cell intercalation, which measures the number of cell neighbor exchanges, also called T1 swaps. We found that both “continuous” and “discrete” measures of cell intercalation are unchanged in twist mutant compared to wild-type embryos (Fig. 6F and 6E, respectively). In contrast, we find that the “cell shape” strain rate is increased in twist mutants (Fig. 5B and Fig. 5S1A). Consistent with this finding, the “tissue” strain rate is also increased in twist mutants (see graph below).

Otherwise, I have no major comments on the experimental approach or the findings of this manuscript. It seems to me a straightforward and systematic approach for determining whether mesoderm invagination affects germband extension. I do have several minor comments that should be addressed prior to publication (below).

*Minor comments: *

*I understand why cells would initially stretch more along the DV axis in wild-type embryos compared with twist embryos, but why do cells become so much more stretched along the AP axis (and become smaller apically) after 10 minutes of GBE in wild type compared with twist (Figure 2C and E). *

*I think this is an interesting and non-intuitive result that would warrant a bit of explanation/conjecture. *

This is not what Fig. 2C and E show, and we realize now that our schematics on the graphs might have been confusing. We will work on those to improve their clarity (or remove them), and also review our text.

Figure 2C shows how cells deform along DV (cell shape strain rate projected onto the DV axis). So the graph does not show that the cells are elongating in AP, as only the DV component of the strain rate is shown in this figure. In the wild type, the DV strain rate is positive (the cells are elongating in DV) at developmental times when the mesoderm invaginate (from about -10 minutes to until 7.5 minutes). The DV strain shows an acceleration until about 5 mins, then decelerates, crossing the x-axis to become negative at 7.5 minutes. From this timepoint and until the end of GBE, the DV strain rate is negative (the cells are contracting along DV). Mirroring the positive section of the curve, the DV contraction of the cells accelerate until about 12 mins and then slows down. The strong rate of DV contraction between 7.5 and 20 mins could in part be due to the endoderm invagination pulling in the orthogonal direction (AP) and helping the cells regaining a more isotropic shape. We could add a mention about this in the discussion.

In Figure 2E, the rate of change in cell area follows a similar time course in the wild type, showing that the cells are increasing their areas until about 10 mins (positive values) and then reduce their areas again until the end of GBE (negative values). Note that the graph does not show raw (instantaneous) cell areas as suggested by the comment, but rather a rate of change.

So in wild type, the cells get stretched by the invaginating mesoderm, and once the mesoderm is not pulling anymore, the cells appear to relax back. As there is no stretching in twist mutants, there is no equivalent relaxation of the cells along DV. Note that in twist, there is a milder increase in cell area in the first 15 mins of GBE (Fig. 2E). This could again be caused by the pull from endoderm invagination stretching the cells along AP, which, as we have shown before, increases both cell shape strain rates along AP and cell areas (Butler et al., 2009). So the pull from endoderm invagination (along AP) will have an impact on cell area rates of change and possibly also, indirectly, on DV cell shape strain rates, in both twist and wild type embryos, during most of GBE. Therefore cell area and DV cell shape strain rates are affected by more than one process during GBE. In this paper, we are focusing on the impact of mesoderm invagination, which happens around the start of GBE, so have focused our analysis of the graphs in the results section to this period, and the differences between wildtype and *twist. *

*I don't understand how you are defining cell orientation in Figure 2G. How are you choosing the cell axis that you are then comparing with the body axis? Is it the long axis, or something more complicated than that? I think you should briefly provide this information in the results section. If it is included in the methods, I wasn't able to locate it. *

Yes, it is the orientation of the long axis of the cell relative to the antero-posterior embryonic axis. We will clarify this in the text, in particular in the Methods, and also try improve our schematics.

Figure 2: Since you have the space, it might help the reader if you simply wrote out "strain rate" for panels B, D, and F, rather that used the abbreviation "SR." Thank you for this suggestion, we will reduce use of abbreviations where space permits.

*Please ensure that all axis labels are fully visible in the final figures. In several figures, the Y-axis labels were cut off (e.g., Fig 2I, 4A, 4D, 6B, 6C). *

These were visible to us in our submitted version, but of course we will ensure everything is visible on the final version.

*Where space permits, I would suggest using fewer abbreviations in axis labels to increase readability of the figures (e.g., in Figures 3H or 4D). *

Thank you for this suggestion, will do.

* In Figure 7, I would move the wild-type panels to the left and the twist panels to the right. I think it is more conventional to describe the normal wild-type scenarios first, and then contrast the mutant state.*

Will do.

To be consistent with the literature, "wildtype" should be hyphenated (wild-type) when used as an adjective, or two separate words (wild type) when used as a noun. Thank you, we will change this.

Review*er #1 (Significance (Required)): *

* Advance: The advances in this manuscript are largely methodological, but the experiments and analyses are quite rigorous and allow the authors to make strong conclusions concerning their hypotheses. Their findings are based on a high-quality collection of movies from control and twist mutant embryos expressing a cell membrane marker and knock-in GFP-tagged myosin. Importantly, I think the researchers were correct in choosing to analyze twist single-mutant embryos (as opposed to snail or twist, snail double-mutant embryos), as the overall embryo geometry of these mutants is fairly similar to wild-type embryos, allowing the researchers to directly compare cell behaviors and myosin dynamics during germband extension. This approach also allows them to avoid indirect effects on the germband due to a completely non-internalized mesoderm. *

*

Audience: The primary audience for this article will be basic science researchers working in the early Drosophila embryo who are interested in the interplay between the germband and neighboring tissues. Secondary audiences will include developmental biologists more broadly who are interested in biomechanical coupling (or in this case decoupling) of neighboring tissues. *

*

Describe your expertise: I have been a Drosophila developmental geneticist for over twenty years, and I have been working directly on Drosophila germband extension for over a decade. I have published numerous papers and reviews in this field, and I am very familiar with the genetic backgrounds and types of experimental analyses used in this manuscript. Therefore, I believe I am highly qualified to serve as a reviewer for this manuscript.*

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

*

In the present manuscript, Lye et al. describe a highly detailed quantification of cell shape changes during germband extension in Drosophila melanogaster early embryo. During this process, ectodermal tissue contracts along the dorso-ventral axis, simultaneously expanding along the perpendicular antero-posterior direction, migrating from the ventral to the dorsal surface of the embryo as it extends. This important morphogenetic event is preceded by ventral furrow formation when mesodermal tissue (located in the ventral part of the embryo) contracts along the dorso-ventral axis and invaginates into the embryonic interior. The study compares cell shape dynamics in the wildtype Drosophila with that in the twist mutant, which largely lacks mesoderm and does not form ventral furrow. The major motivation of the study is to examine whether cellular behaviors and myosin recruitment in the ectoderm is cell autonomous, or if those cellular behaviors depend on mechanical interactions between mesoderm and ectoderm.*

• The authors first examine whether transcriptional patterning of key genes involved in germband extension is different between the wildtype and the twist mutant and find no significant difference. Next, the authors thoroughly quantify cellular behaviors and patterns of myosin recruitment in the two genetic backgrounds. A number of different measures are investigated, notably the rate of change in the degree of cellular asymmetry, rate of cell area change, rate of change of cell orientation, differences in myosin recruitment to cell edges of various orientation, as well as the rates of growth, shrinkage, and re-orientation of the various cellular interfaces. It is thoroughly documented how these quantities change as a function of developmental timing and spatial position within the embryo. These data serve basis for quantitative comparison between cellular dynamics in the two genetic backgrounds considered.*

• Overall, the study shows that cellular behaviors observed in the ectoderm are largely the same during the period of time following ventral furrow formation, as would be expected if those cellular behaviors were predominantly cell autonomous and not dependent on stresses generated in the mesoderm.*

• The data presented in the manuscript are of excellent quality and presentation is very clear.

Minor comments: none *

* Reviewer #2 (Significance (Required)): *

* I find that the study provides a thorough quantification of cell behaviors in a widely studied important model of morphogenesis. The work may be of particular interest for future model-to-data comparison, perhaps providing a basis for future modeling work. I therefore certainly think that this work warrants publication.*

• However, the results of the study largely parallel previous findings and do not appear novel or surprising. It is well established that in snail mutant that lack mesoderm entirely, germband extension proceeds largely normally. This well-established fact suggests that since tissue dynamics in complete absence of mesoderm are largely unaffected, behaviors of individual cells are likely to not be affected either*.

*The work is pretty much entirely observational, and for most part provides a more detailed documentation/quantification of previous findings. I do not think it is appropriate for high profile publication. *

We are not sure which evidence the reviewer is referring to here specifically. We agree that the single mutants twist or snail, or the double twist snail mutants do extend their germ-band. However, the question we are asking here, is how well do they extend their germband and to answer this question, quantitation is needed. The first quantitation of GBE were performed by (Irvine and Wieschaus, 1994). While they quantified GBE in various mutant contexts, they did not perform quantitation for snail, twist, or twist snail mutants. Instead, they refer to these mutants once in p839, with the following sentence: ”Additionally, twist and snail mutant embryos, which lack mesoderm, extend their germbands almost normally (Leptin and Grunewald, 1990; Simpson, 1983)*.” *

Following these earlier qualitative observations, various studies have quantified different aspects of GBE in mesoderm invagination mutants, with contradictory results. For example, some studies, including from our own lab, report a reduction in cell intercalation in the absence of mesoderm invagination (Butler et al., 2009; Wang et al., 2020), but there have also been reports that tissue extension and T1-transistions occur normally (Farrell et al., 2017)(see also introduction of our manuscript). These contradictory results have motivated our present study, and we have implemented rigorous comparison between wild type and mesoderm invagination mutants, being careful i) to check that the regions analyzed were comparable in terms of cell fate, and ii) to control for any confounding effects between experiments (see also response to reviewer 4, main question 2). We have also considered which mesoderm invagination mutants to use. We rejected snail or twist snail mutants because the absence of snail means that the mesodermal cells do not contract and thus stay at the surface of the embryo, which changes the spatial configuration of the embryo considerably and would make a fair quantitative comparison very difficult. Instead, we decided to use twist mutants, as in those, cell contractions still happen so the cells do not take as much space at the surface of the embryo, but the contractions are uncoordinated which means that there is no invagination (and we demonstrate here, no significant pulling on the ectoderm). We note that reviewer 1 highlights the merit of settling the question of the impact of mesoderm invagination on GBE and the pertinence of choosing twist mutants versus the alternatives (see also response to reviewer 4, suggestion 1).