Aposentadoria especial de guardas municipais – ADPF 1.095/DF - Relatores: Ministro Gilmar Mendes - DIREITO ADMINISTRATIVO SERVIDOR PÚBLICO; GUARDA MUNICIPAL; APOSENTADORIA ESPECIAL - RESUMO: As guardas municipais fazem parte do Sistema Único de Segurança Pública (SUSP), mas não possuem direito à aposentadoria especial, visto que o rol constitucional de categorias com direito a esse benefício é taxativo e não as contempla.

Esta Corte, embora tenha reconhecido que as guardas municipais fazem parte do SUSP, não lhes conferiu integral isonomia com os demais órgãos de segurança pública, na medida em que há peculiaridades relevantes quanto ao regime jurídico desses órgãos (1).

A EC nº 103/2019, por sua vez, estabeleceu rol taxativo das categorias em que se pode instituir idade e tempo de contribuição diferenciados mediante lei complementar (2). Como as guardas municipais não f iguram de modo expresso nessa listagem, os respectivos entes federados ficam impedidos de conceder aposentadoria especial para essas carreiras (3).

Também é inaplicável a regra de aposentadoria especial do art. 40, § 4º-C do texto constitucional (4). Isso porque não se admite presunção de exposição a agentes nocivos à saúde pelo mero enquadramento profissional ou ocupacional. Ao contrário, é indispensável que se comprove a efetiva exposição a agentes químicos, físicos e/ou biológicos (5).

Por fim, eventual concessão da aposentadoria especial às guardas municipais sem a elaboração de plano próprio que contenha a devida indicação de fonte de custeio e de medidas compensatórias configura desobediência ao art. 195, § 5º da CF/1988 (6). Com base nesses e em outros entendimentos, o Plenário, por maioria, julgou improcedente a arguição.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

The investigators undertook detailed characterization of a previously proposed membrane targeting sequence (MTS), a short N-terminal peptide, of the bactofilin BacA in Caulobacter crescentus. Using light microscopy, single molecule tracking, liposome binding assays, and molecular dynamics simulations, they provide data to suggest that this sequence indeed does function in membrane targeting and further conclude that membrane targeting is required for polymerization. While the membrane association data are reasonably convincing, there are no direct assays to assess polymerization and some assays used lack proper controls as detailed below. Since the MTS isn't required for bactofilin polymerization in other bacterial homologues, showing that membrane binding facilitates polymerization would be a significant advance for the field.

We agree that additional experiments were required to consolidate our results and conclusions. Please see below for a description of the new data included in the revised version of the manuscript.

Major concerns

(1) This work claims that the N-termina MTS domain of BacA is required for polymerization, but they do not provide sufficient evidence that the ∆2-8 mutant or any of the other MTS variants actually do not polymerize (or form higher order structures). Bactofilins are known to form filaments, bundles of filaments, and lattice sheets in vitro and bundles of filaments have been observed in cells. Whether puncta or diffuse labeling represents different polymerized states or filaments vs. monomers has not been established. Microscopy shows mis-localization away from the stalk, but resolution is limited. Further experiments using higher resolution microscopy and TEM of purified protein would prove that the MTS is required for polymerization.

We do not propose that the MTS is directly involved in the polymerization process and state this more clearly now in the Results and Discussion sections of the revised manuscript. To address this point, we performed transmission electron microscopy studies comparing the polymerization behavior of wild-type and mutant BacA variants. The results clearly show that the MTS-free BacA variant (∆2-8) forms polymers that are indistinguishable from those formed by the wild-type protein, when purified from an E. coli overproduction strain (new Figure 1–figure supplement 1). This finding is consistent with structural work showing that bactofilin polymerization is exclusively mediated by the conserved bactofilin domain (Deng et al, Nat Microbiol, 2019). However, at native expression levels, BacA only accumulates to ~200 molecules per cell (Kühn et al, EMBO J, 2006). Under these conditions, the MTS-mediated increase in the local concentration of BacA at the membrane surface and, potentially, steric constraints imposed by membrane curvature, may facilitate the polymerization process. This hypothesis has now been stated more clearly in the Results and Discussion sections.

For polymer-forming proteins, defined localized signals are typically interpreted as slow-moving or stationary polymeric complexes. A diffuse localization, by contrast, suggests that a protein exists in a monomeric or, at most, (small) oligomeric state in which it diffuses rapidly within the cell and is thus no longer detected as distinct foci by widefield microscopy. Our single-molecule data show that BacA variants that are no longer able to interact with the membrane (as verified by cell fractionation studies and in vitro liposome binding assays) have a high diffusion rate, similar to that measured for the non-polymerizing and non-membrane-bound F130R variant. These results demonstrate that a defect in membrane binding strongly reduces the ability of BacA to form polymeric assemblies. To support this hypothesis, we have now repeated all single-particle tracking experiments and included mVenus as a freely diffusible reference protein. Our data confirm that the mobilities of the ∆2-8 and F130R variants are similar and approach those of free mVenus, supporting the idea that the deficiency to interact with the membrane prevents the formation of extended polymeric structures (which should show much lower mobilities). To underscore the relevance of membrane binding for BacA assembly, we have now included a new experiment, in which we used the PbpC membrane anchor (PbpC_1-132-mcherry) to restore the recruitment of the ∆2-8 variant to the membrane (Figure 9 and Figure 9–figure supplement 1). The results obtained show that the ∆2-8 variant transitions from a diffuse localization to polar foci upon overproduction of PbpC_1-132-mcherry. The polymerization-impaired F130R variant, by contrast, remains evenly distributed throughout the cytoplasm under all conditions. These findings further support the idea that polymerization and membrane-association are mutually interdependent processes.

(2) Liposome binding data would be strengthened with TEM images to show BacA binding to liposomes. From this experiment, gross polymerization structures of MTS variants could also be characterized.

We do not have the possibility to perform cryo-electron microscopy studies of liposomes bound to BacA. However, the results of the cell fractionation and liposome sedimentation assays clearly support a critical role of the MTS in membrane binding.

(3) The use of the BacA F130R mutant throughout the study to probe the effect of polymerization on membrane binding is concerning as there is no evidence showing that this variant cannot polymerize. Looking through the papers the authors referenced, there was no evidence of an identical mutation in BacA that was shown to be depolymerized or any discussion in this study of how the F130R mutation might to analogous to polymerization-deficient variants in other bactofilins mentioned in these references.

Residue F130 in the C-terminal polymerization interface of BacA is conserved among bactofilin homologs, although its absolute position in the protein sequence may vary, depending on the length of the N-terminal unstructured tail. The papers cited in our manuscript show that an exchange of this conserved phenylalanine residue abolishes polymer formation. Nevertheless, we agree that it is important to verify the polymerization defect of the F130R variant in the system under study. We have now included size-exclusion chromatography data showing that BacA-F130R forms a low-molecular-weight complex, whereas the wild-type protein largely elutes in the exclusion volume, indicating the formation of large, polymeric species (new Figure 1–figure supplement 1). In addition, we performed transmission electron microscopy analyses of BacA-F130R, which verified the absence of larger oligomers (new Figure 1–figure supplement 2).

(4) Microscopy shows that a BacA variant lacking the native MTS regains the ability to form puncta, albeit mis-localized, in the cell when fused to a heterologous MTS from MreB. While this swap suggests a link between puncta formation and membrane binding the relationship between puncta and polymerization has not been established (see comment 1).

We show that a BacA variant lacking the MTS (∆2-8) regains the ability to form membrane-associated foci when fused to the MTS of MreB. By contrast, a similar variant that additionally carries the F130R exchange (preventing its polymerization) shows a diffuse cytoplasmic localization. In addition, we show that the F130R exchange leads to a loss of membrane binding and to a considerable increase in the mobility of the variants carrying the MTS of E. coli MreB. As described above, we now provide additional data demonstrating that elevated levels of the PbpC membrane anchor can reinstate polar localization for the ∆2-8 variant, whereas it fails to do so for the polymerization-deficient F130R variant (Figure 9 and Figure 9–figure supplement 1). Together, these results support the hypothesis that membrane association and polymerization act synergistically to establish localized bactofilin assemblies at the stalked cell pole.

(5) The authors provide no primary data for single molecule tracking. There is no tracking mapped onto microscopy images to show membrane localization or lack of localization in MTS deletion/ variants. A known soluble protein (e.g. unfused mVenus) and a known membrane bound protein would serve as valuable controls to interpret the data presented. It also is unclear why the authors chose to report molecular dynamics as mean squared displacement rather than mean squared displacement per unit time, and the number of localizations is not indicated. Extrapolating from the graph in figure 4 D for example, it looks like WT BacA-mVenus would have a mobility of 0.5 (0.02/0.04) micrometers squared per second which is approaching diffusive behavior. Further justification/details of their analysis method is needed. It's also not clear how one should interpret the finding that several of the double point mutants show higher displacement than deleting the entire MTS. These experiments as they stand don't account for any other cause of molecular behavior change and assume that a decrease in movement is synonymous with membrane binding.

We now provide additional information on the single-particle analysis. A new supplemental figure now shows a mapping of single-particle tracks onto the cells in which they were recorded for all proteins analyzed (Figure 2–figure supplement 1). Due to the small size of C. crescentus, it is difficult to clearly differentiate between membrane-associated and cytoplasmic protein species. However, overall, slow-diffusing particles tend to be localized to the cell periphery, supporting the idea that membrane-associated particles form larger assemblies (apart from diffusing more slowly due to their membrane association). In addition, we have included a movie that shows the single-particle diffusion dynamics of all proteins in representative cells (Figure 2-video 1). Finally, we have included a table that gives an overview of the number of cells and tracks analyzed for all proteins investigated (Supplementary file 1). Figure 2A and 4D show the mean squared displacement as a function of time, which makes it possible to assess whether the particles observed move by normal, Brownian diffusion (which is the case here). We repeated the entire single-particle tracking analysis to verify the data obtained previously and obtained very similar results. Among the different mutant proteins, only the K4E-K7E variant consistently shows a higher mobility than the MTS-free ∆2-8 variant, with MSD values similar to that of free mVenus. The underlying reason remains unclear. However, we believe that an in-depth analysis of this phenomenon is beyond the scope of this paper. We re-confirmed the integrity of the construct encoding the K4E/K7E variant by DNA sequencing and once again verified the size and stability of the fusion protein by Western blot analysis, excluding artifacts due to errors during cloning and strain construction.

We agree that the single-molecule tracking data alone are certainly not sufficient to draw firm conclusions on the relationship between membrane binding and protein mobility. However, they are consistent with the results of our other in vivo and in vitro analyses, which together indicate a clear correlation between the mobility of BacA and its ability to interact with the membrane and polymerize (processes that promote each other synergistically).

(6) The experiments that map the interaction surface between the N-terminal unstructured region of PbpC and a specific part of the BacA bactofilin domain seem distinct from the main focus of the paper and the data somewhat preliminary. While the PbpC side has been probed by orthogonal approaches (mutation with localization in cells and affinity in vitro), the BacA region side has only been suggested by the deuterium exchange experiment and needs some kind of validation.

The results of the HDX analysis per se are not preliminary and clearly show a change in the solvent accessibility of backbone amides in the C-terminal region in the bactofilin domain in the presence of the PbpC_1-13 peptide. However, we agree that additional experiments would be required to verify the binding site suggested by these data. We agree that further research is required to precisely map and verify the PbpC binding site. However, as this is not the main focus of the paper, we would like to proceed without conducting further experiments in this area.

We now provide additional data showing that elevated levels of the PbpC membrane anchor are able to recruit the MTS-free BacA variant (∆2-8) to the cytoplasmic membrane and stimulate its assembly at the stalked pole (Figure 9). These results now integrate Figure 8 more effectively into the overall theme of the paper.

Reviewer #2 (Public review):

Summary:

The authors of this study investigated the membrane-binding properties of bactofilin A from Caulobacter crescentus, a classic model organism for bacterial cell biology. BacA was the progenitor of a family of cytoskeletal proteins that have been identified as ubiquitous structural components in bacteria, performing a range of cell biological functions. Association with the cell membrane is a common property of the bactofilins studied and is thought to be important for functionality. However, almost all bactofilins lack a transmembrane domain. While membrane association has been attributed to the unstructured N-terminus, experimental evidence had yet to be provided. As a result, the mode of membrane association and the underlying molecular mechanics remained elusive.

Liu at al. analyze the membrane binding properties of BacA in detail and scrutinize molecular interactions using in-vivo, in-vitro and in-silico techniques. They show that few N-terminal amino acids are important for membrane association or proper localization and suggest that membrane association promotes polymerization. Bioinformatic analyses revealed conserved lineage-specific N-terminal motifs indicating a conserved role in protein localization. Using HDX analysis they also identify a potential interaction site with PbpC, a morphogenic cell wall synthase implicated in Caulobacter stalk synthesis. Complementary, they pinpoint the bactofilin-interacting region within the PbpC C-terminus, known to interact with bactofilin. They further show that BacA localization is independent of PbpC.

Strengths:

These data significantly advance the understanding of the membrane binding determinants of bactofilins and thus their function at the molecular level. The major strength of the comprehensive study is the combination of complementary in vivo, in vitro and bioinformatic/simulation approaches, the results of which are consistent.

Thank you for this positive feedback.

Weaknesses:

The results are limited to protein localization and interaction, as there is no data on phenotypic effects. Therefore, the cell biological significance remains somewhat underrepresented.

We agree that it is interesting to investigate the phenotypic effects caused by the reduced membrane binding activity of BacA variants with defects in the MTS. We have now included phenotypic analyses that shed light on the role of region C1 in the localization of PbpC and its function in stalk elongation under phosphate-limiting conditions (see below).

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

To address the missing estimation of biological relevance, some additional experiments may be carried out.

For example, given that BacA localizes PbpC by direct interaction, one might expect an effect on stalk formation if BacA is unable to bind the membrane or to polymerize. The same applies to PbpC variants lacking the C1 region. As the mutant strains are available, these data are not difficult to obtain but would help to compare the effect of the deletions with previous data (e.g. Kühn et al.) even if the differences are small.

We have now analyzed the effect of the removal of region C1 on the ability of mVenus-PbpC to promote stalk elongation in C. crescentus under phosphate starvation. Interestingly, our results show that the lack of the BacA-interaction motif impairs the recruitment of the fusion protein to the stalked pole, but it does not interfere with its stimulatory effect on stalk biogenesis. Thus, the polar localization of PbpC does not appear to be critical for its function in localized peptidoglycan synthesis at the stalk base. These results are now shown in Figure 8–Figure supplement 4. The results obtained may be explained by residual transient interactions of mVenus-PbpC with proteins other than BacA at the stalked pole. Notably, PbpC has also been implicated in the attachment of the stalk-specific protein StpX to components of the outer membrane at the stalk base. The polar localization of PbpC may therefore be primarily required to ensure proper StpX localization, consistent with previous work by Hughes et al. (Mol Microbiol, 2013) showing that StpX is partially mislocalized in a strain producing an N-terminally truncated PbpC variant that no longer localizes to the stalk base.

We have also attempted to investigate the ability of the Δ2-8 and F130R variants of BacA-mVenus to promote stalk elongation under phosphate starvation. However, the levels of the WT, Δ2-8 and F130R proteins and their stabilities were dramatically different after prolonged incubation of the cells in phosphate-limited medium, so that it was not possible to draw any firm conclusions from the results obtained (not shown).

In addition, the M23-like endopeptidase LdpA is proposed to be a client protein of BacA (in C. crescentus, Billini et al. 2018, and H. neptunium or R. rubrum, Pöhl et al. 2024). In H. neptunium, it is suggested that the interaction is mediated by a cytoplasmic peptide of LmdC reminiscent of PbpC. This should at least be commented on. It would be interesting to see, if LpdA in C. crescentus is also delocalized and if so, this could identify another client protein of BacA.

We agree that it would be interesting to study the role of BacA in LdpA function. However, we have not yet succeeded in generating a stable fluorescent protein fusion to LdpA, which currently makes it impossible to study the interplay between these two proteins in vivo. The focus of the present paper is on the mode of interaction between bactofilins and the cytoplasmic membrane and on the mutual interdependence of membrane binding and bactofilin polymerization. Given that PbpC is so far the only verified interaction partner of BacA in C. crescentus, we would like to limit our analysis to this client protein.

Further comments:

L105: analyze --> analyzed

Done.

L169: Is there any reason why the MTS of E. coli MreB was doubled?

Previous work has shown that two tandem copies of the N-terminal amphiphilic helix of E. coli MreB were required to partially target a heterologous fusion partner protein (GFP) to the cytoplasmic membrane of E. coli cells (Salje et al, 2011).

Fig. S3:

a) Please decide which tag was used (mNG or mVenus) and adapt the figure or legend accordingly.<br /> b) In the legend for panel (C), please describe how the relative amounts were calculated, as the fractions arithmetically cannot add to > 100%. I guess each band was densiometrically rated and independently normalized to the whole-cell signal?

The fluorescent tag used was mNeonGreen, as indicated in the figure. We have now corrected the legend accordingly. Thank you for making us aware of the wrong labeling of the y-axis. We have now corrected the figure and describe the method used to calculate the plotted values in the legend.

Legend of Fig 1b: It is not clear to me, to which part of panel B the somewhat cryptic LY... strain names belong. I suggest putting them either next to the images, to delete them, or at least to unify the layout (compare, e.g. to Fig S7). (I would delete the LY numbers and stay with the genes/mutations throughout. This is just a suggestion).

These names indicate the strains analyzed in panel B, and we have now clarified this in the legend. It is more straightforward to label the images according to the mutations carried by the different strains. Nevertheless, we would like to keep the strain names in the legend, so that the material used for the analysis can be clearly identified.

Fig. 2a: As some of the colors are difficult to distinguish, I suggest sorting the names in the legend within the graph according to the slope of the curves (e.g. K4E K7E (?) on top and WT being at the bottom).

Thank you for this suggestion. We have now rearranged the labels as proposed.

In the legend (L924), correct typo "panel C" to "panel B".

Done.

Fig. 3: In the legend, I suggest deleting the abbreviations "S" and "P" as they do not show up in the image. In line 929, I suggest adding: average "relative" amount... or even more precisely: "average relative signal intensities obtained..."

We have removed the abbreviations and now state that the bars indicate the “average relative signal intensities” obtained for the different fractions.

Fig 4d: same suggestion as for Fig. 2a.

Done.

Fig 8: In the legend (L978), delete 1x "the"

Done.

L258 and Fig. S5: The expression "To account for biases in the coverage of bacterial species" seems somewhat unclear. I suggest rephrasing and adding information from the M+M section here (e.g. from L593, if this is meant).

We now state that this step in the analysis pipeline was performed “To avoid biases arising from the over-representation of certain bacterial species in UniProt”.

I appreciate the outline of the workflow in panel (a) of Fig. S5. It would be even more useful when some more details about the applied criteria for filtering would be provided (e.g. concerning what is meant with "detailed taxonomic information" or "filter out closely related sequences". Does the latter mean that only one bactofilin sequence per species was used? (As quite many bacteria have more than one but similar bactofilins.)

We removed sequences from species with unclear phylogeny (e.g. candidate species whose precise taxonomic position has not yet been determined). For many pathogenic species, numerous strains have been sequenced. To account for this bias, only one sequence from clusters of highly similar bactofilin sequences (>90% identity) was retained per species. This information has now been included in the diagram. It is true that many bacteria have more than one bactofilin homolog. However, the sequences of these proteins are typically quite different. For instance, the BacA and BacB from C. crescentus only share 52% identity. Therefore, our analysis does not systematically eliminate bactofilin paralogs that coexist in the same species.

L281: Although likely, I am not sure if membrane binding has ever been shown for a bactofilin from these phyla. (See also L 380.) Is there an example? Otherwise, membrane binding may not be a property of these bactofilins.

To our knowledge, the ability of bactofilins from these clades to interact with membranes has not been investigated to date. We agree that the absence of an MTS-like motif may indicate that they lack membrane binding activity, and we have now stated this possibility in the Results and Discussion.

L285: See comment above concerning the M23-like peptidase LpdA. Although not yet directly shown for C. crescentus, it seems likely that BacACc does also localize this peptidase in addition to PbpC. I suggest rephrasing, e.g. "known" --> "shown"

We now use the word “reported”.

L295 and Fig S8: PbpC is ubiquitous. Which criteria/filters have been applied to select the shown sequences?

C. crescentus PbpC is different from E. coli Pbp1C. It is characterized by distinctive, conserved N- and C-terminal tails and only found in C. crescentus and close relatives. The C. crescentus homolog of E. coli PbpC is called PbpZ (Yakhnina et al, J Bacteriol, 2013; Strobel et al, J Bacterol, 2014), whereas C. crescentus PbpC is related to E. coli PBP1A. We have now added this information to the text to avoid confusion.

L311: may replace "assembly" by "polymerization"

Done.

L320: bactofilin --> bactofilin domain?

Yes, this was supposed to read “bactofilin domain”. Thank you for spotting this issue.

L324: The HDX analysis of BacA suggests that the exchange is slowed down in the presence of the PbpC peptide, which is indicative of a physical interaction between these two molecules. To corroborate the claim that BacA polymerization is critical for interaction with the peptide (resp. PbpC), this experiment should be carried out with the polymerization defective BacA version F130R.

(Or tone this statement down, e.g. show --> suggest.)

“suggest”

L386: undergoes --> undergo

Done.

L391-400: This idea is tempting but the suggested mechanism then would be restricted to bactofilins of C. crescentus and close relatives. The bactofilin of Rhodomicrobium, for example, was shown to localize dynamically and not to stick to a positively curved membrane.

In the vast majority of species investigated so far, bactofilins were found to associate with specifically curved membrane regions and to contribute to the establishment of membrane curvature. Unfortu­nately, the sequences of the three co-polymerizing bactofilin paralogs of R. vannielii DSM 166 studied by Richter et al (2023) have not been reported and the genome sequence of this strain is not publicly available. However, in related species with three bactofilin paralogs, only one paralog shows an MTS-like N-terminal peptide and another paralog typically contains an unusual cadherin-like domain of unknown function, as also reported for R. vannielii DSM 166. Therefore, the mechanism controlling the localization dynamics of bactofilins may be complex in the Rhodomicrobium lineage. Nevertheless, at native expression levels, the major bactofilin (BacA) of R. vannielii DSM 166 was shown to localize predominantly to the hyphal tips and the (incipient) bud necks, suggesting that regions of distinct membrane curvature could also play a role in its recruitment. We do not claim that all bactofilins recognize positive membrane curvature, which is clearly not the case. It rather appears as though the curvature preference of bactofilins varies depending on their specific function.

L405-406: I agree that localization of BacA has been shown to be independent of PbpC. However, this does not generally preclude an effect on BacA localization by other "client" or interacting proteins. (See also comment above about the putative BacA interactor LpdA). I suggest either to corroborate or to change this statement from "client binding" to "PbpC binding".

Thank you for pointing out the imprecision of this statement. We now conclude that “PbpC binding” is not critical for BacA assembly and positioning.

Suppl. Fig. S11: In the legend, please correct the copy-paste mismatch (...VirB...).

Done.

L482: delete 1x "at"

Done.

L484: may be better "soluble and insoluble fractions"?

We now describe the two fractions as “soluble and membrane-containing insoluble fractions” to make clear to all readers that membrane vesicles are found in the pellet after ultracentrifugation.

L489-490: check spelling immunoglobulin – immuneglobulin

Done.

L500 and 504: º_C --> ºC

Done.

Suppl. file X (HDX data): please check the table headline, table should be included in Suppl. file 1

We have now included a headline in this file (now Supplementary file 3).

Work in progress August 2025

El plasma (la parte líquida de la sangre) y el líquido intersticial (el que rodea las células) tienen composiciones muy parecidas, pero el plasma contiene más proteínas, especialmente albúmina, que no pueden atravesar fácilmente las paredes capilares. Estas proteínas, con carga negativa, atraen iones positivos (cationes) hacia el plasma, fenómeno conocido como equilibrio de Gibbs-Donnan, lo que explica que el plasma tenga una concentración catiónica ligeramente mayor que el líquido intersticial. Además, estas proteínas aumentan la osmolalidad del plasma y generan una presión oncótica que “jala” agua hacia el interior de los capilares, equilibrando la presión hidrostática que empuja el líquido hacia los tejidos. Este balance mantiene estable el intercambio de líquidos entre los vasos sanguíneos y los tejidos; cuando se rompe —por ejemplo, si disminuyen las proteínas plasmáticas—, el líquido se acumula en el espacio intersticial y aparece edema o hinchazón.

• This reinforces the central tragic theme of the entire play—It is a cosmic casualty. All disaster is referred to the divine hand of the gods bringing about judgement upon the pride of the people. The belief are purely Greek in their expressions, especially in how they understood pride to be dealt with the most shameful judgement.

• The hyperbolic opening to set the scene of the destress and mood.

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

Learn more at Review Commons

Reply to the reviewers

We are very grateful for the positive feedback from all three reviewers. Below, we address each point in detail and outline proposed experiments and revision plans, with changes indicated by an underscore.

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

In this paper "Magnesium depletion unleashes two unusual modes of colistin resistance with different fitness costs," the authors examine how Pseudomonas aeruginosa evolves resistance to colistin, a last-resort antibiotic for multidrug-resistant Gram-negative infections. Although colistin resistance is a major clinical challenge, its underlying mechanisms, particularly under nutrient-limited conditions typical of infections, are not fully understood. The study shows that under low magnesium (Mg²_⁺_) conditions-mimicking infection or biofilm stress-P. aeruginosa can develop colistin resistance via two distinct genetic pathways, each with unique fitness costs. The first involves mutations in genes such as htrB2 and lpxO2, granting strong resistance but compromising the outer membrane and increasing susceptibility to other antibiotics. The second involves regulatory mutations (e.g., in the oprH/phoP/phoQ promoter) that confer resistance with minimal membrane defects and generally lower fitness costs. These resistance strategies lead to different trade-offs: membrane-compromising mutations reduce bacterial fitness without colistin, while regulatory mutations typically avoid these penalties, with context-dependent effects. The study underscores clinical relevance, noting that in infections-such as in cystic fibrosis-other microbes like Candida albicans may deplete magnesium, indirectly promoting resistance evolution. Overall, this work offers important insights into antibiotic resistance in nutrient-stressed, polymicrobial environments, highlighting how magnesium availability shapes resistance evolution and fitness costs. The findings suggest new avenues for therapeutic intervention and call for a reevaluation of antibiotic strategies in nutrient-competitive infection settings.

Work is timely and important. Colistin resistance represents an urgent threat as colistin is a last-resort antibiotic used against multidrug-resistant Gram-negative pathogens. Insights into mechanisms evolving under nutrient limitation are highly relevant given the prevalence of such environmental conditions during infection and microbial biofilm growth. The study reveals two previously uncharacterized pathways to colistin resistance in P. aeruginosa triggered by magnesium (Mg²_⁺_) depletion, each with distinct genetic signatures and trade-offs. This finding directly impacts the understanding of polymicrobial infection dynamics, especially where magnesium sequestration by fungi/ or other microbes may occur. The identification of fitness costs and pleiotropic effects associated with specific resistance mutations provides crucial guidance for clinicians considering antibiotic stewardship and combination therapy strategies.

__

We thank the reviewer for their summary of our study and its potential impact.

__Drawbacks

• Experimental scope: While the study is comprehensive for P. aeruginosa, the broader applicability to other Gram-negative pathogens is not directly tested.__

In our revision, we now explicitly point out that the magnesium limitation we have observed broadly applies to Gram-negative bacteria, as we demonstrated in our previous PLOS Biology paper. Therefore, we expect the same themes (and even genes, which are broadly conserved) to apply to Gram-negative bacteria in general. However, a full-fledged experimental study of other Gram-negative pathogens is outside the scope of our current study, which required a 90-day experimental evolution.

__Strengths

• Experimental evolution: This work uses laboratory evolution under controlled Mg²_⁺_-limited conditions to simulate selection pressures relevant to infection microenvironments. • Genetics: Systematic identification and functional validation of key mutations-particularly in htrB2, lpxO2, and the oprH/phoP/phoQ promoter-give mechanistic depth to the findings. • Two distinct resistance modes: Evidence for (i) one pathway leading to colistin resistance via htrB2 mutations, resulting in high resistance but significant membrane integrity loss and increased susceptibility to other antibiotics. (ii) a second pathway providing resistance without compromising membrane integrity, highlighting evolutionary flexibility and ecological implications. • Fitness assessments: measurement of the costs associated with each resistance strategy, both in terms of membrane integrity and susceptibility to other agents. • Relevance: Connection to natural scenarios, such as magnesium sequestration by fungi (e.g. Candida albicans) in polymicrobial environments, underscores the ecological and clinical significance. • This manuscript is well written with clearly logical hypothesis testing__

We thank the reviewer for their appraisal, especially for recognizing the rigor and broader biological implications of our study.

__Drawbacks

• Experimental scope: While the study is comprehensive for P. aeruginosa, the broader applicability to other Gram-negative pathogens is not directly tested.__

We agree with the reviewer's point about broader applicability in other Gram-negative bacteria, as many of the lipid A biosynthesis genes are conserved among diverse bacterial lineages. We will include this point in our revised Discussion to suggest relevance to other Gram-negative bacteria:

"We previously showed that magnesium sequestration by fungi applies not only to P. aeruginosa but to other Gram-negative bacteria as well (ref). Our current study lays a foundation for developing evolution-guided strategies to combat multidrug-resistant P. aeruginosa and other Gram-negative bacteria that can also acquire colistin resistance. Since many other antibiotic mechanisms are similarly dependent on metal ions (refs), our work suggests that nutritional competition for metal ions may alter initial antibiotic resistance in Gram-negative bacteria and potentiate new evolutionary pathways of antibiotic resistance."

• __ Mechanistic depth: Some inferred mechanisms (e.g., the precise molecular impact of late-occurring adaptive mutations) merit deeper biochemical analysis.__ We will emphasize in our Revision that the MS data of endpoint clones and triple mutants reveal that their lipid A structures are identical. This suggests that the role of other late-occurring mutations in enhancing resistance is likely through lipid A-independent pathways.

• __ Results Lines 414- 423: While correlation is most what makes sense for some drugs, causality is implied (membrane defects increase susceptibility), but could be strengthened by directly measuring antibiotic uptake (e.g., fluorescence) or membrane permeability for these 3 antibiotics.__ We thank the reviewer for highlighting the issue of causality. For the three antibiotics tested, the most direct way to measure their effect is by measuring their impact on bacterial growth directly, which is what we have done. Our membrane permeability assay using NpN uptake operates under the same conditions suggested by the reviewer and directly measures molecular uptake. Moreover, only fluorescently labeled vancomycin is commercially available among the three antibiotics tested. Since it binds to the cell wall, its utility to measure membrane defects is more limited than the NpN assay we have already used. However, in response to this comment, we will make clear in our revision that we infer that increased susceptibility to other antibiotics is due to their increased membrane permeability.

__ o Effect is mild and mostly not significant. It is also not clear whether authors only tested a handful of mutants shown in Fig. 7B-D or whether other clones were also tested. The sample of endpoints (P2, P5, P8) covers well-characterized lineages, but additional evolved clones or a broader panel could boost generality about other antibiotics. The authors note "significantly lower MICs" statistical treatment is implied; explicit statistical values and replicate numbers should be given in the text or figures.__

We slightly disagree with the reviewer that the results are not significant. Even two-to-three-fold differences in MICs translate to large differences in microbial competition. These three endpoint clones are representative of all eight evolved strains after 90-day evolution experiments. Moreover, we will emphasize in the Revision that we have tested all the mutations found in the endpoint clones; we know what these are from whole genome sequencing of multiple endpoint clones. In addition, we will explicitly state the p-value in the legend of Figure 7.

• __ The structural or physiological nature of "mild" vs. "severe" membrane defects could be better defined/quantified.__ Although we agree with the reviewer's suggestion, the variability of the SEM assay makes the classification of membrane defects based on cell morphology hard to quantify. We therefore only use the SEM images as representative of the various defects observed. For a more quantitative assay of the membrane defects, we instead rely on the standard NpN uptake assay to quantify membrane permeability as a quantifiable readout for membrane defects.

• __ Quantitative limits: Authors should add in the discussion that statistical robustness could be strengthened-for example, by including longer-term evolutionary predictions.__ We are not sure what the reviewer means and so cannot address this point completely. We ask the reviewer to rephrase this point, and we will address it to the best of our abilities.

• __ in vivo relevance: While the ecological context is discussed, direct in vivo confirmation (e.g., in animal infection models) of the observed resistance trajectories would increase translational impact and relevance.__ We agree with the reviewer's point. However, it is not trivial to directly perform evolution experiments of microbes in animal models. There are only a handful of labs worldwide that have working CF-relevant animal models. However, the colistin resistance mutations we identified provide a tool to look deeper into how colistin-resistant P. aeruginosa can evolve in vivo.

• __ Some sections are repetitive or overly detailed; condense where possible (especially on mutation lists and background for each claim).__

We will condense our manuscript as the reviewer suggested in our revision. Adding a graphical summary as suggested will also allow us to be more succinct in our description.

__Other comments

• Authors should provide clarification on how the Mg²_⁺_ concentrations used in vitro compare to those found in clinically relevant infection settings. This would be helpful to enhance significance.__

We thank the reviewer for raising this good point. Based on our previous work, we know the Mg2+ levels in our model (0.3-0.45mM) are within the physiological range of Mg2+ in infection settings (0.1-0.8mM). We will highlight this point in the introduction.

• __ Authors should explicitly report statistical methods (e.g., types of tests, adjustments for multiple comparisons) in figure legends for reproducibility.__

We will include the details of our statistical tests in each panel of figures both in the main text and the supplement.

• __ Nomenclature for key mutations and their position within the genetic context (e.g., htrB2 mutation specifics) could be more detailed in figures or supplemental materials.__

We will name each of the particular mutations tested to be specific about the nature of all the evolved mutations in our figure legends.

• __ The manuscript could benefit from a graphical summary illustrating the two distinct evolutionary pathways and their respective fitness landscapes.__ We thank the reviewer for this suggestion to enhance the clarity of our work. We will make a new graphical summary highlighting two different evolutionary pathways as a new figure.

• __ A brief discussion of therapeutic implications-such as combining colistin with agents that target membrane integrity-would help bridge the gap from mechanism to clinical management.__ In our discussion, we have suggested that collateral sensitivity (line 446-453) and PhoPQ kinase inhibitors (line 512-515) could be exploited to combat colistin resistance. To make this point more clearly, we will slightly expand our Discussion to include the therapeutic implications of our study.

• __ Additional discussion on whether the fitness costs are reversible or can be compensated by further adaptation would be valuable for long-term dynamics.__ We thank the reviewer for raising this interesting point. The evolution trajectory of P8 suggests that fitness costs can be compensated by later-occurring mutations during evolution. We will further discuss this point to highlight the importance of understanding the mutational dynamics of antibiotic resistance evolution.

• __ It would be valuable for the authors to comment on, or further analyze, whether there is a direct association between specific fitness costs and sensitivity to other antibiotics. Such information could inform on evolutionary constraints and possible trade-offs relevant to clinical settings.__

We will include a supplemental figure showing the correlation between fitness costs and antibiotic susceptibility for P2, P5, and P8.

__ Main figures and support for claims

The main and supplementary figures comprehensively illustrate the evolutionary trajectories, genetic bases, and phenotypic outcomes associated with colistin resistance under magnesium depletion in P. aeruginosa. The figures effectively detail: • Genetic pathways involved including the experimental evolution design (colistin selection under Mg²_⁺_ depletion), whole-genome sequencing results, and timelines of observed mutations (e.g., in htrB2, lpxO2, oprH/phoP/phoQ promoter, PA4824). • Phenotypes and biochemical analyses such as lipid A structure (via mass spectrometry), minimum inhibitory concentration (MIC) assays, and epistasis analyses between mutations are depicted. • Fitness trade-offs are demonstrated using bacterial survival, membrane integrity (e.g., scanning electron microscopy images), membrane permeability assays (NPN uptake), and competitive fitness assays. • Mechanistic claims about the necessity of early mutations, the requirement of the PhoPQ pathway at different evolutionary stages, and the fitness cost imposed by certain resistance mutations. To further enhance the rigor and clarity of the manuscript, the authors should implement the following improvements: • Labelling consistency: In some instances, figure legends could provide more granular detail about specific mutations (e.g., positions of amino acid changes). • Graphical summary: A schematic summary figure that visually integrates the three main evolutionary resistance trajectories, the mutational order, corresponding lipid A changes, and fitness costs, would enhance readability. • Replicates: Plots should more thoroughly indicate the number of replicates and show individual data points (not just means {plus minus} SD), add number of replicates in each experiment. • Supplementary: figures referenced in the text (e.g., lipid A structures or mutation reversion outcomes) should be made more prominent or better cross-referenced from the main results section. Authors should highlight when supplementary data provide critical functional confirmation (e.g., confirming mutation function or fitness reversal).__

We thank the reviewer for their appreciation of our work and constructive feedback.

__Statistics

The authors have appropriately incorporated statistical analyses throughout the figures. To enhance the robustness and credibility of their findings, authors should also cross-check • Tests in legends: Every figure and supplementary figure should clearly state the type of statistical test used, how many biological replicates, and any corrections for multiple comparisons.__

As mentioned above, we will provide more details about the statistical tests of each panel.

• __ Effect sizes: Where appropriate, reporting effect sizes-rather than just p values-would contextualize the biological impact.__ We agree with the reviewer; we will mention the magnitude of MIC changes in the corresponding figure legends.

• __ Raw data accessibility: For full transparency, consider sharing underlying raw data and analysis scripts.

__ We will provide the raw data of each panel.

__Overall, the main and supplementary figures effectively illustrate and substantiate the key claims-particularly the alternative molecular pathways, phenotypic trade-offs, and the role of environmental magnesium in mediating colistin resistance. Statistical analysis is generally robust and appropriately presented throughout, though improvements could include more explicit reporting, additional controls, and accessible raw data. The visual and quantitative data in the figures provide support for the authors' conclusions about the evolution of antibiotic resistance under nutrient limitation in microbial environments. Understanding these alternative pathways is important for designing better treatment strategies and for predicting how resistance might evolve under varying clinical and environmental conditions.

__

We thank the reviewer for their positive assessment.

__ Reviewer #1 (Significance (Required)):

Overall, this work offers important insights into antibiotic resistance in nutrient-stressed, polymicrobial environments, highlighting how magnesium availability shapes resistance evolution and fitness costs. The findings suggest new avenues for therapeutic intervention and call for a reevaluation of antibiotic strategies in nutrient-competitive infection settings.__

We sincerely thank the reviewer for constructive and thoughtful feedback and the acknowledgement of our figure presentation and experimental design. We feel very encouraged by the reviewer's perspective that our study provides unique insights into resistance evolution in polymicrobial environments and may inform therapeutic strategies.

__My expertise: Gut microbiome, gut microbiota resilience, ecology, and evolution in microbial communities, antimicrobial resistance, high-throughput drug-bacteria interactions

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

Summary: The paper by Hsieh and colleagues unravels the molecular basis of colistin resistance in Pseudomonas aeruginosa under low magnesium (Mg2+) conditions. Colistin is a last resort antibiotic that compromises bacterial cell wall integrity. Bacteria can respond (phenotypically and genotypically) to colistin by modifying membrane-anchored lipopolysaccharides. Mg2+ depletion can trigger similar responses. In their study, Hsieh et al. find that Mg2+ depletion (induced by a co-infecting fungal pathogen, Candida albicans) leads to evolutionary trajectories and resistance mechanisms that differ from those observed under Mg-rich conditions. The authors conducted a series of detailed genetic, chemical and fitness-based experiments to elucidate the molecular, physiological and evolutionary basis of these new resistance mechanisms.__

We thank the reviewer for their summary of our study.__

Major comments: __ 1. The authors reconstituted key mutations observed during experimental evolution in the ancestral background. Moreover, they took clones from the final stage of the evolution experiment and restored the ancestral state of the mutated genes. This dual approach is extremely strong and allows to decipher the causal effects of colistin resistance. I like to applaud the authors for this rigorous approach.

We thank the reviewer's appreciation about the rigor and comprehensive analyses of our study.

2. I understand that this work focusses on evolved mutants isolated from a previous experiment. The focus is on Mg2+ limitation. However, it would still have been nice to include a characterised colistin resistent strain featuring more standard resistance mechanisms. How different would such a strain be in the analyses shown in Fig. 3? Would morphological changes (Fig. 5A), fitness trade-offs (Fig. 6) and collateral sensitivity (Fig. 7) also occur in such a mutant. I do not regard it as imperative to include data from such a strain. But putting the new data into context (at least in the discussion) would clearly increase the overall impact of this work.

We thank the reviewer for raising this fascinating and vital point. We will address the point in our Revision using the monoculture (high Mg2+) evolved strains, which acquired many known mutations for colistin resistance, as our reference. We will provide a supplemental figure about the membrane permeability, fitness costs, and collateral sensitivity of monoculture evolved strains. We will also contrast their difference from co-culture evolved strains in the revised Discussion.__

1. I recommend to discuss the findings in the context of the work conducted by Jochumsen et al. 2016 Nature Communications https://doi.org/10.1038/ncomms13002. To me, this is one of the most insightful papers on the genetic basis and epistasis of colistin resistance.__

We thank the reviewer for pointing out this important reference. We will include this reference and its findings in the Discussion.

__Minor comments:

1. First section of results and Fig. 1. It is unclear what parts are repetition from the ref. 37 and what is new. Please clarify.__

We thank the reviewer for this suggestion. Figures 1A and 1B summarize the previous paper; all other panels are new data. We will make this clear in the revised text and figure legend.

5. MIC-data (e.g. Fig. 2) come in discrete categories (based on the underlying dilution series). This comes with some challenges for statistical analysis. First, linear models like ANOVAs are based on normally distributed residuals. This is violated with discrete data distributions. Second, there is often no within-treatment variation (e.g., Fig. 2B), which makes statistical analyses obsolete. These points need to be addressed. Moreover, how is it possible to have subtle variations in MIC (e.g., Fig. 2A, P2 endpoint clone) with classic dilution series (as indicated on the y-axis, 128, 256, 512)? Please explain.

We agree with the reviewer that statistical analysis of MIC data is not straightforward. ANOVAs are not well-suited for this type of discrete data, and the lack of variation within replicates reduces the power of non-parametric tests such as the Mann-Whitney U test. To improve the statistical reporting of MIC data, we will apply non-parametric tests and include effect size measurements, as recommended by Reviewer 1.

Moreover, the design of dilution series may underestimate the true nature of antibiotic susceptibility. To address these issues, we have also performed survival assays to assess colistin resistance in both the endpoint and reversion strains; we will also include statistics to assess the significance of their different survival frequencies.

We thank the reviewer for highlighting the point about subtle variations in a classical dilution series. Our endpoint strains grew robustly in media containing 192 μg/mL colistin-the highest concentration used in our evolution experiment. To more accurately determine and compare their maximum MICs, we expanded the colistin concentration range using finer fold increases (1.5×, 2×, 2.5×, 3×, 3.5×, and 4×) from 192 to 768 μg/mL. We will update these details in the Materials & Methods.

__ Lines 264-269. This analysis focusses on enzyme impairment. However, mutations could also change enzyme activity. Could any of these mutations have such an effect?__

The answer is "yes". As evolved strains with lpxA mutation still have lipid A, we suspect this mutation does not altogether abolish lipid A synthesis. However, this mutation could affect the amount of lipid A or change enzyme specificity. These are interesting ideas for further investigation, but they fall beyond the scope of our current study. We will, however, include the requested detail in the discussion.

__ Figure 5A. Some arrows seem to be out of place and point at void spaces. Please check.__

We thank the reviewer for pointing out this error, which we will correct.

8. The use of polymyxin B is not well justified (Fig. 5 and Fig. S13). Did the authors aim to test whether there is cross-resistance to other antimicrobial peptides?

We will more clearly justify our choice of using polymyxin B for directly assaying binding of polymyxin antibiotics to bacterial cells using fluorescence-labeled polymyxins, since no such reagents exist for colistin and since previous studies (including ours) have shown similarity of susceptibility to colistin and polymyxin B:

"Although P2 and P5 endpoint clones have more permeable membranes, they exhibited greater resistance to polymyxin antibiotics, including colistin (polymyxin E) (Fig. 5D), and polymyxin B (Fig. S13A) than WT cells. To investigate how membrane-compromised cells gain increased resistance to antibiotics that target the outer membrane, we used dansyl-labeled polymyxin B [51] to quantify the binding of polymyxins to P. aeruginosa; dansyl-labeled polymyxin fluoresces upon binding the hydrophobic portion of bacterial membranes. We used polymyxin B binding as a surrogate for how bacterial cells bind to all polymyxin antibiotics, including colistin."

__ Line 564. Please indicate the dilution factor used.__

Thank you for pointing out this inadvertent omission. We will update our Materials & Methods accordingly, as in response to the Reviewer 2's comment 5.

__Reviewer #2 (Significance (Required)):

This is a very strong and well designed study. It provides novel and relevant insights into the resistance mechanisms against an important last resort antibiotic.__

We sincerely thank the reviewer for their thoughtful summary and generous evaluation of our work.

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

This manuscript reports on biologically interesting and clinically-relevant findings, that upon passaging in the presence of spent media from C. albicans, P. aeruginosa develops resistance to colistin through lipid A modifications. The authors thoroughly characterize novel lipid A structures seen in their resistant mutants, and test a variety of genetically constructed mutants to determine the contributions of specific mutant alleles to resistance.__

We thank the reviewer for the appreciation of our experimental design and comprehensive genetic and biochemical analyses of our evolved strains.

However, additional experiments are needed to demonstrate the specific role and necessity of the lipid modifications for colistin resistance.

We are also grateful for the reviewer's feedback and constructive criticisms to improve the clarity and impact of our manuscript. We have listed detailed responses to the reviewer below.

1. __ Evidence that the lipid A mutations are causal for colistin resistance is sparse:
2. Both the htrB2 mutations (in P2 and P5) are posited to be loss-of-function alleles. However, the phenotypes of the individual alleles are different (shown in Fig 2A and 2B). While the mutation in P2 shows a ~2x increase in resistance, the mutation in P5 does not. Thus it is not clear that the specific lipid A modifications seen in the htrB2 mutants are sufficient to confer colistin resistance. Can the authors test a clean deletion mutant of htrB2? Further, reversion of the htrB2 mutation in P2 has only a mild effect on colistin resistance, while reversion in P5 leads to a ~3-4x reduction in colistin resistance (Fig. S3), once again making it hard to parse out the exact effect of the lipid A modifications seen in the htrB2 mutants.
3. Similarly, a single lpxO2 mutation does not have any effect on colistin resistance (in P5), indicating that the modifications seen in this mutant are not sufficient to lead to resistance.__ We thank the reviewer for making this suggestion. The reviewer is correct that a clean deletion will directly assess the effects of htrB2 mutations. We will make htrB2 deletion in WT and the triple mutants and endpoint clones of P2 and P5 to check the effect of htrB2 deletion on colistin resistance.

Additionally, as Reviewer 2 pointed out, both mutation reconstruction and reversion experiments are required for understanding the roles of each mutation and interactions among different mutations in contributing to resistance. Combining all the results of htrB2 and lpxO2 mutations in these two orthogonal genetic experiments, it is the synergistic interactions among these mutations that lead to enhanced resistance after evolution. This explains why we saw genetic background effects of htrB2 mutation (P2 vs P5) and why each single mutation is required for resistance but doesn't contribute to resistance significantly by itself.

- In P8, the effect of a single lpxA mutation is not tested. Further, the resistance of a P-oprH + lpxA mutant is the same as that of just the P-oprH mutant, indicating that the lpxA mutation likely does not directly alter colistin resistance. It is possible that mutations in lpxA were selected to compensate for fitness defects resulting from the other mutations, or for adaptation to some other component of the media conditions.

This is an excellent suggestion. We will assess the MIC and fitness of reconstructed strains with the lpxA mutation to update the role of this mutation.

- While reversion of the htrB2 and lpxO2 mutations do lead to ~3-4x reduced resistance in P5 indicating some contribution of these mutations, it is specific to this population, and thus not clear whether it is due to the specific lipid A modifications (some of which are seen in the other populations too). A specific combination of lipid A modifications may confer colistin resistance, but this needs to be demonstrated by generating just those clean deletion mutants and showing an effect on resistance.

In response to this comment and comment 1, we will make lpxO2 deletions in WT, the triple mutant and the endpoint clone of P5 to test colistin resistance. However, our results of reverting single htrB2 or lpxO2 mutation to WT are robust and use two independent assays, including the standard MIC test and colistin survival assay. So, we are confident that each mutation is necessary for enhancing colistin resistance.

__ Overall, given the high levels of colistin resistance still exhibited by single mutant revertants (Fig. S3) and the absence of double or triple revertants, it is hard to come to any conclusions regarding causality. This is especially the case for P8 but also true of P2 and P5. What are the other mutations in these populations, and what role do they play in colistin resistance?__

We respectfully disagree with the reviewer on this point. One point that we have made and will re-emphasize in our Revision is that we have assayed all the mutations in these populations; this is one of the advantages of our experimental evolution and genome sequencing strategy. All the mutations that could play a role in colistin resistance have therefore been tested. Furthermore, due to genetic epistasis of mutations in different evolutionary lineages, we do not necessarily expect that a single revertant would altogether abolish colistin resistance, as has been demonstrated in several previous studies. As Reviewer 2 pointed out, combining mutation reconstruction and reversion is the best way to establish causality, and we have done so. Therefore, it is not correct to say that we cannot come to 'any conclusions regarding causality'.

__ Figure 4 is titled "The PhoPQ pathway synergizes with early-arising mutations to confer colistin resistance.", but instead what this figure shows is that the mutation upstream of oprH increases PhoP activity. I'm not sure what the synergy here is. The same is true for the section starting on line 276. Further, the first sentence of that section states "We next investigated why the mutations conferring robust colistin resistance in low Mg2+ conditions are not observed in Mg2+ replete conditions.". However, there are no experiments there testing whether the mutations conferred resistance in Mg2+ conditions, instead the authors just test whether the mutations they are studying increase PhoP activity, and require PhoPQ to confer resistance.__

We thank the reviewer for raising this point. We apologize for the unclear writing. We will use this opportunity to improve the clarity of this section by rewriting it to focus on two points: 1. Evolved resistance is PhoPQ-dependent, instead of PmrAB-dependent. 2. Two lineages evolved enhanced resistance by boosting PhoPQ activity in both high and low Mg2+ conditions. We will also remove the statement highlighted by the reviewer from this section that obfuscates the motivation of this section. We feel this approach will more clearly show how lipid A-related mutations contribute to resistance in low Mg2+.

__ The authors claim that the identified mutations did not appear in the high magnesium conditions because they had a fitness cost under those conditions, but figure 6A shows that the evolved strains have fitness costs in low magnesium conditions as well. Further, the authors suggest that because the studied mutations act via increased PhoPQ activity, they do not lead to resistance under high magnesium conditions (lines 376-379). However, the increased PhoPQ activity is mediated by the P-oprH mutation in the isolates which likely increases PhoPQ activity even in high magnesium conditions. Overall, it is not clear why the mutations in the low magnesium condition were not selected for under high magnesium conditions.__

The reviewer is correct about the fitness cost in high Mg2+ and low Mg2+ conditions. These fitness experiments were carried out in the absence of colistin, which explains the finding that there are fitness defects in both conditions. As is well known, evolution for antibiotic resistance will ultimately select for resistant mutants, despite their fitness costs. In contrast, colistin MIC of these endpoint strains in high Mg2+ conditions was still much lower than the colistin concentration we applied during evolution (Fig. S15), indicating it is much less likely for these mutations to be selected for in high Mg2+. We will clarify this point in our revised Results and Discussion.

We agree with the reviewer about the P-oprH mutations (PhoPQ expression) and will note that, unlike the other mutations, it is not clear why these emerge only in the low Mg2+ condition.

__ The authors used C. albicans spent BHI media as their low magnesium condition, but this condition has a lot of other C. albicans metabolites that may be affecting the results. It is possible that what the authors are observing is not related to magnesium at all, and the authors should test the phenotypes in normal BHI medium depleted for magnesium or some defined medium where magnesium levels can be controlled.__

We thank the reviewer for mentioning this important point. In our prior PLOS Biology paper (https://doi.org/10.1371/journal.pbio.3002694.g005), we demonstrated that supplementing Mg2+ in evolved co-culture populations reduces colistin resistance, suggesting this evolved resistance is Mg2+ dependent. We also know that the MIC of our endpoint strains in C. albicans-spent BHI with supplemented Mg2+ (MIC of all three endpoint clones is less than 48 mg/mL colistin) is much lower than in C. albicans-spent BHI. We will mention this detail in the paper and include the data in our revision if the reviewer and editor require it.

Other comments: - The authors use MIC assays as well as % survival to measure resistance against colistin, and sometimes use both in the same figure (e.g. Figure 2). This makes direct comparisons difficult. It would be better to consistently use one assay, preferably the MIC, at least in all the main figures. If the survival data needs to be included, it could go in the supplementary figures.

We thank the reviewer for this suggestion. We will move the MIC data of mutation-reversion strains to the main Fig. 2D-F.

- While the mutations seen in the low and high magnesium conditions were shown in the previous manuscript, given the extensive dissection here, it would be useful for readers if the authors gave some details about the serial passaging and evolution experiment, identification of mutations, and some mention of what mutations were seen in high Mg populations.

We will add these details in the introduction.

- Given that oprH is present in an operon, it would be more accurate to call that mutation as being in the promoter of the oprH-phoP-phoQ operon rather than it being an oprH mutation (at least in the text, e.g. lines 127-129).

We agree. We will change this as the reviewer requested.

- Unlike what is stated on lines 287-290, deletion of oprH in P2 leads to a greater than 2x reduction in colistin MIC, suggesting that OprH is playing a role (albeit a smaller role than phoP) - Line 50 has a typo, remove "160". - Line 122: Specify which Pa and Ca strain backgrounds were used. - Line 132: Were representative isolates derived from terminal passages? This should be defined.

We will change these points according to the reviewer's suggestions; we thank them for these suggestions.

- Line 215-219: It is interesting that Pa WT grown in spent medium additionally results in lipid A that is hexa-acylated. Is this sufficient to alter colistin resistance on its own?

We find that WT PAO1 in low Mg2+ conditions has PagP-mediated acylation, which can slightly increase colistin resistance, but not to the extent of resistance as our evolved strains.

- It would be useful to see a PCA plot for the samples shown in figures S6 and S7.

We will include such a plot in Figures S6 and S7

- Fig. S11: What are the colistin MICs of pmrA and phoP deletions in the WT background?

MIC of pmrA and phoP deletions in WT is 1.5ug/mL. We will include these data in the Revision.

- Instead of qualitative data, can the authors quantify cell length and perhaps some measure of cell shape (instead of just showing images in Fig. 5A and S12).

We thank the reviewer for raising this point. A similar comment was raised by Reviewer 1. As it's challenging to quantify membrane changes from the morphological data obtained through SEM (a point which we will now clarify in our Revision), we used a quantifiable NpN uptake assay to quantify membrane defects of our evolved strains.

- What is the WT MIC in high magnesium conditions? Please show that in Fig. S15.

We will include this detail in Fig. S15

- I am not an expert in lipid modifications and structures, but in figure S5, P2 and P4 show high peaks with lower m/z that seem specific to low magnesium conditions, but they are not labeled or discussed. What are these peaks?

We thank the reviewer for bringing up this concern. The unlabeled lipids in these spectra are cardiolipin, not lipid A. These peaks are present in all the samples, and the reason they appear larger in the P1 and P4 low magnesium conditions is that both spectra are scaled to the relative intensity of one another. It is important to note that MALDI-TOF MS is not a quantitative technique, and the relative intensity of the peak heights between two samples should not be used to compare the amounts of lipids in one sample versus another. Therefore, we cannot say that these lipids are present in greater quantities in low magnesium conditions versus high magnesium conditions.

- Lines 357-358 state that "mutant cells minimally bind polymyxin B (Fig. S13B)", but the figure shows increased binding compared to the WT. The legend of the figure also says something similar. Are the phoP pmrA mutants expected to bind more polymyxin B because they can't modify lipid A?

We thank the reviewer for pointing out this substantial error. We will change 'minimally bind' to 'demonstrate increased binding'.

- Given the fitness defects in just regular medium, is the data shown in Figure 7 specific collateral sensitivity to the antibiotics tested? Are there other conditions where P2 and P5 do not show increased sensitivity?

These are all the antibiotics we have tested. It is conceivable that P2 and P5 might not show increased sensitivity to other antibiotics that use the same mode of action as colistin or polymyxin B.

__Reviewer #3 (Significance (Required)):

This study aims to dissect novel mechanisms of colistin resistance in P. aeruginosa that arise upon passaging in C. albicans spent media. While the authors identify novel lipid A modifications associated with the evolved strains, the significance of the modifications for resistance, and the mechanisms for why these evolutionary trajectories were not selected for in high magnesium are not clear from the data presented.__

We thank the reviewer for recognizing the integrity of our work and for the constructive feedback on improving the clarity of our writing. We understand that some concerns may stem from a lack of clarity in our original submission, but that additional genetic experiments are necessary. We have already identified all mutations that arose independently across different lineages and characterized their contributions to resistance, which we believe supports a robust inference of causality. To strengthen our conclusions, we will incorporate additional experiments, including htrB2 deletion, lpxO2 deletion, and lpxA mutation, to better dissect the roles of these genes and mutations in colistin resistance. We hope this revision plan will ameliorate the reviewer's concerns.

Hola grupo, ¿cómo están?

I just learned a new to me meaning of he word essay

https://hypothes.is/a/88pf1H3ZEfCpcgvmBb9Weg

Learning a new word, or just a new meaning of a familiar word, for me

is a psyche-dellic mind-expanding experience

Most i've been doing in my own space and annotations on the margin was I tried to say what I mean to work on tried to do

Just write down what comes to mind as my mind is focusing on some tacit intent, purpose, exploring ways

-the.meaning.of?essay-is

to try something

I Say to do that

Author response:

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

Reviewer #1 (Public review):

Sumary:

This study evaluates whether species can shift geographically, temporally, or both ways in response to climate change. It also teases out the relative importance of geographic context, temperature variability, and functional traits in predicting the shifts. The study system is large occurrence datasets for dragonflies and damselflies split between two time periods and two continents. Results indicate that more species exhibited both shifts than one or the other or neither, and that geographic context and temp variability were more influential than traits. The results have implications for future analyses (e.g. incorporating habitat availability) and for choosing winner and loser species under climate change. The methodology would be useful for other taxa and study regions with strong community/citizen science and extensive occurrence data.

We thank Reviewer 1 for their time and expertise in reviewing our study. The suggestions are very helpful and will improve the quality of our manuscript.

Strengths:

This is an organized and well-written paper that builds on a popular topic and moves it forward. It has the right idea and approach, and the results are useful answers to the predictions and for conservation planning (i.e. identifying climate winners and losers). There is technical proficiency and analytical rigor driven by an understanding of the data and its limitations.

We thank Reviewer 1 for this assessment.

Weaknesses:

(1) The habitat classifications (Table S3) are often wrong. "Both" is overused. In North America, for example, Anax junius, Cordulia shurtleffii, Epitheca cynosura, Erythemis simplicicollis, Libellula pulchella, Pachydiplax longipennis, Pantala flavescens, Perithemis tenera, Ischnura posita, the Lestes species, and several Enallagma species are not lotic breeding. These species rarely occur let alone successfully reproduce at lotic sites. Other species are arguably "both", like Rhionaeschna multicolor which is mostly lentic. Not saying this would have altered the conclusions, but it may have exacerbated the weak trait effects.

We thank the reviewer for their expertise on this topic. We obtained these habitat classifications from field guides and trait databases, and reviewed our primary sources to clarify the trait classifications. We reclassified the species according to the expertise of this reviewer and perform our analysis again; please see details below.

(2) The conservative spatial resolution (100 x 100 km) limits the analysis to wide- ranging and generalist species. There's no rationale given, so not sure if this was by design or necessity, but it limits the number of analyzable species and potentially changes the inference.

It is really helpful to have the opportunity to contextualize study design decisions like this one, and we thank the reviewer for the query. Sampling intensity is always a meaningful issue in research conducted at this scale, and we addressed it head-on in this work.

Very small quadrats covering massive geographical areas will be critically and increasingly afflicted by sampling weaknesses, as well as creating a potentially large problem with pseudoreplication. There is no simple solution to this problem. It would be possible to create interpolated predictions of species’ distributions using Species Distribution Models, Joint Species Distribution Models, or various kinds of Occupancy Models. None of these approaches then leads to analyses that rely on directly observed patterns. Instead, they are extrapolations, and those extrapolations typically fail when tested, although they have still been tested (for example, papers by Lee-Yaw demonstrate that it is rare for SDMs to predict things well; occupancy models often perform less well than SDMs and do not capture how things change over time - Briscoe et al. 2021, Global Change Biology). The result of employing such techniques would certainly be to make all conclusions speculative, rather than directly observable. 

Rather than employing extrapolative models, we relied on transparent techniques that are used successfully in the core macroecology literature that address spatial variation in sampling explicitly and simply. Moreover, we constructed extensive null models that show that range and phenology changes, respectively, are contrary to expectations that arise from sampling difference. 100km quadrats make for a reasonable “middle-ground” in terms of the effects of sampling, and we added a reference to the methods section to clarify this (see details below).

(3) The objective includes a prediction about generalists vs specialists (L99-103) yet there is no further mention of this dichotomy in the abstract, methods, results, or discussion.

Thank you for pointing this out - it is an editing error that should have been resolved prior to submission. We replaced the terms specialist and generalist with specific predictions based on traits (see details below).

(4) Key references were overlooked or dismissed, like in the new edition of Dragonflies & Damselflies model organisms book, especially chapters 24 and 27.

We thank Reviewer 1 for making us aware of this excellent reference. We have reviewed the text and include it as a reference, in addition to other references recommended by Reviewer 1 and other reviewers (see details below).

Reviewer #2 (Public review):

Summary:

This paper explores a highly interesting question regarding how species migration success relates to phenology shifts, and it finds a positive relationship. The findings are significant, and the strength of the evidence is solid. However, there are substantial issues with the writing, presentation, and analyses that need to be addressed. First, I disagree with the conclusion that species that don't migrate are "losers" - some species might not migrate simply because they have broad climatic niches and are less sensitive to climate change. Second, the results concerning species' southern range limits could provide valuable insights. These could be used to assess whether sampling bias has influenced the results. If species are truly migrating, we should observe northward shifts in their southern range limits. However, if this is an artifact of increased sampling over time, we would expect broader distributions both north and south. Finally, Figure 1 is missed panel B, which needs to be addressed.

We thank Reviewer 2 for their time and expertise in reviewing our study.

It is possible that some species with broad niches may not need to migrate, although in general failing to move with climate change is considered an indicator of “climate debt”, signaling that a species may be of concern for conservation (ex. Duchenne et al. 2021, Ecology Letters). We revised the discussion to acknowledge potential differences in outcomes (please see details below).

We used null models to test whether our results regarding range shifts were robust, and if they varied due to increased sampling over time. We found that observed northern range limit shifts are not consistent with expectations derived from changes in sampling intensity (Figure S1, S2). 

We thank Reviewer 2 for pointing out this error in Figure 1. This conceptual figure was a challenge to construct, as it must illustrate how phenology and range shifts can occur simultaneously or uniquely to enable a hypothetic odonate to track its thermal niche over time. In a previous version of the figure, we had a second panel and we failed to remove the reference to that panel when we simplified the figure. We have updated the figure and figure caption (please see details below).

Reviewer #3 (Public review):

Summary:

In their article "Range geographies, not functional traits, explain convergent range and phenology shifts under climate change," the authors rigorously investigate the temporal shifts in odonate species and their potential predictors. Specifically, they examine whether species shift their geographic ranges poleward or alter their phenology to avoid extreme conditions. Leveraging opportunistic observations of European and North American odonates, they find that species showing significant range shifts also exhibited earlier phenological shifts. Considering a broad range of potential predictors, their results reveal that geographical factors, but not functional traits, are associated with these shifts.

We thank Reviewer 3 for their expertise and the time they spent reviewing our study. Their suggestions are very helpful and will improve the quality of our manuscript.

Strengths:

The article addresses an important topic in ecology and conservation that is particularly timely in the face of reports of substantial insect declines in North America and Europe over the past decades. Through data integration the authors leverage the rich natural history record for odonates, broadening the taxonomic scope of analyses of temporal trends in phenology and distribution to this taxon. The combination of phenological and range shifts in one framework presents an elegant way to reconcile previous findings improving our understanding of the drivers of biodiversity loss.

We thank Reviewer 3 for this assessment.

Weaknesses:

The introduction and discussion of the article would benefit from a stronger contextualization of recent studies on biological responses to climate change and the underpinning mechanism.

The presentation of the results (particularly in figures) should be improved to address the integrative character of the work and help readers extract the main results. While the writing of the article is generally good, particularly the captions and results contain many inconsistencies and lack important detail. With the multitude of the relationships that were tested (the influence of traits) the article needs more coherence.

We thank Reviewer 3 for these suggestions. We revised the introduction and discussion to better contextualize species’ responses to climate change and the mechanisms behind them (see details below). We carefully reviewed all figures and captions, and made changes to improve the clarity of the text and the presentation of results (see details below).

Reviewer #1 (Recommendations for the authors):

Comment:

(1) Following weakness #1 in the public review, the authors should review the habitat classifications, consult with an odonatologist, and reclassify many species from Both to Lentic and redo the analysis.

Thank you for pointing out this disagreement among expert habitat classifications that we cited and other literature. We reclassified species’ habitat preferences based on classifications by Hof et al., a source that was consistent with your suggestions, and identified additional species as Lentic that our other references had identified as Both. We performed our analysis with this new dataset and, as you suspected, our results did not change qualitatively: species habitat preferences did not predict their range shifts.

Hof, Christian, Martin Brändle, and Roland Brandl. "Lentic odonates have larger and more northern ranges than lotic species." Journal of Biogeography 33.1 (2006): 63-70.

Comment:

(2) Following weakness #2, would it be worthwhile or interesting to analyze a smaller ranging group (e.g. cut the quad size in half, 50 x 50 km) to bring in more species and potentially change the inference? Or is the paper too tightly constructed to allow this, even as a secondary piece?

Thank you for this comment, as it highlights an important consideration for macroecological analyses, and the importance of balancing multiple factors for determining quadrat size. Issues exist with identifying drivers of range boundaries among species with narrow ranges when they are analyzed separately from wide-ranging species, and examining larger quadrats can actually help clarify drivers (Szabo, Algar, and Kerr 2009). The smaller quadrats are, the higher the likelihood that the species is actually there but was never observed, or that the quadrat only covers unsuitable habitat and the species is absent from the entire (or almost entire) quadrat. Too many absences creates issues with violating model assumptions, and creates noise that makes it difficult to identify drivers of species’ range and phenology shifts.

Moreover, we constructed extensive null models that show that range and phenology changes, respectively, are contrary to expectations that arise from sampling difference. 100km quadrats make for a reasonable “middle-ground”, and we have included a brief explanation of this in the text: “We assigned species presences to 100×100 km quadrats, a scale that is large enough to maintain adequate sampling intensity but still relevant to conservation and policy (Soroye et al., 2020), to identify the best sampled species.”  (Lines 170-172).

Szabo, Nora D., Adam C. Algar, and Jeremy T. Kerr. "Reconciling topographic and climatic effects on widespread and range‐restricted species richness." Global Ecology and Biogeography 18.6 (2009): 735-744.

Comment:

(3) Following weakness #3, are specialists the ones that "failed to shift" (L18)? If so please specify. The prediction about generalists vs specialists needs to be removed or incorporated in other parts of the paper.

Thank you for pointing this out, we intended to suggest that species with more generalist habitat requirements might be better able to shift, but ultimately found that traits did not predict species’ shifts. We corrected our prediction regarding habitat generalists as follows: “We predicted that species able to use both lentic and lotic habitats would shift their phenologies and geographies more than those able to use just one habitat type, as generalists outperform specialists as climate and land uses change (Ball-Damerow et al., 2015, 2014; Hassall and Thompson, 2008; Powney et al., 2015; Rapacciuolo et al., 2017).” (Lines 128-132).

Comment:

(4) Following weakness #4, cite Pinkert et al at lines 70-73 and Rocha-Ortega et al at lines 73-77 along with https://doi.org/10.1098/rspb.2019.2645. Add Sandall et al https:// doi.org/10.1111/jbi.14457 to L69 references.

Thank you for the excellent reference suggestions, we have added them as suggested (Lines 80, 86, 77).

Comment:

Other comments/suggestions:

(1) Title: consider adding temp variability 'Range geography and temperature variability, not functional traits,...'.

Thank you for this suggestion, we have added temperature variability to the title: “Range geography and temperature variability explain cross-continental convergence in range and phenology shifts in a model insect taxon”.

Comment:

(2) L125: is (northern) Mexico included in North America?

Yes, we did include observations from Northern Mexico, and have specified this in the text: “We retained ~1,100,000 records from Canada, the United States, and Northern Mexico, comprising 76 species (Figure 2).” (Lines 174-176).

Comment:

(3) L128: I'd label this section 'Temperature variability' rather than 'Climate data'.

Thank you, we agree that this is a more appropriate title for this section, and have replaced ‘Climate data’ with ‘Temperature variability’ (Line 185).

Comment:

(4) Table 2: why are there no estimates for the traits?

We apologise, this information should have been included in the main body of the manuscript, but was only explained in the Table 2 caption. We have added the following explanation: “Non-significant variables, specifically all functional traits, were excluded from the final models.”. (Line 312-323).

Comment:

(5) Figure 2: need to identify the A-D panels.

We apologise for this error and have clarified the differences between panels in the figure caption:

“Figure 2: Richness of 76 odonate species sampled in North America and Europe in the historic period (1980-2002; panes A and C) and the recent period (2008-2018; panes B and D). Species richness per 100 × 100 km quadrat is shown in panes A and B, while panes C and D show species richness per 200 × 200 km quadrat. Dark red indicates high species richness, while light pink indicates low species richness.” (Lines 1002-1006).

Comment:

(6) L163-173: I am not familiar with this analysis but it sounds interesting and promising, I am not sure if this can be clarified further. Why the -25 to 25, and -30 to 30, doesn't the -35 to 35 cover these? And what is meant by "include only phenology shifts that could be biologically meaningful", that larger shifts would not be meaningful or tied to climate change?

We used different cutoffs for phenology shifts to inspect for outliers that were likely to be errors, potentially do to insufficient sampling to calculate phenology. We clarified in the text as follows:

“We retained emergence estimates between March 1st and September 1st, as well as species and quadrats that showed a difference in emergence phenology of -25 to 25 days, -30 to 30 days, or -35 to 35 days between both time periods, to include only phenology shifts that could be biologically meaningful to environmental climate change (i.e. exclude errors).” (Lines 169-173).

Comment:

(7) L193-200: I agree but would make a distinction between ecological vs functional traits, as other studies view geographic traits as ecological manifestations of functional biology, e.g. https://doi.org/10.1016/j.biocon.2019.07.001 and https://doi.org/10.1016/ j.biocon.2023.110098.

Thank you for this suggestion, and for making us aware of the thinking around range geographies as ecological traits. We have specified throughout the manuscript that the ‘traits’ we are considering are ‘functional traits’, changed the methods subsection title to “Range geographies and functional traits” (Line 252), and added a brief discussion of ecological traits: “Geographic range and associated climatic characteristics are often considered ecological traits, as they are consequences of functional traits and their interactions with geographic features (Bried and Rocha-Ortega, 2023; Chichorro et al., 2019).” (Lines 256-259).

Comment:

(8) L203: What's the rationale for egg-laying habitat as "biologically relevant to spatial and temporal responses to climate change"? That one's not as obvious as the others and needs a sentence more. Also, I am wondering why other traits were not considered here, like color lightness and voltinism. And why not wing size instead of body size, or better yet the two combined (wing loading) as a proxy for dispersal ability?

We agree that our rationale for using this trait should be better explained, and we have included the following explanation: “Egg laying habitat was assigned according to whether species use exophytic egg-laying habitat (i.e. eggs laid in water or on land, relatively larger in number), or endophytic egg-laying habitat (i.e. eggs laid inside plants, usually fewer in number); species using exophytic habitats are associated with greater northward range limit shifts (Angert et al., 2011).” (Lines 271-275).

We considered traits that have been found to be important for range and phenology shifts among odonates, as well as being key traits for expectations for species responses to climate change. Flight duration and body size are correlated with dispersal ability (Powney et al. 2015). Body size is also correlated with competitive ability (Powney et al. 2015), potentially making it an important predictor of a species’ ability to establish and maintain populations in expanding range areas. Traits correlated with range shifts also include breeding habitat type (Powney et al. 2015; Bowler et al. 2021) and egg laying habitat (Angert et al. 2011). Ideally, we would have used dispersal data from mark/release/recapture studies, but it was not available for many of the species included in this study. After finding that none of the functional traits we included were related to range shifts, there was no reason to believe that a further investigation of traits would be meaningful.

Angert AL, Crozier LG, Rissler LJ, Gilman SE, Tewksbury JJ, Chunco AJ. 2011. Do species’ traits predict recent shifts at expanding range edges? Ecology Letters 14:677–689. doi:10.1111/j.1461-0248.2011.01620.x

Bowler DE, Eichenberg D, Conze K-J, Suhling F, Baumann K, Benken T, Bönsel A, Bittner T, Drews A, Günther A, Isaac NJB, Petzold F, Seyring M, Spengler T, Trockur B, Willigalla C, Bruelheide H, Jansen F, Bonn A. 2021. Winners and losers over 35 years of dragonfly and damselfly distributional change in Germany.Diversity and Distributions 27:1353–1366. doi:10.1111/ddi.13274

Powney GD, Cham SSA, Smallshire D, Isaac NJB. 2015. Trait correlates of distribution trends in the Odonata ofBritain and Ireland. PeerJ 3:e1410. doi:10.7717/peerj.1410

Comment:

(9) L210: I count at least 5 migratory species in table S3, so although maybe not enough to analyze it's misleading to say "nearly all" were non-migratory, revise to "most" or "vast majority".

Thank you for pointing this out, we have made the suggested correction (Line 277).

Comment:

(10) L252-254: save this for the Discussion and write a more generalized statement for results to avoid citations in the results.

Thank you for this suggestion, we have moved this to the discussion (Lines 517-527).

Comment:

(11) Figures S5 & S6: these are pretty important, I'd consider elevating them to the main document as one figure with two panels.

Thank you for this suggestion, we agree these figures should be elevated to the main text, and have made them into a panel figure (Figure 4).

Comment:

(12) L305-307: great point and recommendation!

Thank you very much for this positive feedback!

Comment:

(13) L335-336: another place to cite https://doi.org/10.1098/rspb.2019.2645 which includes a thermal sensitivity index and would add an odonate citation behind the statement.

Thank you for this excellent suggestion, we have added this citation (line 480). (Rocha-Ortega et al. 2020)

Comment:

(14) L352-353: again see also https://doi.org/10.1098/rspb.2019.2645.

Thank you for highlighting this reference, we have added it to Line 505 as suggested.

Comment:

(15) L355: revise "populations that coexist" to "species that co-occur" (big difference between population and species levels and between coexistence and co-occurrence).

Thank you very much for pointing this out, we have made the suggested change (Line 507).

Comment:

(16) L359-365: are the winners and losers depicted in Figures S5 & S6? If so reference the figure (which I suggest combining and promoting to the main text), if not create a table listing the analyzed species and their winner/loser status.

We agree that this is an excellent place to bring up Figures S5 and S6 from the supplemental. We have moved them to the main document as one figure and referenced it at line 510.

Reviewer #2 (Recommendations for the authors):

Comment:

(1) Line 53-55: The claim that "These relationships generalize poorly taxonomically and geographically" is valid, but the study only tests Odonata on two continents.

Thank you for this comment – the word ‘generalize’ may imply that our study tries to find a general pattern across many groups. We have changed the language to: “However, these relationships are inconsistent across taxa and regions, and cross-continental tests have not been attempted (Angert et al., 2011; Buckley and Kingsolver, 2012; Estrada et al., 2016; MacLean and Beissinger, 2017).” (Lines 57-59).

Comment:

(2) Line 58-59: Is this statement only true for Odonata? It does not seem to hold for plants, for example.

Thank you for this comment – this statement references a meta-analysis of multiple animal and plant taxa, but the evidence for the importance of range location comes from animal taxa. We have specified that we are referring to animal species to clarify (Line 60).

Comment:

(3) Line 87-91: This section is difficult to understand and needs clarification.

We have clarified this section as follows: “While warm-adapted species with more equatorial distributions could expand their ranges poleward following warming (Devictor et al., 2008), they could also increase in abundance in this new range area relative to species that historically occupied those areas and are less heat-tolerant (Powney et al., 2015).” (Lines 95-121).

Comment:

(4) Line 99-100: Please define "generalist" and "specialist" more clearly here (e.g., based on climate niche?).

Thank you for pointing this out, we intended to suggest that species with more generalist habitat requirements might be better able to shift, but ultimately found that traits did not predict species’ shifts. We corrected our prediction regarding habitat generalists as follows: “We predicted that species able to use both lentic and lotic habitats would shift their phenologies and geographies more than those able to use just one habitat type, as generalists outperform specialists as climate and land uses change (Ball-Damerow et al., 2015, 2014; Hassall and Thompson, 2008; Powney et al., 2015; Rapacciuolo et al., 2017).” (Lines 128-132).

Comment:

(5) Line 122: Replace the English letter "X" in "100x100 km" with the correct mathematical symbol.

We have made the suggested replacement throughout the manuscript.

Comment:

(6) Line 148: To address sampling effects, you could check the paper: https://onlinelibrary.wiley.com/doi/full/10.1111/gcb.15524. Additionally, maximum and minimum values are sensitive to extreme data points, so using 95% percentiles might be more robust.

Thank you for sharing this paper, as it offers a valuable perspective on the study of species’ ranges. While our dataset is substantially composed of observations from adult sampling protocols, unlike the suggested paper which compares adults and juveniles, this is an interesting alternative approach.

For our purposes it is meaningful to include outliers, as otherwise we may have missed individuals at the leading edge of range expansions. Our intent here was to detect range limits, as opposed to finding the central tendency of species distributions. This approach is widely accepted in the macroecology literature (i.e. Devictor et al., 2012, 2008; Kerr et al. 2015).

We have included the following discussion of our approach in the methods section:

“We followed widely accepted methods to determine species range boundaries (Devictor et al., 2012, 2008; Kerr et al., 2015), although other methods exist that are appropriate for different data types and research questions i.e. (Ni and Vellend, 2021). We assigned species presences to 100×100 km quadrats, a scale that is large enough to maintain adequate sampling intensity but still relevant to conservation and policy (Soroye et al., 2020), to identify the best sampled species.” (Lines 168-173).

Kerr JT, Pindar A, Galpern P, Packer L, Potts SG, Roberts SM, Rasmont P, Schweiger O, Colla SR, Richardson LL,Wagner DL, Gall LF, Sikes DS, Pantoja A. 2015. Climate change impacts on bumblebees converge across continents. Science 349:177–180. doi:10.1126/science.aaa7031

Soroye P, Newbold T, Kerr J. 2020. Climate change contributes to widespread declines among bumble bees across continents. Science 367:685–688. doi:10.1126/science.aax8591

Devictor V, Julliard R, Couvet D, Jiguet F. 2008. Birds are tracking climate warming, but not fast enough.Proceedings of the Royal Society B: Biological Sciences 275:2743–2748. doi:10.1098/rspb.2008.0878

Devictor V, van Swaay C, Brereton T, Brotons L, Chamberlain D, Heliölä J, Herrando S, Julliard R, Kuussaari M,Lindström Å, Reif J, Roy DB, Schweiger O, Settele J, Stefanescu C, Van Strien A, Van Turnhout C,

Vermouzek Z, WallisDeVries M, Wynhoff I, Jiguet F. 2012. Differences in the climatic debts of birds and butterflies at a continental scale. Nature Clim Change 2:121–124. doi:10.1038/nclimate1347

Comment:

(7) Line 195: The species' climate niche should also be considered a product of evolution.

Thank you for this suggestion. To address this comment and a comment from another reviewer, we changed the text to the following: “Geographic range and associated climatic characteristics are often considered ecological traits, as they are consequences of functional traits and their interactions with geographic features (Bried and Rocha-Ortega, 2023; Chichorro et al., 2019).” (Lines 256-259).

Comment:

(8) Line 244: This speculative statement belongs in the Discussion section.

Thank you for this suggestion, we have moved this statement to the discussion (Lines 451-453).

Comment:

(9) Line 252-254: The projection of Coenagrion mercuriale's range contraction is not part of your results and should be clarified or removed.

Following this suggestion and a similar suggestion from another reviewer, we moved this text to the discussion (Line 517-527).

Comment:

(10) Line 314-316: If the species can tolerate warmer temperatures better, why would they migrate?

We apologize for the confusion, and we have reworded the section as follows: “Emerging mean conditions in areas adjacent to the ranges of southern species may offer opportunities for range expansions of these relative climate specialists, which can then tolerate climate warming in areas of range expansion better than more cool-adapted historical occupants (Day et al., 2018).” (Lines 445-448).

Comment:

(11) Line 334-335: Species' tolerance to temperature likely depends on their traits, which were not tested in this study. This should be noted.

We agree, and we have removed the wording “rather than traits” from this sentence (Line 479).

Reviewer #3 (Recommendations for the authors):

Comment:

(1) Title: The title is too general not specifying that your results are on odonates only, but also stressing the implicit role of climate change to a degree the tests do not support.

Following this comment and a suggestion from another reviewer we changed the title to the following: “Range geography and temperature variability explain cross-continental convergence in range and phenology shifts in a model insect taxon”. We wanted to emphasize our use of Odonates as a model species that we used to ask broad questions, while being more specific about the climatic variable that we examined (temperature variability).

Comment:

(2) L32: consider including Novella-Fernandez et al. 2023 (NatCommun) which addresses this topic in Odonates.

Thank you for suggesting this very interesting paper, we have added it as a citation (Line 31-32).

Comment:

(3) L35: consider including Grewe et al. 2013 (GEB) and Engelhardt et al. 2022(GCB).

Thank you for these excellent suggestions, we have added the citations (Line 35).

Comment:

(4) L47: rather write 'result from' instead of 'driven by'.

We agree this is a better characterization and have corrected the wording (Line 48-49).

Comment:

(5) L49-52: There has been a recent study on this topic for birds (Neate-Clegg et al., 2024 NEE). However, specifying this to insects would make it not less relevant. This review for odonates might be helpful in this regard (Pinkert et al.. 2022, Chapter: "Odonata as focal taxa for biological responses to climate change" IN Dragonflies & Damselflies: Córdoba-Aguilar et al. (2022) Model Organisms for Ecological and Evolutionary Research.

Thank you for again suggesting excellent references, we have added them to line 52-53, as well as adding the Pinkert citation to lines 61 and 82.

Comment:

(6) L53-66: Combine into one paragraph about drivers. With traits first and the environment second. The natural land cover perspective may be too complicated in this context. Consider focusing on generalities of the impact of changes within species' ranges.

As suggested we have combined these into one paragraph about drivers (Line 59).

Comment:

(7) L67-69: The book from before would be a much stronger reference for this claim. Kalkmann et al (2018) do not address the emphasis of global change research in insects on bees and butterflies. Also, I would highlight that most of the current work is at a national scale, rather than cross-continental.

Thank you for this suggestion, we have added the suggested reference and included that “…recently assembled databases of odonate observations provide a rare opportunity to investigate species’ spatiotemporal responses at larger taxonomic and spatial scales, particularly as most work has been done at national scales.” (Lines 75-77).

Comment:

(8) L68: consider rephrasing this part to '..provide a rare opportunity to investigate spatiotemporal biotic responses at larger taxonomic and spatial scales'

We appreciate this suggestion and really like the wording. We have changed the phrase to read as follows: “While global change research on insects often emphasizes butterfly and bee taxa, recently assembled databases of odonate observations provide a rare opportunity to investigate species’ spatiotemporal responses at larger taxonomic and spatial scales, particularly as most work has been done at national scales.” (Lines 74-77).

Comment:

(9) L69: This characteristic is not unique to odonates and would hamper drawing general conclusions. Honestly, I think the detailed and comprehensive data on them is the selling point.

Thank you for this suggestion, we have edited the sentence to emphasize their use as an indicator species: “Due to their use of aquatic and terrestrial habitat across life different stages, dragonflies and damselflies are also considered indicator species for both terrestrial and aquatic insect responses to changing climates (Hassall, 2015; Pinkert et al., 2022; Šigutová et al., 2025), giving the study of these species broad relevance for conservation.” (Lines 78-81)

Comment:

(10) L73: Indicator for what? The first part of the sentence would suggest lesser surrogacy for responses of other taxa. Reconsider this statement. They are well- established indicators for habitat intactness and freshwater biodiversity. Darwell et al. suggested their diversity can serve as a surrogate for the diversity of both terrestrial and aquatic taxa.

Thank you for this suggestion, we have edited the sentence to emphasize their use as an indicator species: “Due to their use of aquatic and terrestrial habitat across life different stages, dragonflies and damselflies are also considered indicator species for both terrestrial and aquatic insect responses to changing climates (Hassall, 2015; Pinkert et al., 2022; Šigutová et al., 2025), giving the study of these species broad relevance for conservation.” (Lines 78-81)

Comment:

(11) L76: Fritz et al., is a study on mammals, not odonates.

Thank you for pointing out this error, the reference has been removed (Line 84-85).

Comment:

(12) L84: Lotic habitats are generally better connected than lentic ones. Lentic species are considered to have a greater propensity for dispersal DUE to the lower inherent spatiotemporal stability (implying lower connectivity) compared to lotic habitats.

Thank you for your comment, we have rewritten this section as follows: “For example, differences in habitat connectivity and dispersal ability may constrain range shifts for lentic species (those species that breed in slow moving water like lakes or ponds) and lotic species (those living in fast moving-water) in different ways (Kalkman et al., 2018). More southerly lentic species may expand their range boundaries more than lotic species, as species accustomed to ephemeral lentic habitats better dispersers (Grewe et al., 2013), yet lotic species have also been found to expand their ranges more often than lentic species, potentially due to the loss of lentic habitat in some areas (Bowler et al., 2021).” (Lines 88-95).

Comment:

(13) L90: I would be cautious with this interpretation. If only part of the range is considered (here a country in the northern Hemisphere) southern species are moving more of their range into and northern species more of their range out of the study area in response to warming (implying northward shifts).

We have clarified this section as follows: “While warm-adapted species with more equatorial distributions could expand their ranges poleward following warming (Devictor et al., 2008), they could also increase in abundance in this new range area relative to species that historically occupied those areas and are less heat-tolerant (Powney et al., 2015).” (Lines 95-121)

Comment:

(14) L117: Odonata Central contains many county centroids as occurrence records. These could be an issue for your use case. I may have overlooked the steps you took to address this, but I think this requires at least more detail and possibly further removal/checks using for instance CoordinateCleaner. The functions implemented in this package allow you to filter records based on political units to avoid exactly this source of error.

Thank you for this suggestion, we weren’t aware of this issue with Odonata Central. We used the CoordinaterCleaner tool in R to filter all odonate records that we used in our analyses. Less than 1% of observations in our dataset were identified as having potential problems by the tool, so we would not expect this to affect our inferences. However, in future we will employ this tool when using similar datasets.

Comment:

(15) L119: Please add a brief explanation of why this was necessary. I am ok with something along the lines in the supplement.

We moved this information from the supplemental to the main text as follows: “If a species was found on both continents, we only retained observations from the continent that was the most densely sampled. If we merged data for one species found on both continents, we could not perform a cross-continental comparison. However, if the same species on different continents was treated as different species, this would lead to uninterpretable outcomes (and the creation of pseudo-replication) in the context of phylogenetic analyses. In addition, species found on both continents did not have sufficient data to meet criteria for the phenology analysis.” (Lines 161-167).

Comment:

(16) L132: This is the letters 'X' or 'x' are not multiplier symbols! Please change to the math symbol (×), everywhere.

Thank you for pointing out this error, we have made the correction throughout the manuscript.

Comment:

(17) L133: add 'main' before 'flight period'

Thank you for this suggestion, we have made the change. (Line 190)

Comment:

(18) L135: I suggest using the coefficient of variation, as it is controlled for the mean. Otherwise, what you see is partly the signature of temperature and not of its variation. For me, it's very difficult to understand what this variation of the variation means and at least needs more explanation.

Thank you very much for this suggestion, we agree that using the coefficient of variation is a better fit for the question that we’re asking. We re-ran out analyses with the coefficient of variation as the measure of climate variability: all the results reported in the manuscript are now updated for that analysis (Line 377, Table 2), and we have also updated the methods section (Line 191). The results are qualitatively the same to our previous analysis, but we agree that they are now easier to interpret.            

Comment:

(19) L155: Please adequately reference all R packages (state the name, and a reference for them including the authors' names, title, and version).

Thank you for pointing out this omission, we have added reference information for the glm function in base R (Line 298) and ensured all other packages are properly referenced.

Comment:

(20) L207: Mention the literature sources here (again).

We agree that they should be referenced here again, and we have done so (Lines 267-268).

Comment:

(21) L209: You could use the number of grid cells as a proxy for range size.

Following this excellent suggestion, we re-analysed our data using range size, calculated as the number of quadrats occupied by a species in the historical time period, as a predictor. Range size was not significant in our models, but we believe this is the best way to analyze our data, and so have updated our methods (Lines 261-263) and results (375-378).

Comment:

(22) L218: It would be preferable to say 'species-level' instead of 'by-species'.

Thank you for this suggestion, we agree that this is clearer and made the change (Line 298).

Comment:

(23) L219-220: this is unclear. Please rephrase.

We have clarified as follows: “We used both species-level frequentist (GLM; glm function in R) and Bayesian (Markov Chain Monte Carlo generalized linear mixed model, MCMCglmm; Hadfield, 2010) models to improve the robustness of the results.” (Lines 298-300).

Comment:

(24) L224: At least for Europe there is a molecular phylogeny available, which you should preferably use (Pinkert et al. 2018, Ecography). Otherwise, I am ok with using what is available

We apologize that the nature of the phylogeny that we used was not clear; the phylogeny that we used was built similarly to that in Pinkert et al. 2018, Ecography. It created a molecular phylogeny with a morphological/taxonomic tree as the backbone tree, so that species could only move within their named genera or families. We clarified this in the manuscript as follows:

“We used the molecular phylogenetic tree published by the Odonate Phenotypic Database (Waller et al., 2019), which used a morphological and taxonomic phylogeny as the backbone tree, allowing species to move within their named genera or families according to molecular evidence (Waller and Svensson, 2017).” (Lines 302-305).

Comment:

(25) L233: You said so earlier (1st sentence of this paragraph).

Thank you for pointing this out, we removed the repetitive sentence (Line 323).

Comment:

(26) L236-238: To me, it makes more sense to test this prior to fitting the phylogenetic models.

MCMC-GLMM is considerably less familiar to most researchers than general linear models or there derivatives/descendants, such as PGLS. We report models both with and without phylogenetic relationships included for the sake of transparency, and we are happy to acknowledge that no interpretation here changes substantially relative to these decisions. However, failing to report models that included possible (if small) effects of phylogenetic relatedness might cause some readers to question what those models might have implied. For the moment, we are opting for the most transparent reporting approach here.

Comment:

(27) L241: Rather say directly XX of XX species in our data....

(28) L245: Same here. Provide the actual numbers, please.

Thank you for this suggestion, we made this change on Line 332 and Line 334.

Comment:

(29) L247-249: Then not necessary.

This issue highlights a challenge in the global biology literature and around the issue of biodiversity monitoring for understanding global change impacts on species. Almost no studies have been able to report simultaneous range and phenology shifts, and the literature addresses these biotic responses to global change predominantly as distinct phenomena. Differences in numbers of species for which these observations exist, even among the extremely widely-observed odonates, seems to us to be a meaningful issue to report on. If the reviewer prefers that we abbreviate or remove this sentence, we are happy to do so.

Comment:

(30) L251:261: That is discussion as you interpret your results.

Following your suggestion and the suggestion of another reviewer, we moved the following lines to the discussion section: “Species that did not shift their ranges northwards or advance their phenology included Coenagrion mercuriale, a European species that is listed as near threatened by the IUCN Red List (IUCN, 2021), and is projected to lose 68% of its range by 2035 (Jaeschke et al., 2013).” (Lines 517-527).

Comment:

(31) 252: Good to mention, but why is the discussion limited to C. mercurial?

We feel that it is important to link the broad-scale results to the specific biological characteristics of individual species, and C. mercurial is an IUCN threatened species. We are happy to expand links to natural history of this group and have added the following: “This group also includes Coenagrion resolutum, a common North American damselfly (Swaegers et al., 2014), for which we could not find evidence of decline. This may be due in part to the greater area of intact habitat available in North American compared to Europe, enabling C. resolutum to maintain larger populations that are less vulnerable to stochastic climate events. Still, this and other species failing to shift in range or phenology should be assessed for population health, as this species could be carrying an unobserved extinction debt.” (Lines 527-533).

Comment:

(32) L264: Insert 'being' before 'consistently'.

Thank you for the suggestion, we made this change (Line 373).

Comment:

(33) L271: .'. However,'.

Thank you for pointing out this grammatical error, we have corrected it (Line 382).

Comment:

(34) L273: 'affected' instead of 'predicted'

Thank you for the suggestion, we made this change (Line 383).

Comment:

(35) L279: 'despite pronounced recent warming' sounds not relevant in this context.

Thank you for this suggestion, we removed this portion of the sentence (Line 408).

Comment:

(36) L281: Rather 'the model performance did not improve....'

Thank you for the suggestion, we made this change (Line 409).

Comment:

(37) L288: Add 'but' before 'not'.

Thank you for the suggestion, we made this change (Line 416).

Comment:

(38) L311-316: Reconsider the causality here. maybe rather rephrase to are associated instead. Greater dispersal ability and developmental plasticity might well lead to higher growth rates, rather than the other way around.

We agree that plasticity/evolution at range edges is important to consider and have included it as an alternative explanation: “Adaptive evolution and plasticity may enable higher population growth rates in newly-colonized areas (Angert et al., 2020; Usui et al., 2023), but this possibility can only be directly tested with long term population trend data.” (Line 449-451).  

Comment:

(39) L313-316: Maybe delete the second 'should be able to'.

This phrase has been changed in response to other reviewer comments and now reads as follows:

“Emerging mean conditions in areas adjacent to the ranges of southern species may offer opportunities for range expansions of these relative climate specialists, which can then tolerate climate warming in areas of range expansion better than more cool-adapted historical occupants (Day et al., 2018).” (Lines 445-448).

Comment:

(40) L331: Limit this statement ending with 'in North American and European Odonata'.

Thank you for this suggestion, we made this addition (Lines 475-476).

Comment:

(41) L346-347: There are too many of these more-research-is-needed statements in the discussion (at least three in the last paragraphs). Please consider finishing the paragraphs rather with a significance statement.

Thank you for this suggestion, we have changed the final sentence here to the following: “The extent to which species’ traits actually determine rates of range and phenological shifts, rather than occasionally correlated with them, is worth considering further, but functional traits do not systematically drive patterns in these shifts among Odonates in North America and Europe.” (Lines 480-483).

We also made additional changes, removing a ‘more-research is needed’ statement from the following paragraph (Line 443), as well as from line 499.

Comment:

(42) L349: See also Franke et al. (2022, Ecology and Evolution).

Thank you for highlighting this excellent reference! We have added it to Line 501.

Comment:

(43) L363: Maybe a bit late in the text, but it is important to note that there is the third dimension 'abundance trends' or rather a common factor related to range and phenology shifts. I feel this fits better with the discussion of population growth.

Thank you for this suggestion, we have addressed the importance of abundance trends in the following sentences: “Further mechanistic understanding of these processes requires abundance data.” (Lines 442-443); “It remains unclear if range and phenology shifts relate to trends in abundance, but our results suggest that there are clear ‘winners’ and ‘losers’ under climate change.” (Lines 509-510).

Comment:

(44) L375-377: This last sentence is very similar to L371-373. Please reduce the redundancy. Focus more on specifically stating the process instead of vaguely saying 'new insights into patterns' and 'suggesting processes'. Rather, deliver a strong concluding message here.

Thank you for this suggestion, we feel that we now have a much stronger concluding message: “By considering both the seasonal and range dynamics of species, emergent and convergent climate change responses across continents become clear for this well-studied group of predatory insects.” (Lines 545-547).

Comment:

(45) Table 1: To me, the few estimates presented here do not justify a table. rather include them in the text. OR combine them with Table 2. Also, why not include the traits as predictors (from the range shift models) in these models as well?

We have clarified in the text that the results displayed in Table 1 are from the analysis of the relationship between range and phenology shifts: “The effect of species’ range shifts on phenology range shifts was significant in our model investigating the relationship between these responses, indicating that species shifting their northern range limits to higher latitudes also showed stronger advances in their emergence phenology (Figure 3).” (Lines 341-344).

As there were no significant effects in the model of phenology change drivers, we have not shown results of this model: “Emergence phenology shifts were not affected by species’ traits, range geography, nor climate variability; due to this, model results are not displayed here.” (Lines 383-384).

Comment:

(46) Table 2: L712-713: What does this mean? Are phenology shifts not used as a predictor of range shifts? (why then this comment?). Or do you want to say phenological shifts are not related to Southern range etc? Why do you present a phylosig here but not in Table 1? Why not include the traits as predictors (from the range shift models) in these models as well? Consider using the range size as a continuous predictor instead of 'Widespread'.

We are glad the reviewer pointed this out to us. We did not emphasize this issue sufficiently. We DID evaluate traits as predictors both of geographical range and phenological shifts, and species-specific biological traits did not significantly affect models predicting either of those sets of responses. We state this on Lines 312-323, but we have also noted in the discussion (Lines 473-476) that the most commonly assessed traits, like body size, do not alter observed trends here. Instead, where species are found, rather than the characteristics of species, is the key determinant of their overall responses.

Following this excellent suggestion, we re-analysed our data using range size, calculated as the number of quadrats occupied by a species in the historical time period, as a predictor. Range size was not significant in our models, but we believe this is the best way to analyze our data, and so have updated our methods (Lines 261-263) and results (375-378).

Comment:

(47) Figure 1: I don't see any grey points in the figure. Also, there is no A or B. If you are referring to the symbols then write cross and triangle instead and not use capital letters which usually refer to component plots of composite figures. Also, I highly recommend providing a similar figure based on your data (maybe each species as a dot for T1 and another symbol for T2). Given the small number of species, you could try to connect these points with arrows. For the set with only range shifts maybe play the T2-dots at the center of the 'Emergence' axis.

Thank you for pointing out this error: a previous version of Figure 1 included grey points and multiple panels. We have removed this text from the figure caption to be consistent with the final version of the figure (Line 989).

The graphical depictions of the conceptual and empirical discoveries in this paper were challenging to create. The reviewer might be suggesting effectively decomposing Figure 3 (change in range on the y axis vs change in phenology among all species into two sets of points on the same graph, where each pair of points is a before and after value for each species. This would make for a very busy figure indeed. We have modified the conceptual Figure 1 to illustrate more clearly, we believe, that species can (in principle) remain within tolerable niche spaces by shifting their activity periods in time (phenology) or in space (geographical range) or both.

Comment:

(48) Figure 2: Please add a legend. Also black is a poor background color. The maps appear to be stretched. Please check aspect ratios. Now here are capital letters without an explanation in the caption. From the context I assume the upper panel maps are for the data used to calculate range shifts at the bottom panel maps are for data used to calculate the phenological shifts.

We apologise for the error in the figure caption and have clarified the differences between panels in the text, as well as changing the map background colour and fixing the aspect ratio:

“Figure 2: Richness of 76 odonate species sampled in North America and Europe in the historic period (1980-2002; panes A and C) and the recent period (2008-2018; panes B and D). Species richness per 100 × 100 km quadrat is shown in panes A and B, while panes C and D show species richness per 200 × 200 km quadrat. Dark red indicates high species richness, while light pink indicates low species richness.” (Lines 1002-1006).

Comment:

(49) Figure 3: Why this citation? Of terrestrial taxa? Please explain. Consider adding some stats here, such as the r-squared value for each of the relationships.

We have better explained the citation in the figure caption, as well as adding r-squared values:

“Figure 3: Relationship between range shifts and emergence phenology shifts among North American and European odonate species (N = 66; model R2 = 17.08 for glm, 14.9% for MCMCglmm). For reference, the shaded area shows mean latitudinal range shifts of terrestrial taxa as reported by Lenoir et al. (2020; calculated as the yearly mean dispersal rate of 1.11 +/- 0.96 km per year over 38 years).” (Lines 679-682)

Comment:

(50) L801: What are these underscored references?

This was an issue with the reference software and has been resolved.

Comment:

(51) Table S1: L848: Consider starting with 'Samples of 76 North American and European odonate species from between ...'. Please use a horizontal line to separate the content from the table header. Add a horizontal line below the last row. Same for all tables.

Thank you for this suggestion, we have edited the caption for Figure S1 as suggested (Line 1124). We have also made the suggested line additions to Table S1, S2, and S3.

Comment:

(52) Table S3: This is confusing. In Table 1 (main text) both 'southern range' and 'widespread' are used as predictors. Please explain.

We originally included information on species range geography, including southern versus northern range, and widespread versus not, into one categorical variable. Following additional comments we re-analysed our data using range size, calculated as the number of quadrats occupied by a species in the historical time period, as a predictor. Now the methods section text (Lines 261-263) and Table 1 report results of that variable with distribution options northern, southern, or both. 

Comment:

(53) Figure S5 and S6: It would be more coherent if the colors refer to the continents and the suborders are indicated by shading. I would love to see a combination of the two figures with species ordered by the phylogenetic relationship and a dot matrix indicating the traits in the main text! This could really be a good starting point for a synthesis figure.

The reviewer presents an interesting challenge for us. We have a choice, as we understand things, to present a figure showing phylogeny and traits (as requested here), or an ordered list of species relative to effect sizes in the two main responses to global change. The latter choice centers on the discoveries of the paper, while the former would be valuable for dragonfly biology but would depict information that proved to be biologically uninformative relative to our discovery. That is to say, there is no phylogenetic trend and biological traits among species did not affect results. We have gone some way toward illustrating that issue by retaining phylogeny in the MCMC-GLMM models, but we feel that a figure illustrating phylogeny and traits would (for most readers, at least) illustrate noise, rather than signal. For this reason, we have opted to take on the previous reviewer’s suggestion for a modified, main-text Figure 4, which we include below.

Figure 4: Distribution of Northern range limit shifts (Panel A, kilometers) and emergence phenology shift (Panel B, Julian day) of 76 European and North American odonate species between a recent time period (2008 - 2018) and a historical time period (1980 - 2002). Anisoptera (dragonflies) are shown in pink, Zygoptera (damselflies) are shown in blue.

Change last: Figure 3: Relationship between range shifts and emergence phenology shifts among North American and European odonate species (N = 66; model R2 = 17.08 for glm, 14.9% for MCMCglmm). For reference, the shaded area shows mean latitudinal range shifts of terrestrial taxa as reported by Lenoir et al. (2020; calculated as the yearly mean dispersal rate of 1.11 +/- 0.96 km per year over 38 years).

DOI: 10.1101/2025.07.31.667991

Resource: RRID:BDSC_24371

Curator: @scibot

SciCrunch record: RRID:BDSC_24371

What is this?

This reminds me of how most movies/shows are/were based on fictional stories, and how these films often inspire(d) new movies or rewrites, just like how earlier TV comedy borrowed from older forms of humor from the radio.

So much is found in this paragraph. The reference to families that were not welcome into the middle class is striking. The fact that advertising of the same product had to be made separately, one for white families and another for black families, is a reminder of how different times were in comparison to where we are today. We still have a long way to go.

I like to emphasize the word "replace." How many Beethovens did the world lose? Families were replacing pianos with TV sets. It became easier to watch someone on TV play the piano than to learn how to play the piano.

ponto de nao retorno

• Informativo nº 688
• 15 de março de 2021.
• RECURSOS REPETITIVOS
• Processo: REsp 1.769.306-AL, Rel. Min. Benedito Gonçalves, Primeira Seção, por unanimidade, julgado em 10/03/2021. (Tema 1009).

Ramo do Direito DIREITO ADMINISTRATIVO

Tema - Servidor público. Devolução de valores recebidos. Artigo 46, caput, da Lei n. 8.112/1990. Revisão da tese definida no Tema repetitivo 531/STJ. Ausência de alcance nos casos de pagamento indevido decorrente de erro de cálculo ou operacional da administração pública. Possibilidade de devolução. Salvo inequívoca presença da boa-fé objetiva. Tema 1009.

DESTAQUE - Os pagamentos indevidos aos servidores públicos decorrentes de erro administrativo (operacional ou de cálculo), não embasado em interpretação errônea ou equivocada da lei pela Administração, estão sujeitos à devolução, ressalvadas as hipóteses em que o servidor, diante do caso concreto, comprova sua boa-fé objetiva, sobretudo com demonstração de que não lhe era possível constatar o pagamento indevido.

INFORMAÇÕES DO INTEIRO TEOR - A controvérsia consiste em definir se a tese firmada no Tema 531/STJ seria igualmente aplicável aos casos de erro operacional ou de cálculo, para igualmente desobrigar o servidor público, de boa-fé, a restituir ao erário a quantia recebida a maior.

• No julgamento do Recurso Especial Repetitivo n. 1.244.182/PB (Tema 531/STJ), definiu-se que quando a Administração Pública interpreta erroneamente uma lei, resultando em pagamento indevido ao servidor, de boa-fé, cria-se uma falsa expectativa de que os valores recebidos são legais e definitivos, impedindo, assim, que ocorra desconto dos mesmos, o que está em conformidade com a Súmula 34 da Advocacia Geral da União - AGU.

• Assim, acerca da impossibilidade de devolução ao erário de valores recebidos indevidamente por servidor público, de boa-fé, em decorrência de equívoco na interpretação de lei pela Administração Pública, constata-se que o tema está pacificado.

• O artigo 46, caput, da Lei n. 8.112/1990 estabelece a possibilidade de reposições e indenizações ao erário. Trata-se de disposição legal expressa, plenamente válida, embora com interpretação dada pela jurisprudência com alguns temperamentos, especialmente em observância aos princípios gerais do direito, como boa-fé, a fim de impedir que valores pagos indevidamente sejam devolvidos ao erário.

• Diferentemente dos casos de errônea ou má aplicação de lei, onde o elemento objetivo é, por si, suficiente para levar à conclusão de que o beneficiário recebeu o valor de boa-fé, assegurando-lhe o direito da não devolução do valor recebido indevidamente, na hipótese de erro material ou operacional deve-se analisar caso a caso, de modo a averiguar se o servido tinha condições de compreender a ilicitude no recebimento dos valores, de modo a se lhe exigir comportamento diverso, diante do seu dever de lealdade para com a Administração Pública.

• Impossibilitar a devolução dos valores recebidos indevidamente por erro perceptível da Administração Pública, sem a análise do caso concreto da boa-fé objetiva, permitiria o enriquecimento sem causa por parte do servidor, em flagrante violação do artigo 884 do Código Civil.

• Por tudo isso, não há que se confundir erro na interpretação de lei com erro operacional, de modo àquele não se estende o entendimento fixado no Recurso Especial Repetitivo n. 1.244.182/PB, sem a observância da boa-fé objetiva do servidor público, o que possibilita a restituição ao Erário dos valores pagos indevidamente decorrente de erro de cálculo ou operacional da Administração Pública.

• Isto é, se houve erro de interpretação ou de aplicação de lei; não deve haver devolução;

• Se se tratar de erro de cálculo ou operacional, deve haver devolução.

dweb.link@ http://bafybeihda4gloeygr5moflptlfedkbhkuysutw7ulbomreqfx6fywro4xa.ipfs.localhost:8080/?filename=%EF%BC%82display%20metaphor%20scripting%20language%EF%BC%82%20gyuri%20lajos%20dime%20-%20Brave%20Search%20(8_12_2025%209%EF%BC%9A35%EF%BC%9A01%20AM).html

for = wikify myself

CSP vs Actor model for concurrency -

my comment

my comment on this

Aunque los biocombustibles suelen considerarse una opción ecológica, su producción a partir de cultivos agrícolas provoca más daños que beneficios: favorece la deforestación, incrementa las emisiones de CO₂ de manera indirecta, encarece los alimentos y amenaza a los ecosistemas. Solo tendrían un verdadero valor sustentable si se elaboran con residuos o mediante tecnologías que no compitan con la agricultura ni demanden grandes extensiones de tierra.

• 1. Indeferimento do pedido de aditamento da inicial para incluir as alterações trazidas pela Lei Estadual nº 12.978/2005. A jurisprudência desta Corte é no sentido de que o aditamento à inicial somente é possível nas hipóteses em que a inclusão da nova impugnação (i) dispense a requisição de novas informações e manifestações; e (ii) não prejudique o cerne da ação, o que não ocorre no presente caso. Precedente.

(ADI 1926, Relator(a): ROBERTO BARROSO, Tribunal Pleno, julgado em 20-04-2020, PROCESSO ELETRÔNICO DJe-136 DIVULG 01-06-2020 PUBLIC 02-06-2020)

• 1. Entendimento desta CORTE no sentido de que o aditamento da inicial só é possível, observados os princípios da economia e da celeridade processuais, quando a inclusão de nova impugnação dispensa a requisição de novas informações. No presente caso, não é possível tal aditamento com a finalidade de corrigir vício relativo à legislação não impugnada do complexo normativo. 4. Agravo Regimental a que se nega provimento.

(ADI 4.265, Plenário, Rel. Min. Alexandre de Moraes, j. em 09.04.2018)

Taxa de segurança preventiva relativa a eventos não gratuitos e a emissão de certidões para defesa de direitos - ADI 3.717/PR Relator: Ministro Nunes Marques

RESUMO - É constitucional a instituição de taxa por serviços prestados por órgãos de segurança pública relativos (i) à segurança preventiva em eventos esportivos e de lazer com cobrança de ingresso, bem como (ii) à emissão de certidões e atestados, desde que não se destinem à defesa de direitos ou ao esclarecimento de interesse pessoal (CF/1988, art. 5º, XXXIV, b).

• Conforme jurisprudência desta Corte (1), o serviço de segurança pública e as atividades a ela inerentes, como policiamento ostensivo e vigilância, não podem ser financiados mediante taxas, dada a impossibilidade de que sua prestação ocorra de forma individualizada. Assim, por constituir serviço geral e indivisível, prestado a toda a coletividade, este deve ser remunerado por meio de impostos. Contudo, há situações em que os serviços, apesar de prestados por órgãos de segurança pública, são efetivamente oferecidos de modo específico e divisível. Nesse contexto, prestações oferecidas atipicamente pelos órgãos de segurança pública e que são usufruídas de modo particular pelos administrados podem ser custeadas por meio de taxas (2).

• Na espécie, a operação logística necessária para garantir a segurança em eventos de grande porte, com finalidade lucrativa, não pode ser imputada à sociedade como um todo através de um financiamento indistinto, arrecadado pelo poder público via impostos. Também não é cabível partilhar, entre toda a sociedade, os custos de serviços prestados pelos órgãos da Administração Policial Militar estadual para fornecimento, entre outros, de “cópias (xerox) autenticadas (por folha)”, “diárias/permanência de veículos apreendidos nas unidades policiais militares”, fotografias e inscrição em cursos e exames. Por expressa vedação constitucional (3), a cobrança de taxa não é válida apenas para o fornecimento de certidões e atestados direcionados à defesa de direitos ou ao esclarecimento de interesse pessoal (4). Com base nesses entendimentos, o Plenário, por unanimidade, julgou parcialmente procedente a ação para (i) declarar a inconstitucionalidade dos itens 1.1.1 e 1.2 (1.2.1 a 1.2.5) da tabela anexa à Lei nº 10.236/1992 do Estado do Paraná; e (ii) dar interpretação conforme aos itens 2.1 e 2.3 da mesma lista, no sentido de impossibilitar a cobrança de taxa para emissão de certidões/atestados solicitados com o propósito de defender direitos e esclarecer situações de interesse pessoal.

• Também não é cabível partilhar, entre toda a sociedade, os custos de serviços prestados pelos órgãos da Administração Policial Militar estadual para fornecimento, entre outros, de “cópias (xerox) autenticadas (por folha)”, “diárias/permanência de veículos apreendidos nas unidades policiais militares”, fotografias e inscrição em cursos e exames. Por expressa vedação constitucional (3), a cobrança de taxa não é válida apenas para o fornecimento de certidões e atestados direcionados à defesa de direitos ou ao esclarecimento de interesse pessoal (4). Com base nesses entendimentos, o Plenário, por unanimidade, julgou parcialmente procedente a ação para (i) declarar a inconstitucionalidade dos itens 1.1.1 e 1.2 (1.2.1 a 1.2.5) da tabela anexa à Lei nº 10.236/1992 do Estado do Paraná; e (ii) dar interpretação conforme aos itens 2.1 e 2.3 da mesma lista, no sentido de impossibilitar a cobrança de taxa para emissão de certidões/atestados solicitados com o propósito de defender direitos e esclarecer situações de interesse pessoal.

• Informativo nº 857
• 12 de agosto de 2025.
• PRIMEIRA TURMA
• Processo: REsp 1.931.196-RS, Rel. Ministro Paulo Sérgio Domingues, Primeira Turma, por unanimidade, julgado em 5/8/2025.

Ramo do Direito DIREITO PROCESSUAL CIVIL, DIREITO TRIBUTÁRIO

TemaPaz, Justiça e Instituições Eficazes <br /> Execução fiscal. Juntada de título executivo relativo a terceiro. Emenda à inicial. Art. 240, § 1º, do CPC/2015. Retroação da interrupção da prescrição à data da propositura da ação. Impossibilidade.

Destaque - Não é possível considerar como válida, para fins de interrupção da prescrição, a propositura de execução fiscal fundada em certidão de dívida ativa (CDA) de contribuinte diverso.

Informações do Inteiro Teor - A controvérsia consiste em decidir se a emenda à inicial pela juntada do título executivo incorreto afasta a regra do art. 240, § 1º, do Código de Processo Civil (CPC), segundo a qual a interrupção da prescrição, operada pelo despacho que ordena a citação, retroage à data da propositura da ação.

• No caso, por ocasião do ajuizamento da execução fiscal, a Fazenda Nacional procedeu à juntada de título executivo cujo sujeito passivo não era a parte executada, ou seja, referente a empresa distinta. Intimado, o ente fazendário apresentou o documento correto, prosseguindo, assim, a tramitação do processo.

• Inicialmente, afasta-se a aplicação da Súmula n. 392 do Superior Tribunal de Justiça, que admite a substituição da certidão de dívida ativa (CDA) até a prolação da sentença de embargos, nos casos de correção de erro material ou formal, vedada a modificação do sujeito passivo da execução. Isso porque, na hipótese, não se discute a validade da CDA em si, mas sim a juntada equivocada de título executivo alheio à parte executada, o que comprometeria a regularidade da petição inicial.

• A correção desse vício atrai a incidência do Código de Processo Civil, aplicável de forma subsidiária à execução fiscal, nos termos do art. 1º da Lei n. 6.830/1980.

• Assim, é cabível a aplicação do art. 321 do CPC para permitir a emenda da petição inicial, com a correção dos documentos que a instruem. Apenas em caso de inércia da parte após a intimação é que se justifica o indeferimento da inicial.

• Contudo, embora haja previsão legal para a correção de defeitos ou irregularidades na petição inicial, para o Superior Tribunal de Justiça, quando a petição inicial é protocolada em desacordo com o disposto no art. 319 do CPC, de modo a impedir o desenvolvimento válido e regular do processo, a interrupção da prescrição, nos termos do art. 240, § 1º, do CPC, somente retroage à data da emenda da inicial (AgInt no REsp n. 1.749.085/DF, rel. Ministro Marco Buzzi, Quarta Turma, julgado em 2/10/2023, DJe de 5/10/2023; e AgInt no REsp n. 1.746.781/PE, rel. Ministro Napoleão Nunes Maia Filho, Primeira Turma, julgado em 25/5/2020, DJe de 28/5/2020).

• Aplica-se esse entendimento ao caso, visto que seria completamente inviável o prosseguimento da execução fiscal cujo sujeito passivo do título executivo não correspondesse à parte executada.

• SEGUNDA TURMA

• Processo: AREsp 2.523.152-CE, Rel. Ministro Francisco Falcão, Segunda Turma, por unanimidade, julgado em 21/5/2024, DJe 23/5/2024.

• Ramo do Direito <br /> DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL, DIREITO TRIBUTÁRIO

TemaPaz, Justiça e Instituições Eficazes <br /> Embargo à execução. Desistência do embargado. Adesão ao REFIS. Previsão de pagamento de honorários. Nova cobrança. Bis in idem.

DESTAQUE - Havendo a previsão de pagamento, na esfera administrativa, dos honorários advocatícios, na ocasião da adesão do contribuinte ao Programa de Parcelamento Fiscal, a imposição de pagamento da verba honorária, quando da extinção da execução fiscal, configura bis in idem, sendo vedada nova fixação da verba.

INFORMAÇÕES DO INTEIRO TEOR - Havendo a previsão de pagamento, na esfera administrativa, dos honorários advocatícios, na ocasião da adesão do contribuinte ao Programa de Parcelamento Fiscal, a imposição de pagamento da verba honorária, quando da extinção da execução fiscal, configura bis in idem, sendo vedada nova fixação da verba. Tal entendimento, inclusive, foi cristalizado no enunciado do Tema repetitivo n. 400/STJ.

• Nesse mesmo sentido, destaca-se: [...] V. Na esteira do entendimento firmado nesta Corte, em regra, a desistência da Ação Anulatória ou dos Embargos à Execução, decorrente da adesão do contribuinte ao Programa de Parcelamento, não implica o afastamento da condenação aos honorários advocatícios. [...] VI. Todavia, a jurisprudência desta Corte orienta-se no sentido de que, havendo a previsão de pagamento, na esfera administrativa, dos honorários advocatícios, quando da adesão do contribuinte ao Programa de Parcelamento Fiscal, a imposição de pagamento da verba honorária, quando da extinção da Execução Fiscal, configura bis in idem. [...] (AgInt no REsp n. 1.994.559/MG, relatora Ministra Assusete Magalhães, Segunda Turma, julgado em 14/11/2022, DJe de 22/11/2022).

• Informativo nº 856
• 5 de agosto de 2025.

• PRIMEIRA TURMA

• Processo: REsp 2.032.814-RS, Rel. Ministro Gurgel de Faria, Rel. para acórdão Ministro Paulo Sérgio Domingues, Primeira Turma, por maioria, julgado em 10/6/2025, DJEN 30/6/2025.

Ramo do Direito DIREITO TRIBUTÁRIO

TemaPaz, Justiça e Instituições Eficazes <br /> Transação tributária. Renúncia para fins de adesão. Silêncio da legislação. Condenação em honorários advocatícios com base no art. 90 do CPC/2015. Não cabimento. Princípios da segurança jurídica, da boa-fé, da proteção e da confiança. Violação.

Destaque - A cobrança, pela Fazenda Pública, de honorários advocatícios sem previsão na legislação que instituiu as condições da transação tributária viola os princípios da segurança jurídica, da boa-fé do administrado e da proteção da confiança.

Informações do Inteiro Teor - Discute-se, no caso, se a parte recorrida, que renunciou ao direito pleiteado na presente ação como condição para aderir à transação tributária prevista na Lei n. 13.988/2020, deve ser condenada ao pagamento de honorários advocatícios, com base no artigo 90 do Código de Processo Civil/2015, aplicado subsidiariamente.

• Dentre os compromissos a serem assumidos pelo administrado/contribuinte na celebração da transação, previstos no art. 3º da Lei n. 13.988/2020, está a renúncia do direito objeto do litígio, independentemente de qual ação judicial está sendo utilizada para discutir o valor cobrado pelo fisco. O parágrafo 1º deixa evidente que o administrado/contribuinte deve aceitar todas as condições estabelecidas na Lei e sua regulamentação, confessando o débito.

• Diferentemente de outros acordos que possam ser realizados, os dispositivos legais transcritos deixam clara a supremacia da Fazenda Nacional na celebração da transação, ao fixar suas condições no edital que a parte aderirá ou não. Não há negociação e sim o aceite ou não pelo administrado/contribuinte das condições impostas, ou seja, não há horizontalidade na relação.

• Por sua vez, quanto à incidência dos honorários advocatícios na renúncia, pelo contribuinte, das ações judiciais nas quais o valor transacionado está sendo discutido a Lei n. 13.988/2020 é omissa. Assim, essa é a questão que se coloca: realizada a adesão do contribuinte à transação, em caso de silêncio da respectiva lei regente, deve ser aplicado subsidiariamente o CPC/2015, como lei geral, para arbitramento de honorários quando da renúncia ao direito em que se fundam ações judiciais em andamento?

• A transação apresenta verdadeira novação em relação ao crédito tributário que estava sendo discutido judicialmente. Toma-se o valor do crédito, divide-se pelo número de parcelas, e eis o valor que será cobrado do contribuinte.

• Não é possível admitir que, após a transação, se venha a incluir no montante transacionado novos valores não previstos na lei que a instituiu nem no edital com o qual o contribuinte concordou. A cobrança de honorários advocatícios não previstos no instrumento de transação - elaborado pela própria Fazenda Nacional - viola os princípios da boa-fé e da não-surpresa.

• Nessa esteira de raciocínio está o venire contra factum proprium, implícito na cláusula geral da boa-fé objetiva, pois não há previsão de honorários na lei que rege a matéria nem na Portaria da transação elaborada pela própria Fazenda Nacional. Assim, não cabe a ela requerer ao Poder Judiciário que supra uma lacuna que ela mesma criou.

• Não se trata aqui de negar vigência ao art. 90 do CPC/2015, que versa sobre a incidência de honorários sucumbenciais em caso de renúncia ao direito sobre o qual se funda a ação. É que a renúncia, em geral, é o ato unilateral da parte, a qual havia ingressado em Juízo e, por qualquer razão, desejou deixar de litigar. Aplica-se a regra geral do CPC/2015.

• Contudo, no caso da transação tributária, o negócio jurídico realizado tem todas as suas condições estabelecidas na nova lei que a instituiu. E elas estão todas previstas no artigo 3º da Lei n. 13.988/2020 e respectivas regulamentações.

• No tocante ao princípio da segurança jurídica, o Superior Tribunal de Justiça possui entendimento de que: "a proteção da confiança no âmbito tributário, uma das faces do princípio da segurança jurídica, prestigiado pelo CTN, deve ser homenageada, sob pena de olvidar-se a boa-fé do contribuinte, que aderiu à política fiscal de inclusão social, concebida mediante condições onerosas para o gozo da alíquota zero de tributos." (REsp 1.928.635/SP, rel. Ministra Regina Helena Costa, Primeira Turma, julgado em 10/8/2021, DJe de 16/8/2021).

• Aqui a renúncia não é totalmente voluntária. É uma condição para a realização da transação a que o contribuinte aderiu, como a própria Fazenda Nacional alega em seu recurso especial. Por isso, somente podem ser incluídos no instrumento de transação as verbas expressamente previstas na legislação que a permitiu.

• Ou seja, a situação foge ao que ordinariamente se encontra, e não se pode aplicar a regra do CPC/2015 de forma subsidiária. Aplica-se o art. 171 do Código Tributário Nacional: somente valem as condições expressas na lei.

• Desse modo, sem previsão na legislação que instituiu as condições da transação, a Fazenda Pública não pode cobrar honorários sem violar os princípios da segurança jurídica, da boa-fé do administrado e da proteção da confiança. O silêncio da norma quanto à aplicação de honorários advocatícios não permite a aplicação do artigo 90 do CPC/2015 ao caso, pelas razões já expostas.

• Sendo assim, o fato de a Lei n. 13.988/2020 e a Portaria PGFN n. 14.402/2020 silenciarem a respeito da inclusão de honorários sucumbenciais por ocasião da renúncia em ações em andamento não constitui uma omissão a ser suprida pela aplicação subsidiária do CPC/2015. É um silêncio deliberado, que leva à aplicação da lei especial, o art. 171 do CTN e a lei específica que regula a transação e exclui a aplicação da lei geral.

• Informativo nº 857
• 12 de agosto de 2025.
• QUARTA TURMA
• Processo: REsp 2.200.180-SP, Rel. Ministro Antonio Carlos Ferreira, Quarta Turma, por unanimidade, julgado em 5/8/2025.

Ramo do Direito DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Cumprimento de sentença. Adjudicação de bens. Penhora prévia. Necessidade. Devido processo legal.

Destaque - A penhora é ato processual prévio e necessário à adjudicação de bens.

Informações do Inteiro Teor - A controvérsia jurídica diz respeito à possibilidade de se deferir a adjudicação de bem no processo de execução sem sua penhora prévia e formal.

• Os artigos 523, § 3º e 825, inciso I, do Código de Processo Civil estabelecem que a penhora é ato processual prévio e necessário à adjudicação de bens. Essa sequência lógica e cronológica decorre da própria natureza da execução forçada e do sistema de expropriação nela previsto.

• A exigência da penhora prévia como pressuposto para a adjudicação não representa mera formalidade processual, mas concretiza a garantia fundamental do devido processo legal prevista no art. 5º, LIV, da Constituição Federal, segundo o qual " ninguém será privado da liberdade ou de seus bens sem o devido processo legal".

• Dessa forma, a sequência procedimental estabelecida pelo legislador processual (penhora-avaliação-expropriação) reforça o comando constitucional, estruturando um processo executivo que equilibra a efetividade da tutela jurisdicional com as garantias do executado.

• A penhora, nessa perspectiva constitucional, representa uma etapa processual qualificada, que não pode ser suprimida por decisão judicial sem que isso implique violação à própria garantia do devido processo legal.

• A supressão da penhora viola, portanto, não apenas as disposições infraconstitucionais que regulam o procedimento executivo, mas também o núcleo essencial da garantia constitucional do devido processo legal, na medida em que permite a privação de bens do executado sem a observância do procedimento legalmente estabelecido.

• A inobservância deste pressuposto processual caracteriza nulidade absoluta, dispensando a comprovação de dano efetivo. Nesse contexto, o prejuízo é presumido ex lege, uma vez que vulnera princípios fundamentais como a segurança jurídica e o devido processo legal.

• Por fim, é relevante observar que a necessidade da penhora antecedente não se restringe à adjudicação, mas constitui requisito inafastável em qualquer modalidade de expropriação prevista no art. 825 do CPC, seja ela a adjudicação (inciso I), a alienação (inciso II) ou a apropriação de frutos e rendimentos (inciso III).

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

Learn more at Review Commons

Reply to the reviewers

Manuscript number: RC-2025-02879 Corresponding author(s): Matteo Allegretti; Alia dos Santos

1. General Statements

In this study, we investigated the effects of paclitaxel on both healthy and cancerous cells, focusing on alterations in nuclear architecture. Our novel findings show that:

• Paclitaxel-induced microtubule reorganisation during interphase alters the perinuclear distribution of actin and vimentin. The formation of extensive microtubule bundles, in paclitaxel or following GFP-Tau overexpression, coincides with nuclear shape deformation, loss of regulation of nuclear envelope spacing, and alteration of the nuclear lamina.

• Paclitaxel treatment reduces Lamin A/C protein levels via a SUN2-dependent mechanism. SUN2, which links the lamina to the cytoskeleton, undergoes ubiquitination and consequent degradation following paclitaxel exposure.

• Lamin A/C expression, frequently dysregulated in cancer cells, is a key determinant of cellular sensitivity to, and recovery from, paclitaxel treatment.

Collectively, our data support a model in which paclitaxel disrupts nuclear architecture through two mechanisms: (i) aberrant nuclear-cytoskeletal coupling during interphase, and (ii) multimicronucleation following defective mitotic exit. This represents an additional mode of action for paclitaxel beyond its well-established mechanism of mitotic arrest.

We thank the reviewers for their time and constructive feedback. We have carefully considered all comments and have carried out a full revision. The updated manuscript now includes additional data showing:

• Overexpression of microtubule-associated protein Tau causes similar nuclear aberration phenotypes to paclitaxel. This supports our hypothesis that increased microtubule bundling directly leads to nuclear disruption in paclitaxel during interphase.

• Paclitaxel's effects on nuclear shape and Lamin A/C and SUN2 expression levels occur independently of cell division.

• Reduced levels of Lamin A/C and SUN2 upon paclitaxel treatment occur at the protein level via ubiquitination of SUN2.

• The effects of paclitaxel on the nucleus are conserved in breast cancer cells.

Full Revision

We have also edited our text and added further detail to clarify points raised by the reviewers. We believe that our revised manuscript is overall more complete, solid and compelling thanks to the reviewers' comments.

1. Point-by-point description of the revisions

Reviewer #1 Evidence, reproducibility and clarity

This description of the down-regulation of the expression of lamin A/C upon treatment with paclitaxel and its sensitivity to SUN2 is quite interesting but still somehow preliminary. It is unclear whether this effect involves the regulation of gene expression, or of the stability of the proteins. How SUN2 mediates this effect is still unknown.

We thank the reviewer for this valuable comment. To elucidate the mechanism behind the decrease in Lamin A/C and SUN2 levels, we have now performed several additional experiments. First, we performed RT-qPCR to quantify mRNA levels of these genes, relative to the housekeeping gene GAPDH (Supplementary Figure 3B and O). The levels of SUN2 and LMNA mRNA remained the same between control and paclitaxel-treated cells, indicating that this effect instead occurs at the protein level. We have also tested post-translational modifications as a potential regulatory mechanism for Lamin A/C and SUN2. In addition to the phosphorylation of Ser404 which we had already tested (Supplementary Figure 3C), we have now included additional Phos-tag gel and Western blotting data showing that the overall phosphorylation status of Lamin A/C is not affected by paclitaxel (Supplementary Figure 3E and F). We also pulled-down Lamin A/C from cell lysates and then Western blotted for polyubiquitin and acetyl-lysine, which showed that the ubiquitination and acetylation states of Lamin A/C are also not affected by paclitaxel (Supplementary Figure 3G-I). However, Western blots for polyubiquitin of SUN2 pulled down from cell lysates showed that paclitaxel treatment results in significant SUN2 ubiquitination (Figure 3M and N). Therefore, we propose that the downregulation of SUN2 following paclitaxel treatment occurs by ubiquitin-mediated proteolysis.

The roles of free tubulins and polymerized microtubules, and thus the potential role of paclitaxel, need to be uncovered.

We addressed this important point by using an alternative method to stabilise/bundle microtubules in interphase, namely by overexpressing GFP-Tau, as suggested by reviewer 2. Following GFP- Tau overexpression, large microtubule bundles were observed throughout the cytoplasm (Figure 4A), and this resulted in a significant decrease in nuclear solidity (Figure 4B). Furthermore, in cells where microtubule bundles extensively contacted the nucleus, the nuclear lamina became unevenly distributed and appeared patchy (Figure 4C). This supports our hypothesis that the aberrations to nuclear shape and Lamin A/C localisation in paclitaxel-treated cells are due to the presence of microtubules bundles surrounding the nucleus.

The doses of paclitaxel at which occur the effects described in the paper are not fully consistent with all the conclusions. Most experiments have been done at 5 nM. However, at this dose the effect of lamin A/C over or down expression on the growth (differences in the slopes of the curves in Figure 4A) are not fully convincing and not fully consistent with the clear effect on viability as well (in addition, duration of treatments before assessing vialbility are not specified). At 1 nM, cell growth is reduced and the rescuing effect of lamin over-expression is much more clear (Fig 4A), and the nucleus deformation clear (Fig 2A) but this dose has no effect on lamin A/C expression (Fig 3C), which questions how lamins impact nucleus shape and cell survival. Cytoskeleton reorganisation in these conditions is not described although it could clarify the respective role of force production (suggested in figure 1) and nuclei resistance (shown in figure 2) in paclitaxel sensitivity.

We thank the reviewer for raising this important point. We have addressed this by conducting additional repeats for the cell confluency measurements to increase the statistical power of our experiments (Figure 5A). Our data now show that GFP-lamin A/C had a statistically significant effect on rescuing cell growth at both 1 nM and 5 nM paclitaxel, while Lamin A/C knockdown exacerbated the inhibition of cell growth at 5 nM paclitaxel but not 1 nM paclitaxel (Figure 5A). In addition, we note that the duration of paclitaxel treatment before assessing viability was specified in the figure legend: "Bar graph comparing cell viability between wild-type (red), GFP-Lamin A/C overexpression (green), and Lamin A/C knockdown (blue) cells following 20 h incubation in 0, 1, 5, or 10 nM paclitaxel." We also repeated cell viability analysis after 48 h incubation in paclitaxel instead of 20 h to allow for a longer time for differences to take effect (Figure 5B).

We also added figures showing the cytoskeletal reorganisation at both 1 and 10 nM in addition to 0 and 5 nM (Supplementary Figure 1A) showing that microtubule bundling and condensation of actin into puncta correlated with increased paclitaxel concentration. Vimentin colocalised well with microtubules at all concentrations.

We have also included in our results section further clarification for the use of 5nM paclitaxel in this study. The new section reads as follows: "Experiments were performed at 5 nM paclitaxel (with additional experiments to determine dose relationships at 1 and 10 nM) because this aligns with previous studies7,14,24. Furthermore, previous analysis of patient plasma reveals that typical concentrations are within the low nanomolar range8, and concentrations of 5-10 nM are required in cell culture to reach the same intracellular concentrations observed in vivo in patient tumours9. This aligns with in vitro cytotoxic studies of paclitaxel in eight human tumour cell lines which show that paclitaxel's IC50 ranges between 2.5 and 7.5 nM41."

Finally, although the absence of role of mitotic arrest is clear from the data, the defective reorganisation of the nucleus after mitosis still suggest that the effect of paclitaxel is not independent of mitosis.

We thank the reviewer for pointing out the need for clarification in the wording of our manuscript. We have reworded the title and relevant sections of our abstract, introduction, and discussion to make it clearer that the effects of paclitaxel on the nucleus are due to a combination of aberrant nuclear cytoskeletal coupling during interphase and multimicronucleation following mitotic slippage. We have also added additional data in support of the effect of paclitaxel on nuclear architecture during interphase. For this, we used serum-starved cells (which divide only very slowly such that the majority of cells do not pass through mitosis during the 16 h incubation in paclitaxel [Supplementary Figure 2D]). Our new data confirmed that paclitaxel's effects on nuclear solidity, and Lamin A/C and SUN2 proteins levels can occur independently of cell division (Figure 2C; Figure 3H-J). Finally, when we overexpressed GFP-Tau (as discussed above) we observed similar aberrations to nuclear solidity and Lamin A/C localisation. This indicates that these effects occur due to microtubule bundling in interphase, especially as in our study GFP-Tau did not lead to multimicronucleation or appear to affect mitosis (Figure 4).

Below are the main changes to the text regarding the interphase effect of paclitaxel:

• Title: "Paclitaxel compromises nuclear integrity in interphase through SUN2-mediated cytoskeletal coupling"

• Abstract: "Overall, our data supports nuclear architecture disruption, caused by both aberrant nuclear-cytoskeletal coupling during interphase and exit from defective mitosis, as an additional mechanism for paclitaxel beyond mitotic arrest."

• Introduction: "Here we propose that cancer cells have increased vulnerability to paclitaxel both during interphase and following aberrant mitosis due to pre-existing defects in their NE and nuclear lamina."

• Discussion: "Overall, our work builds on previous studies investigating loss of nuclear integrity as an anti-cancer mechanism of paclitaxel separate from mitotic arrest14,20,21. We propose that cancer cells show increased sensitivity to nuclear deformation induced by aberrant nuclear-cytoskeletal coupling and multimicronucleation following mitotic slippage. Therefore, we conclude that paclitaxel functions in interphase as well as mitosis, elucidating how slowly growing tumours are targeted."

minor: a more thorough introduction of known data about dose response of cells in culture and in vivo would help understanding the range of concentrations used in this study.

As mentioned above, we have now included additional information in our Results section to clarify our paclitaxel dose range: "Experiments were performed at 5 nM paclitaxel (with additional experiments to determine dose relationships at 1 and 10 nM) because this aligns with previous studies7,14,24. Furthermore, previous analysis of patient plasma reveals that typical concentrations are within the low nanomolar range8, and concentrations of 5-10 nM are required in cell culture to reach the same intracellular concentrations observed in vivo in patient tumours9. This aligns with in vitro cytotoxic studies of paclitaxel in eight human tumour cell lines which show that paclitaxel's IC50 ranges between 2.5 and 7.5 nM41."

Significance

In this manuscript, Hale and colleagues describe the effect of paclitaxel on nucleus deformation and cell survival. They showed that 5nM of paclitaxel induces nucleus fragmentation, cytoskeleton reorganisation, reduced expression of LaminA/C and SUN2, and reduced cell growth and viability. They also showed that these effects could be at least partly compensated by the over-expression of lamin A/C. As fairly acknowledged by the authors, the induction of nuclear deformation in paclitaxel-treated cells, and the increased sensitivity to paclitaxel of cells expressing low level of lamin A/C are not novel (reference #14). Here the authors provided more details on the cytoskeleton changes and nuclear membrane deformation upon paclitaxel treatment. The effect of lamin A/C over and down expression on cell growth and survival are not fully convincing, as further discussed below. The most novel part is the observation that paclitaxel can induce the down-regulation of the expression of lamin A/C and that this effect is mediated by SUN2.

We appreciate the reviewer's summary and thank them for their time. We believe our comprehensive revisions have addressed all comments, strengthening the manuscript and making it more robust and compelling.

Reviewer #2 Evidence, reproducibility and clarity This study investigates the effects of the chemotherapeutic drug paclitaxel on nuclear-cytoskeletal coupling during interphase, claiming a novel mechanism for its anti-cancer activity. The study uses hTERT-immortalized human fibroblasts. After paclitaxel exposure, a suite of state- of-the-art imaging modalities visualizes changes in the cytoskeleton and nuclear architecture. These include STORM imaging and a large number of FIB-SEM tomograms.

We thank the reviewer for the summary and for highlighting our efforts in using the latest imaging technical advances.

Major comments:

The authors make a major claim that in addition to the somewhat well-described mechanism of paclitaxel on mitosis, they have discovered 'an alternative, poorly characterised mechanism in interphase'.

However, none of the data proves that the effects shown are independent of mitosis. To the contrary, measurements are presented 48 hours after paclitaxel treatment starts, after which it can be assumed that 100% of cells have completed at least one mitotic event. The appearance of micronuclei evidences this, as discussed by the authors shortly. It looks like most of the results shown are based on botched mitosis or, more specifically, errors on nuclear assembly upon exit from mitosis rather than a specific effect of paclitaxel on interphase. The readouts the authors show just happen to be measurements while the cells are in interphase.

Alternative hypotheses are missing throughout the manuscript, and so are critical controls and interpretations.

We thank the reviewer for highlighting the lack of clarity in our wording. We have revised the title, abstract and relevant sections of the introduction and discussion to clarify our message that the effects of paclitaxel on the nucleus arise from a combination of aberrant nuclear-cytoskeletal coupling during interphase and multimicronucleation following exit from defective mitosis. We have also included additional data where we used slow-dividing, serum-starved cells (under these conditions, the majority of cells do not undergo mitosis during the 16 h incubation in paclitaxel [Supplementary Figure 2D]). Our new data show that even in these cells there is a clear effect of paclitaxel on nuclear solidity, and Lamin A/C and SUN2 protein levels, further supporting our hypothesis that these phenotypes can occur independently of cell division (Figure 2C; Figure 3H-J). Furthermore, we performed additional experiments where we used overexpression of GFP-Tau as an alternative method of stabilising microtubules in interphase and observed similar aberrations to nuclear solidity and Lamin A/C localisation. As GFP-Tau overexpression did not lead to micronucleation or appear to affect mitosis, these data support the hypothesis that nuclear aberrations occur due to microtubule bundling in interphase (Figure 4). We discuss these experiments in more detail below. Finally, we have reworded the introduction to better introduce alternative hypotheses and mechanisms for paclitaxel's activity.

The authors claim that 'Previously, the anti-cancer activity of paclitaxel was thought to rely mostly on the activation of the mitotic checkpoint through disruption of microtubule dynamics, ultimately resulting in apoptosis.' The authors may have overlooked much of the existing literature on the topic, including many recent manuscripts from Xiang-Xi Xu's and another lab.

We would like to note that the paper from Xiang-Xi Xu's lab (Smith et al, 2021) was cited in our original manuscript (reference 14 in both the original and revised manuscripts). We have now also included additional review articles from the Xiang-Xi Xu lab (PMID:36368286 20 and PMID: 35048083 21). Furthermore, we have clarified the wording in both the introduction and discussion to better reflect the current understanding of paclitaxel's mechanism and alternative hypotheses.

The data, e.g. in Figure 1, does not hold up to the first alternative hypothesis, e.g. that paclitaxel stabilizes microtubules and that excessive mechanical bundling of microtubules induces major changes to cell shape and mechanical stress on the nucleus. Even the simplest controls for this effect (the application of an alternative MT stabilizing drug or the overexpression of an MT stabilizer, e.g., tau).

We thank the reviewer for suggesting this control experiment using the microtubule stabiliser Tau. We have now included these experiments in the revised version of the manuscript (Figure 4). The overexpression of GFP-Tau supports our hypothesis that cytoskeletal reorganisation in paclitaxel exerts mechanical stress on the nucleus during interphase, resulting in nuclear deformation and aberrations to the nuclear lamina. In particular, GFP-Tau overexpression resulted in large microtubule bundles throughout the cytoplasm (Figure 4A). Notably, in cells where these bundles extensively contacted the nucleus, we observed a significant decrease in nuclear solidity (Figure 4B) accompanied by changes in nuclear lamina organisation, including a patchy lamina phenotype, similar to that induced by paclitaxel (Figure 4C).

The focus on nuclear lamina seems somewhat arbitrary and adjacent to previously published work by other groups. What would happen if the authors stained for focal adhesion markers? There would probably be a major change in number and distribution. Would the authors conclude that paclitaxel exerts a specific effect on focal adhesions? Or would the conclusion be that microtubule stabilization and the following mechanical disruption induce pleiotropic effects in cells? Which effects are significant for paclitaxel function on cancer cells?

We thank the reviewer for raising important points regarding the specificity of paclitaxel's effects. We agree that microtubule stabilisation can induce myriad cellular changes, including alterations to focal adhesions and other cytoskeletal components. Our focus on Lamin A/C and nuclear morphology is grounded both in the established clinical relevance of nuclear mechanics in cancer and builds on mechanistic work from other groups.

Lamin A/C expression is commonly altered in cancer, and nuclear morphology is frequently used in cancer diagnosis35. Lamin A/C also plays a crucial role in regulating nuclear mechanics32 and, importantly, determines cell sensitivity to paclitaxel14. However, the mechanism by which Lamin A/C determines sensitivity of cancer cells to paclitaxel is unclear.

Our data are consistent with Lamin A/C being a determinant of paclitaxel survival sensitivity. We also provide evidence that paclitaxel itself reduces Lamin A/C protein levels and disrupts its organisation at the nuclear envelope. We directly link these effects to microtubule bundling around the nucleus and degradation of force-sensing LINC component SUN2, highlighting the importance of nuclear architecture and mechanics to overall cellular function. Furthermore, we show that recovery from paclitaxel treatment depends on Lamin A/C expression levels. This has clinical relevance, as unlike cancer cells, healthy tissue with non-aberrant lamina would be able to selectively recover from paclitaxel treatment.

Minor comments:

While I understand the difficulty of the experiments and the effort the authors have put into producing FIB-SEM tomograms, I am not sure they are helping their study or adding anything beyond the light microscopy images. Some of the images may even be in the way, such as supplementary Figure 6, which lacks in quality, controls, and interpretation. Do I see a lot of mitochondria in that slice?

We agree with the reviewer that Supplementary Figure 6 does not add significant value to the manuscript and thank the reviewer for pointing this out. We have removed it from the manuscript accordingly.

I may have overlooked it, but has the number of cells from which lamellae have been produced been stated?

We thank the reviewer for pointing out the missing information. For our cryo-ET experiments, we collected data from 9 lamellae from paclitaxel-treated cells and 6 lamellae from control cells, with each lamella derived from a single cell. This information has now been added to the figure legend (Figure 2F).

Significance

The significance of studying the effect of paclitaxel, the most successful chemotherapy drug, should be broad and of interest to basic researchers and clinicians.

As outlined above, I believe that major concerns about the design and interpretation of the study hamper its significance and advancements.

We appreciate the reviewer's concerns and have performed major revisions to strengthen the significance of our study. Specifically, we conducted two key sets of experiments to validate our original conclusions: serum starvation to control for the effects of cell division, and overexpression of the microtubule stabiliser Tau to demonstrate that paclitaxel can affect the nucleus via its microtubule bundling activity in interphase.

By elucidating the mechanistic link between microtubule stabilisation and nuclear-cytoskeletal coupling, our findings contribute to our understanding of paclitaxel's multifaceted actions in cancer cells.

My areas of expertise could be broadly defined as Cell Biology, Cytoskeleton, Microtubules, and Structural Biology.

Reviewer #3 Evidence, reproducibility and clarity The manuscript presents interesting new ideas for the mechanism of an old drug, taxol, which has been studied for the last 40 years.

We thank the reviewer for the positive feedback.

Although similar ideas are published, which may be suitable to be cited? • Paclitaxel resistance related to nuclear envelope structural sturdiness. Smith ER, Wang JQ, Yang DH, Xu XX. Drug Resist Updat. 2022 Dec;65:100881. doi: 10.1016/j.drup.2022.100881. Epub 2022 Oct 15. PMID: 36368286 Review. • Breaking malignant nuclei as a non-mitotic mechanism of taxol/paclitaxel. Smith ER, Xu XX. J Cancer Biol. 2021;2(4):86-93. doi: 10.46439/cancerbiology.2.031. PMID: 35048083 Free PMC article.

We thank the reviewer for bringing to our attention these important review articles. In our initial manuscript, we only cited the original paper (14, also reference 14 in the original manuscript). We have now included citations to the suggested publications (20,21).

We would also like to emphasise how our manuscript distinguishes itself from the work of Smith et al.14,20,21:

• Cell-type focus: In their study 14, Smith et al. examined the effect of paclitaxel on malignant ovarian cancer cells and proposed that paclitaxel's effects on the nucleus are limited to cancer cells. However, our data extends these findings by demonstrating paclitaxel's effects in both cancerous and non-cancerous backgrounds.

• Cytoskeletal reorganisation: Smith et al. show reorganisation of microtubules in paclitaxel-treated cells14. Our data show re-organisation of other cytoskeletal components, including F-actin and vimentin.

• Multimicronucleation: Smith et al. propose that paclitaxel-induced multimicronucleation occurs independently of cell division14. Although we observe progressive nuclear abnormalities during interphase over the course of paclitaxel treatment, our data do not support this conclusion; we find that multimicronucleation occurs only following mitosis.

• Direct link between microtubule bundling and nuclear aberrations: We show that nuclear aberrations caused by paclitaxel during interphase (distinct from multimicronucleation) are directly linked to microtubule bundling around the nucleus, suggesting they result from mechanical disruption and altered force propagation.

• Lamin A/C regulation: Consistent with Smith et al.14, we show that Lamin A/C depletion leads to increased sensitivity to paclitaxel treatment. However, we further demonstrate that paclitaxel itself leads to reduced levels of Lamin A/C and that this effect occurs independently of mitosis and is mediated via force-sensing LINC component SUN2. Upon SUN2 knockdown, Lamin A/C levels are no longer affected by paclitaxel treatment.

• Recovery: Finally, our work reveals that cells expressing low levels of Lamin A/C recover less efficiently after paclitaxel removal. This might help explain how cancer cells could be more susceptible to paclitaxel.

Only one cell line was used in all the experiments? "Human telomerase reverse transcriptase (hTERT) immortalised human fibroblasts" ? The cells used are not very relevant to cancer cells (carcinomas) that are treated with paclitaxel. It is not clear if the observations and conclusions will be able to be generalized to cancer cells.

We thank the reviewer for this comment. Our initial study aimed to understand the effects of paclitaxel on nuclear architecture in non-aberrant backgrounds. To show that the observed effects of paclitaxel are also applicable to cancer cells, we have now repeated our main experiments using MDA-MB-231 human breast cancer cells (Supplementary Figure 1B; Supplementary Figure 3P-T). Similar to our findings in human fibroblasts, paclitaxel treatment of MDA-MB-231 led to cytoskeletal reorganisation (Supplementary Figure 1B), a decrease in nuclear solidity (Supplementary Figure 3P), aberrant (patchy) localisation of Lamin A/C (Supplementary Figure 3Q), and a reduction in Lamin A/C and SUN2 levels (Supplementary Figure 3R-T).

"Fig. 1. (B) STORM imaging of α-tubulin immunofluorescence in cells fixed after 16 h incubation in control media or 5 nM paclitaxel. Lower panels show α-tubulin clusters generated with HDBSCAN analysis. Scale bars = 10 μm." It needs explanation of what is meaning of the different color lines in the lower panels, just different filaments?

We have added further detail to the figure legend for clarification: "Lower panels show α-tubulin clusters generated with HDBSCAN analysis. Different colours distinguish individual α-tubulin clusters, representing individual microtubule filaments or filament bundles."

Generally, the figures need additional description to be clear.

We have added further clarification and detail to our figure legends.

"Figure 3 - Paclitaxel results in aberrations to the nuclear lamina." The sentence seems not to be well constructed. "Paclitaxel treatment causes ..."?

We changed this sentence to: "Figure 3 - Paclitaxel treatment results in aberrant organisation of the nuclear lamina and decreased Lamin A/C levels via SUN2."

Lamin A and C levels are different in different images (Fig. 3B, H): some Lamin A is higher, and sometime Lamin C is higher? This may possibly due to culture condition or subtle difference in sample handling?.

We thank the reviewer for pointing this out and we agree that the ratio of Lamin A to Lamin C can vary with culture conditions. To confirm that paclitaxel treatment reduces total Lamin A/C levels regardless of this ratio, we repeated the Western blot analysis in three additional biological replicates using cells in which Lamin C levels exceeded Lamin A levels. These experiments confirmed a comparable decrease in total Lamin A/C levels. Figure 3B and 3C have been updated accordingly.

Also, the effect on Lamin A/C and SUN2 levels are not significant of robust.

Decreased Lamin A/C and SUN2 levels following paclitaxel treatment were consistently seen across three or more biological repeats (Figure 3B-C), and this could be replicated in a different cell type (MDA-MB-231) (Supplementary Figure 3R-T). Furthermore, Western blotting results are consistent with the patchy Lamin A/C distribution observed using confocal and STORM following paclitaxel treatment (Figure 3A; Supplementary Figure 3A), where Lamin A/C appears to be absent from discrete areas of the lamina.

Any mechanisms are speculated for the reason for the reduction?

We have now included additional data which aims to shed light on the mechanism behind the decrease in Lamin A/C and SUN2 levels following paclitaxel treatment. We found that SUN2 is selectively degraded during paclitaxel treatment. Immunoprecipitation of SUN2 followed by Western blotting against Polyubiquitin C showed increased SUN2 ubiquitination in paclitaxel (Figure 3M and N). Furthermore, in our original manuscript, we showed that Lamina A/C levels remained unaltered during paclitaxel treatment in cells where SUN2 had been knocked down. We propose that changes in microtubule organisation affect force propagation to Lamin A/C specifically via SUN2 and that this leads to Lamina A/C removal and depletion. Future work will be needed to fully understand this mechanism.

In addition to the findings described above, we report no significant changes in mRNA levels for LMNA or SUN2 in paclitaxel (Supplementary Figure 3B and O). Phos-tag gels followed by Western blotting analysis for Lamin A/C also did not detect changes to the overall phosphorylation status of Lamin A/C due to paclitaxel treatment. This is in agreement with our initial data showing no changes to Lamin A/C Ser 404 phosphorylation levels (Supplementary Figure 3E and F). Finally, Lamin A/C immunoprecipitation experiments followed by Western blotting for Polyubiquitin C and acetyl-lysine showed no significant changes in the ubiquitination and acetylation state of Lamin A/C in paclitaxel-treated cells (Supplementary Figure 3G-I).

Also, the about 50% reduction in protein level is difficult to be convincing as an explanation of nuclear disruption.

The nuclear lamina and LINC complex proteins play a critical role in regulating nuclear integrity, stiffness and mechanical responsiveness to external forces28,31-33,54,75, as well as in maintaining the nuclear intermembrane distance69,74. In particular, SUN-domain proteins physically bridge the nuclear lamina to the cytoskeleton through interactions with Nesprins, thereby preserving the perinuclear space distance30,69,74. Mutations in Lamins have been shown to disrupt chromatin organization, alter gene expression, and compromise nuclear structural integrity, and experiments with LMNA knockout cells reveal that nuclear mechanical fragility is closely coupled to nuclear deformation47. Furthermore, nuclear-cytoskeletal coupling is essential during processes such as cell migration, where cells undergo stretching and compression of the nucleus; weakening or loss of the lamina in such cases compromises cell movement47,73. In our work, we show that alterations to nuclear Lamin A/C and SUN2 by paclitaxel treatment coincide with nuclear deformations (Figure 2A-D, F, G; Figure 3A-D, F, G; Supplementary Figure 3A, P-T) and that these deformations are reversible following paclitaxel removal (Supplementary Figure 4B-D). Our experiments also demonstrate that Lamin A/C expression levels significantly influence cell growth, cell viability, and cell recovery in paclitaxel (Figure 5). Therefore, drawing on current literature and our results, we propose that, during interphase, paclitaxel induces severe nuclear aberrations through the combined effects of: i) increased cytoskeletal forces on the NE caused by microtubule bundling; ii) loss of ~50% Lamin A/C and SUN2; iii) reorganisation of nucleo-cytoskeletal components.

Significance

The manuscript presents interesting new ideas for the mechanism of an old drug, taxol, which has been studied for the last 40 years.

The data may be improved to provide stronger support.

Additional cell lines (of cancer or epithelial origin) may be repeated to confirm the generality of the observation and conclusions.?

We thank the reviewer for the feedback and valuable suggestions. In response, we have included experiments using human breast cancer cell line MDA-MB-231 to further corroborate our findings and interpretations. We believe these additions have improved the clarity, robustness and impact of our manuscript, and we are grateful for the reviewer's contributions to its improvement.

DOI: 10.1083/jcb.201804018

Resource: None

Curator: @scibot

SciCrunch record: RRID:BDSC_9060

What is this?

Acá sería bueno señalar que en términos generales, la mayoría de los logros en ciencias sociales en América Latina se concentran en el crecimiento de fuentes y publicaciones abiertas como Scielo o Redalyc: https://www.ouvrirlascience.fr/latin-america-could-become-a-world-leader-in-non-commercial-open-science/

Sería bueno incluir la fuente acá: https://www.science.org/content/article/university-california-boycotts-publishing-giant-elsevier-over-journal-costs-and-open o https://www.nature.com/articles/d41586-019-00758-x

DOI: 10.1016/j.celrep.2025.116130

Resource: (Cell Signaling Technology Cat# 4441, RRID:AB_2198445)

Curator: @scibot

SciCrunch record: RRID:AB_2198445

What is this?

Prezados autores, lido o texto há por destacar o seguinte: - o texto é relevante e aponta para uma temática pouco explorada em Moçambique; - Possui uma descrição clara do objecto e indica seu foco, relevância e inserção na perspectiva teórico metodológica adoptada; - É rico na descrição e atualização autoral. Entretanto, alguns aspectos podem merecer reparo: - A necessidade de uma correção linguística e gramatical; - A necessidade de, logo na introdução, no âmbito da busca por mapear o contexto da fala, afinar o foco para a proposta da pesquisa. Por exemplo, evitando grande menção feita sobre o fotojornalismo. Att., Roberto Chaua

Prezados autores, segue a apreciação do artigo

• O título do artigo está adequadamente alinhado à temática desenvolvida, refletindo com clareza o conteúdo e a proposta da investigação. O texto cumpre o objetivo delineado, ao abordar o problema a partir das contradições de classe, demonstrando coerência entre a metodologia adotada e a perspectiva teórica do materialismo histórico-dialético que sustenta a análise.

• Gostaria de destacar o mérito do trabalho desenvolvido. A originalidade da abordagem e a qualidade do conteúdo o tornam uma contribuição valiosa para o campo da História da Educação. Por isso, considero que essa produção tem pleno potencial para ser publicada e dialogar com a comunidade acadêmica de forma significativa.

• Entretanto, sugiro a realização de uma revisão ortográfica e gramatical, à adequada regência verbal e ao uso preciso da pontuação, a fim de aprimorar a clareza e a fluidez do texto.

Atenciosamente, Lucia Mara de Lima Padilha

Pilas. Basicamente infecciones.

lighthouse

a map of this new territory

unique wise choices together

inserted my page in the middle of their site

new page comes up

walls become joint property

link open doors instead of putting up walls

but if the link is into a walled garden?

Relações contraditórias que o público escolar estabelece com o livro didático

Modo dos professores usarem o livro didático

Um dos problemas considerados mais graves em relação ao livro didático: a forma pela qual apresenta os conteúdos históricos. O conhecimento produzido por ele é categórico, caracteristica perceptivel pelo discurso unitário e simplificado que reproduz, sem possibilidade de ser contestado, como afirmam vários dos seus críticos. Trata-se de textos que dificilmente são passíveis de contestação ou confronto, pois expressam "uma verdade" de maneira bastante impositiva.

Importância do livro didático: Reside na explicitação e sistematização de conteúdos históricos provenientes das propostas curriculares e da produção historiográfica. *Autores e editoras tem sempre, na elaboração dos livros, o desafio de criar esses vínculos. - O livro didático tem sido o principal responsável pela concretização dos conteúdos históricos escolares.

Livro didático: produto da industria cultural Enquanto mercadoria: lógica de vendagem e requer definições sobre preço e formas de consumo Destinatário principal: professor (mas se esse livro foi distribuido pela rede municipal de Floripa, quem comprou? Aluno: consumidor compulsório Sua confeccção segue os princípios do sistema de avaliação, obedecendo às normas definidas pelo poder estatal, que assim interfere indiretamente na sua produção e é o principal comprador desse material

o livro de Floripa é um livro paradidático. Obra interessante para o mercado editorial

O livro didático de história não é só uma lista de datas e nomes. Ele é uma ferramenta que ajuda a fixar uma versão da história na nossa mente, escolhendo alguns fatos como os mais importantes. Mas o autor, Valentine, avisa que o livro não faz isso sozinho. Na verdade, ele pega as ideias dos historiadores e as repete, reforçando as mesmas histórias e explicações que já eram aceitas. Assim, o livro continua a usar os mesmos eventos principais como base para contar toda a história.

O livro didático é um objeto de intensos debates e críticas. Sua complexidade o coloca no centro de discussões que vão além da escola, envolvendo educadores, pais e alunos, e se estendem a jornais, revistas e encontros acadêmicos. Autores, editores, políticos e intelectuais de diversas áreas participam dessas polêmicas, que têm seu alcance exemplificado pelo processo de avaliação do MEC. Apesar de sua importância cultural e social no Brasil, o livro didático também tem um papel econômico crucial, sustentando um vasto setor da indústria editorial do país.

Questionar e saber analisar o conteúdo que contém naquele livro, julgando se faz sentido com o que você acredita ou até na veracidade das informações ali contidas.

Livro didático como um suporte de conhecimentos escolares propostos pelos currículos educacionais. Essa característica faz que o estado esteja sempre presente na existência do livro didático: interfere indiretamente na elaboração dos conteúdos escolares veiculados por ele e posteriormente estabelece critérios para avaliá-lo, seguindo, na maior parte das vezes, os pressupostos dos currículos escolares institucionais. Como os conteúdos propostos pelos currículos são expressos pelos textos didáticos, o livro torna-se um instrumento fundamental na própria constituição dos saberes escolares.

Livro didático: produto cultural fabricado por técnicos que determinam seus aspectos materiais, o livro didático caracteriza-se nessa dimensão material, por ser uma mercadoria ligada ao mundo editorial e à lógica da indústria cultural do sistema capitalista.

críticas aos livros didáticos apontam para muitas de suas deficiências de conteúdo, suas lacunas e erros conceituais ou informativos e mostra que o problema dessas análises reside na concepção de que seja possível existir um livro didático ideal.

Livros didáticos de História como um tema polêmico, onde diversas pesquisas tem revelado que são um instrumento a serviço da ideologia e da perpetuação de um "ensino tradicional". Porém, ainda continuam sendo usados no trabalho diário das escolas em todo o País. Ao serem maior analisados dentro de uma perspectiva histórica, demonstram ter sofrido mudanças em seus aspectos formais e de um ganho de possibilidades de uso diferenciado por parte de professores e estudantes.

Autora traz que para a constante interação entre grupos, existe a opção de ter materiais didáticos adequados e em seguida afirma que a escolha desses materiais, depende das nossas concepções sobre o conhecimento, de como o estudante vai apreender e do tipo de formação que nós, professoras, estamos oferecendo.

This refers to academic RAG systems only.

e.g. Correctness and Quality of References generated by AI-based Research Assistant Tools: The Case of Scopus AI, Elicit, SciSpace and Scite in the Field of Business Administration shows no references from Scopus AI etc were entirely fabricated or non-existent though minor inaccuracies exist

For non-academic RAG, A 2025 study in Nature Communications showed that 99.3% and 96% of links from GPT4o RAG and Gemini 1.o ultra RAG were not broken. Not all these would technically be hallucinations, as some broken links are invalid urls given by an web API.

Generally though, because of the bounded nature of academic RAG, systems can easily do a post-processing check to ensure any suggested citation exists and if any are flagged as non0existent, the response can be regenerated. Note, this does not help with the source faithfulness issues.

The Weimar Republic, officially known as the German Reich, was the German state from 1919 to 1933, during which it was a constitutional republic for the first time in history; hence it is also referred to, and unofficially proclaimed itself, as the German Republic.

all sounds like Structured comunication - ideating = formulative thinking articulation descriptions Affective not just effective

Structured communication

structured communication

20% o value

Is everything is a spec?

No

∀ is a Formulation

DOI: 10.1186/s12964-025-02357-y

Resource: None

Curator: @olekpark

SciCrunch record: RRID:Addgene_46025

What is this?

DOI: 10.1016/j.celrep.2025.115966

Resource: RRID:Addgene_52961

Curator: @scibot

SciCrunch record: RRID:Addgene_52961

What is this?

dweb.link.self: http://bafybeickgzy5g3mi4plziw4nav4bxlcglkzfkrc74xt4hjdguzdhqdpnue.ipfs.localhost:8080/?filename=Autonomy%20Autopoiesis%20Enactive%20%EF%BD%9CIvo%20%20Substack%20(8_8_2025%201%EF%BC%9A54%EF%BC%9A54%20PM).html

from: https://hypothes.is/a/z-4f-HRPEfCCXzPaqCeZFQ

Autonomy

Autopoiesis

are the by words for all my work

Interested to learn about the Enactive approach

may well be it chimes in with my own experiential exploratory experimental approach?

to autopoiesis of the Next Web design to promote autonomy and autopoiesis on the Web?

to Autonomy

Autopoiesis

are the by words for all my work

Interested to learn about the Enactive approach

may well be it chimes in with my own experiential exploratory experimental approach?

to autopoiesis of the Next Web design to promote autonomy and autopoiesis on the Web?

Colocar o fundo todo branco

DOI: 10.7554/eLife.101918

Resource: RRID:Addgene_173953

Curator: @scibot

SciCrunch record: RRID:Addgene_173953

What is this?

D-azúcares pero no L-azúcares, L-aminoácidos pero no D-aminoácidos— o un pequeño conjunto de sustratos con relaciones estrechas

Hosting should be discussed. This breaks alignment with W3C Verifiable Credentials because it puts privacy at risk. Also, hosting is not part of the 3.0 specifictaion.

To discuss: if platforms want o host open badges, they should use 2.0 not 3.0.

DOI: 10.3389/fonc.2025.1582996

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_10746877

What is this?

DOI: 10.1158/1535-7163.MCT-24-0684

Resource: (Thermo Fisher Scientific Cat# 02-6502, RRID:AB_2532951)

Curator: @scibot

SciCrunch record: RRID:AB_2532951

What is this?

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

Learn more at Review Commons

Reply to the reviewers

REVIEWER 1

This is an important and solid study that identified sequences that can improve circRNA translation and that as or more importantly are very short and hence are suitable for generating of efficient protein expressing circRNAs. This manuscript fills an important gap in the field, and it is highly significant. The study is well controlled, the rationale clear and the results conclusive with no major flaws.

• While this is a minor concern as the vector has been used before, it will greatly improve the quality of the paper if the authors could just verify that the vector only generates circRNA molecules and not linear concatenamers. To do so the authors can focus only in their control and the most optimal transcripts and perform northern blot or well controlled RNAseR experiments to show that all RNA molecules containing the back splicing junction are circular We thank the reviewer for raising this point. As suggested, we performed RNaseR resistance assays on our three most efficient candidates driving cGFP translation (VCIP, T3-glo, and T3-U3) to confirm that all derived RNA molecules containing the back-splicing junction are circular. As proof of this, cGFP proved strongly resistant to RNase R (new Fig. S1N), confirming its circular structure. We further ruled out the possibility that molecules other than the circRNA encoding GFP serve as templates for translation from our vectors. Specifically, ad hoc PCR amplifications performed for this purpose (new Fig. S1M) showed no bands that would indicate the presence of concatemers. Indeed, ad hoc PCR amplifications (new Fig. S1M) revealed no bands indicative of concatemer formation. The primers used and the expected sizes of the amplicons are schematically represented in new Fig. S1M. In brief, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF, thus detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed (new Fig. S1M). Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively. These results are expected for a circRNA, as also indicated by the fact that the circZNF609 positive control behaves in a similar manner. Collectively, these results confirm the circular nature of our transcript and exclude translation originating from possible concatemers.

• These results are shown in new Fig. S1M and S1N and described in the text as follows: “Importantly, we ruled out the possibility that templates other than the GFP-encoding circRNA drive translation from our best performing constructs (V-cGFP, T3-glo-cGFP and T3-U3-cGFP). Ad hoc PCRs amplifications (Fig. S1M) revealed no bands indicative of concatemer formation. The left panel of Fig. S1M schematically illustrates the primer sets and expected amplicons sizes. In particular, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed. Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively (Fig. S1M). These results are consistent with the circularity of the transcripts tested and coherent with the results obtained for circZNF609, used as control (Fig. S1M). Finally, cGFP resulted resistant to RNAseR treatment (Fig. S1N), further supporting its circular nature.”*

• There is a repetition of the world "a" in the abstract. We thank the reviewer for the attention paid to our text, we removed the extra “a” from the abstract.

• All circRNA translation studies should be cited when describing translation of circRNAs. We thank the reviewer for the suggestions, we corrected the mistake present in the text and included extra referenced about circRNA translation.

*Specifically, we included: *

• Fan, X., Yang, Y., Chen, C. et al. Pervasive translation of circular RNAs driven by short IRES-like elements. Nat Commun 13, 3751 (2022). https://doi.org/10.1038/s41467-022-31327-y
• Chen CK et al. Structured elements drive extensive circular RNA translation. Mol Cell. 2021 Oct 21; 81(20):4300-4318.e13.doi: 10.1016/j.molcel.2021.07.042. Epub 2021 Aug 25. PMID: 34437836; PMCID: PMC8567535.
• Obi P, Chen YG. The design and synthesis of circular RNAs. Methods. 2021 Dec;196:85-103. doi: 10.1016/j.ymeth.2021.02.020. Epub 2021 Mar 2. PMID: 33662562; PMCID: PMC8670866.
• Fukuchi, K., Nakashima, Y., Abe, N. et al. Internal cap-initiated translation for efficient protein production from circular mRNA. Nat Biotechnol (2025). https://doi.org/10.1038/s41587-025-02561-8
• Du, Y., Zuber, P.K., Xiao, H. et al. Efficient circular RNA synthesis for potent rolling circle translation. Nat. Biomed. Eng 9, 1062–1074 (2025). https://doi.org/10.1038/s41551-024-01306-3
• Wang F, Cai G, Wang Y, Zhuang Q, Cai Z, Li Y, Gao S, Li F, Zhang C, Zhao B, Liu X. Circular RNA-based neoantigen vaccine for hepatocellular carcinoma immunotherapy. MedComm (2020). 2024 Jul 29;5(8):e667. doi: 10.1002/mco2.667. PMID: 39081513; PMCID: PMC11286538.
• Andries O, Mc Cafferty S, De Smedt SC, Weiss R, Sanders NN, Kitada T. N(1)-methylpseudouridine-incorporated mRNA outperforms pseudouridine-incorporated mRNA by providing enhanced protein expression and reduced immunogenicity in mammalian cell lines and mice. J Control Release. 2015 Nov 10;217:337-44. doi: 10.1016/j.jconrel.2015.08.051. Epub 2015 Sep 3. PMID: 26342664.
• Yang Y, Fan X, Mao M, Song X, Wu P, Zhang Y, Jin Y, Yang Y, Chen LL, Wang Y, Wong CC, Xiao X, Wang Z. Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Res. 2017 May;27(5):626-641. doi: 10.1038/cr.2017.31. Epub 2017 Mar 10. PMID: 28281539; PMCID: PMC5520850. REVIEWER 2

Circular RNAs (circRNAs) have attracted significant interest due to their unique properties, which make them promising tools for expressing exogenous proteins of therapeutic value. However, several limitations must be addressed before circRNAscan become a biologically and economically viable platform for the biotech industry.One of the main challenges is the reliance on large, highly structured sequences withinternal ribosome entry site (IRES) activity to initiate translation of the downstream open reading frame. In this study, the authors propose an alternative strategy that combines the 5′ untranslated region (5′UTR) of a previously characterized natural circRNA(circZNF609) with a short 13-nt nucleotide sequence shown to act as a translational enhancer. By evaluating the activity of various constructs containing a reporter geneacross multiple cell lines, they identify the most efficient and compact sequence, 63-nt long, capable of boosting translation within a circular RNA context.

Major Comments:

• This study is well-executed and relies on standard in vitro molecular biology techniques, which are adequate to support the conclusions drawn. *We thank the reviewer for the very positive opinion on the execution of our study. *

• The experimental procedures are clearly described, and the statistical analyses have been performed according to accepted standards. *We thank the reviewer for the very positive comment about the analyses we performed. *

Minor Comments:

• The manuscript would greatly benefit from a comprehensive revision to improve clarity and language. Involving a native English speaker during the editing process could significantly enhance the manuscript's readability and overall quality. The Results section would benefi t from closer attention, as certain parts of the description are attimes confusing and could be clarifi ed for better reader comprehension. We thank the reviewer for the input. We performed a huge revision of the text to improve language quality and enhance readability. We extended the descriptions in the results sections in order to explicit and clarify our data.

• The references should be carefully reviewed for accuracy and consistency-forinstance, references 9 and 10 appear to require correction or clarifi cation. We thank the reviewer for the careful reading of our paper. We amended the reference section, and we expanded it.

Reviewer #2 (Significance (Required)):

This study addresses a critical bottleneck in RNA therapeutics. The use of the proposed short sequences could significantly enhance the in vivo activity of protein-encoding circular RNAs. A highly efficient, compact translational enhancer has thepotential to substantially improve the therapeutic applicability of circRNAs and broaden their range of applications. Given the potential utility of these findings, we would anticipate pursuing intellectual property (IP) protection. To further strengthen the study, future work should include additional data on polysome association and a detailed analysis of the secondary structure of the 66-nt enhancer sequence. This work should be of broad interest to molecular biologists working on RNA biology, translation, and RNA-based therapeutics. I expect the identified sequence will betested by multiple laboratories to evaluate its strength and versatility, further underscoring the potential impact of this study. For context, I am actively engaged in research on non-coding RNAs.

• *

REVIEWER 3

In this brief report, the authors take advantage of circular RNA expression plasmids to define elements that can be used to enable efficient translation. They test a handful of known IRES elements as well as short translation enhancing elements (TEEs) for their ability to promote translation of circular GFP and c-ZNF609 reporters. They focus on one particular element that is of a short length and seems to work as well as longer IRES elements. My major concern relates to possible alternative sources of the translated proteins, which the authors have not ruled out (see below). I find themanuscript to be too preliminary in its current state.

• Work from the Meister group (Ho-Xuan et al 2020 Nucleic Acids Res 48:10368) has shown that apparent translation from circRNA over-expression plasmids is not from circular RNAs, but instead from trans-splicing linear by-products. The authors have not ruled out such alternative explanations here, e.g. by using deletion constructs that prevent backsplicing. We thank the reviewer for raising this point. *We ruled out the possibility that molecules other than the circRNA encoding GFP serve as templates for translation from our vectors. Specifically, ad hoc PCR amplifications performed for this purpose (new Fig. S1M) showed no bands that would indicate the presence of concatemers. Indeed, ad hoc PCR amplifications (new Fig. S1M) revealed no bands indicative of concatemer formation. The primers used and the expected sizes of the amplicons are schematically represented in new Fig. S1M. In particular, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed. Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively. These results are expected for a circRNA, as also indicated by the fact that the circZNF609 positive control behaves in a similar manner. Collectively, these results confirmed the circular nature of our transcript and excluded translation originating from possible concatemers. *

These results are shown in new Fig. S1M and S1N and described in the text as follows: “Importantly, we ruled out the possibility that templates other than the GFP-encoding circRNA drive translation from our top constructs (V-cGFP, T3-glo-cGFP and T3-U3-cGFP). Ad hoc PCRs amplifications (Fig. S1M) revealed no bands indicative of concatemer formation. The left panel of Fig. S1M schematically illustrates the primer sets and expected amplicons sizes. In brief, we used a divergent primers set spanning the BSJ (3-4) to specifically detect the mature circRNA and a set of convergent primers (1-2) pairing on the GFP ORF detecting both the circRNA and its linear precursor as well as the putative concatemer expected. Although a ~1 kb band was expected if a trans-splicing by-product was present, no such band was observed (new Fig. S1M). Moreover, RT-PCR amplification of the cGFP back-splice junction was markedly more efficient when reverse transcription was primed with random hexamers than with oligo(dT), priming total RNA or preferentially polyA+ RNA, respectively (Fig. S1M). These results are consistent with the circularity of the transcripts tested (Fig. S1M). Importantly, cGFP PCR amplifications showed similar results as a validated endogenous circRNA, namely circZNF609, used as control (Fig. S1M, right panel), confirming the circular nature of cGFP. Finally, cGFP resulted resistant to RNAseR treatment (Fig. S1N), further supporting its circular nature.”* *

• Echoing the point above, the overall results would be stronger if the authors couldconfirm IRES activity using highly pure, in vitro transcribed RNAs that are transfected into cells * We thank the reviewer for this suggestion. Unfortunately, we are currently unable to produce synthetic circular molecules in-house, and the cost and time for purchasing synthetic ones are prohibitive. Nevertheless, we have performed the experiments described above to ensure the circularity of the transcripts tested.*

• The authors should also confirm their IRES activity using standard dual luciferase reporter (linear) constructs which have long been a standard approach in the field. We thank the reviewer for raising this point. As recommended, we cloned our three best candidates (VCIP, T3-glo, and T3-U3) into the pRL-TK/pGL3 dual-luciferase vector to assess their IRES activity (producing the vectors VCIP-Luc, T3-glo-Luc, and T3-U3-Luc), transfected them into RD cells, and, after 24 h of incubation, measured luciferase activity to assess the IRES performance of each candidate. From our analyses, VCIP and T3-U3 confirmed their IRES activity, although showing different relative efficiency, whereas T3-glo was inactive in the linear luciferase context. This finding is consistent with previous observations (Legnini et al., 2017) showing that the performance of IRES sequences in a linear luciferase reporter may differ from their activity when driving translation from a circRNA template. Overall, these results highlight the need for further investigation into the sequences and contexts specifically governing circRNA translation, rather than relying solely on knowledge derived from linear RNAs. *The results are shown below. We did not include them in the text to not overcomplicate the readability. However, we are happy to add and discuss them if required. *

***

***

Bar plot representing the relative luciferase activity deriving from VCIP-Luc (“V”), T3-glo-Luc (“T3-glo”), and T3-U3-Luc (“T3-U3”)*. Dual luciferase assay was performed and Renilla luciferase activity from each candidate was normalized against the Firefly luciferase. An empty ptKRL-pgl3 vector was used as reference. The ratio of each sample versus its experimental control was tested by two-tailed Student’s t test. * indicates a Student’s t test-derived p-value * *

• Methods, Plasmids Construction Section: Rather than including long lists of oligos and forcing a reader to figure out the final product that was cloned, it would be more intuitive if the authors provided the full sequences of the ORF and IRES sequencesthat were tested. We thank the reviewer for the comment, we added the sequences to the methods (Supplementary Table 1).

• The manuscript needs extensive English editing. Parts of it are also formatted in anunusual style, especially the introduction where it seems like each paragraph is a single sentence. As requested by the reviewer, we edited the text to make the language and content more accessible to readers.

• References included by the authors are selective and surprisingly do not include Chen et al (2021) Mol Cell 20:4300-4318 which already defined IRES elements for circRNAs that are fairly small. *Thank you for pointing this out. We have now cited the elegant work of Chen et al. (2021, Mol Cell 20:4300–4318) in the revised manuscript. While Chen and colleagues screened IRES-like elements of roughly 200 nt, our study was designed to uncover an even more minimal motif. The elements we report are therefore markedly shorter, highlighting a complementary, rather than overlapping, aspect of IRES available for driving circRNA translation. However, we now refer to Chen et al. in our text. *

• Error bars in Fig 2, especially Fig 2B, are huge. It seems impossible to make any conclusion given the large variety across these experiments. Thank you for your input. Although the error bars appear relatively large, the overall conclusions remain robust, as also noted by the other reviewers: both T3-glo and T3-U3 are intrinsically compact elements, yet they drive translation as efficiently as larger canonical IRESs. The error bars largely reflect the inherent variability of transient transfection assays, which naturally increases with the number of constructs examined. To strengthen our dataset without discarding existing replicates, we chose not to repeat experiments in the previously tested lines. Instead, we assessed our vectors in an additional model, the D283 medulloblastoma cell line. In this setting, we unexpectedly observed that the EMCV IRES surpasses the VCIP IRES, opposite to what we saw in the other lines, yet even here the short elements we identified remain strong competitors (new Fig. 2C, S2G, S2H). The evaluation of multiple CDSs across several cell lines, make our findings to be solid and well supported.

Add IPFS! Desktop

Can do it manually

DOI: 10.1038/s41598-025-12511-8

Resource: ggplot2 (RRID:SCR_014601)

Curator: @scibot

SciCrunch record: RRID:SCR_014601

What is this?

imperialism to think that melanesia was the most fragmented small, and isolated places, while in reality it's one o f the most connected given the multilingual roots as well as connections.

This work has been peer reviewed in GigaScience (see https://doi.org/10.1093/gigascience/giaf077), which carries out open, named peer-review. These reviews are published under a CC-BY 4.0 license and were as follows:

Reviewer name: Laura Caquelin

1. Summary of the Study This study provides the first multi-omics investigation of the innate immune response in turbot (Scophthalmus maximus). By integrating RNA-Seq, ATAC-Seq, and ChIP-Seq data, researchers identified changes in gene expression, chromatin accessibility, and histone modifications after viral and bacterial stimulation. The findings reveal a significant coupling between the transcriptome and chromatin state, offering insights for the selection of disease resistance in aquaculture.

2. Scope of reproducibility

According to our assessment the primary objective is: Association of ATAC-Seq and ChIP-Seq data with RNA-Seq data

● Outcome: Overlap of promoter DARs and DHMRs with DEG promoters ● Analysis method outcome: Hypergeometric test ● Main result: "DARs and DHMRs were much more overrepresented at the promoter regions of upregulated rather than downregulated DEGs" (Table 4, Supplementary Table 11; Lines 403-405, Page 9)

1. Availability of Materials a. Data ● Data availability: Raw data are available, but generated data from the study are shared with the journal and not yet publicly available ● Data completeness: Complete ● Access Method: Manuscript's supplementary files/Private journal dropbox ● Repository: - ● Data quality: Structured, but lacks variable definitions in supplementary files, making it difficult to interpret and use. b. Code ● Code availability: Not available for the primary result ● Programming Language(s): Excel ● Repository link: - ● License: - ● Repository status: - ● Documentation: README lacks information on hypergeometric test.

2. Computational environment of reproduction analysis

● Operating system for reproduction: MacOS 14.7.4 ● Programming Language(s): Excel ● Code implementation approach: Excel formulas based on methodology description provided by authors ● Version environment for reproduction: Excel version 16.94

1. Results

5.1 Original study results ● Results 1: Table 4 and supplementary table 11

5.3 Steps for reproduction

 Reproduce supplementary table 11 to perform hypergeometric test * Issue 1: No code or instructions for constructing Table 4 in manuscript and README text. ▪ Resolved: Authors shared methodology upon request Authors' Clarification: The hypergeometric test wasn't carried out with any particular script but with the following public online tool, that can be replicated in excel: https://systems.crump.ucla.edu/hypergeometric/ The tool basically runs the following excel formulas: Cumulative distribution function (CDF) of the hypergeometric distribution in Excel =IF(k>=expected,1-HYPGEOM.DIST(k-1,s,M,N,TRUE),HYPGEOM.DIST(k,s,M,N,TRUE)) =IF(k>=((sM)/N),1-HYPGEOM.DIST(k-1,s,M,N,TRUE),HYPGEOM.DIST(k,s,M,N,TRUE)) expected = (sM)/N direction =IF(k=expected,"match",IF(k<expected,"de-enriched","enriched")) fold change =IF(k<expected,expected/k,k/expected)

where k is the number of successes (intersection of DAR/DHMR in promoters + DEG), s the sample size (DEG), M the number of successes in the population (DAR/DHMR in promoters) and N the population size (28.602 genes). For each condition, the count of downregulated and upregulated DEG (s) was taken from supplementary table 4. Similarly, the count of downregulated and upregulated DAR/DHMR (M) was taken from supplementary table 10, considering only differential peaks that are annotated as "promoter-TSS" in the annotation column (column M). The population size (N) was the total list of genes that were DEG, DAR or DHMR (combining the data on supplementary tables 4 and 11, eliminating duplicates). Finally, the intersection of of DAR and DEG (k) for each condition was retrieved with the following venn diagram online tool: https://bioinformatics.psb.ugent.be/webtools/Venn/" * Issue 2: Discrepancies in DEG counts from supplementary table 11 ▪ Resolved: Investigated variable definitions (using the wrong variable - strand), confirmed that log2FoldChange determines up/down-regulation * Issue 3: Filling in DAR/DHMR values ▪ Unresolved: Unclear correspondence between "promoters" rows and excel file sheets. Does H3K27me3 correspond to the promoters? * Issue 4: Using the Venn diagram tool to find intersections ▪ Unresolved: Worked for one condition (ATC vivo poly (down)) but failed for ATAC vitro-vibrio and ATAC-vivo-vibrio. Tool returns a "Request Entity Too Large" error. * Issue 5: Define the population size ▪ Unresolved: The instructions for defining the population size are not clear. In supplementary table 4, it seems that the variable "Gene ID (ENSEMBL)" should be used, but in supplementary table 10, should the variable "Nearest PromoterID" or "Gene symbol" be used?  Using supplementary table 11 values to perform hypergeometric test Having failed to obtain the values required to reproduce supplementary table 11, the data already provided were used to obtain the "enrichment" and "p-value" values using the excel function provided. * Issue 1: Comparison of p-values ▪ Resolved: For Up condition, extremely small p-values are not displayed correctly due to Excel's limitations in scientific notation. Excel may either display them as zero or in an incomplete scientific format (e.g., 0.00E+00). Using the tool on the web.

5.4 Statistical comparison Original vs Reproduced results ● Results: Based on the available data in supplementary table 11, the "enrichment" and "p-value" values have been successfully reproduced in most cases. ● Comments: The full table could not be reproduced, particularly the data corresponding to DAR/DHMR, DAR/DHMR+DEG and population size values, due to missing information or unclear definitions in the supplementary files. ● Errors detected: The enrichment value for the Up condition of promoters-vitro-vibrio was incorrectly reported in the manuscript/table. Based on the Excel formula and the online tool used, the correct value appears to be 2.28 instead of 2.82. ● Statistical Consistency: All the values that could be reproduced from the available data matched the original results, except for the detected error.

1. Conclusion

2. Summary of the computational reproducibility review The study's results were partially reproduced. Key values such as enrichment and p-values were successfully replicated, but some dataset elements (DAR/DHMR, DAR/DHMR+DEG, and size population) could not be verified due to insufficient methodological details provided in the manuscript. An error in the enrichment value for the Up condition of promoters-vitro-vibrio was identified (2.28 instead of 2.82). The p values used for statistical inference were however successfully reproduced.

3. Recommendations for authors o Improve data documentation: Define variables in supplementary files. o Provide all code and scripts: Share the excel formulas used for table 4/supplementary table 11. o Clarify statistical methodology: Include detailed methods description for the hypergeometric test. o Enhance reproducibility workflow: Provide a structured README with all necessary steps.

Let's not use this, as true as it is. There are lots and lots of examples of mistaken translation in the Fornander version of Kelou Kamakau.

justificar esta decisión

se mencionan subconjuntos o se muestran?

como que no? abajo tenes series están planchadas y luego suben y tenes una que oscila

no esta claro con que conjunto de parámetros te quedaste. Quiero decir, si uno hace una exploración es para decidir algo, no me resulta claro ver que configuración fue la que terminaste usando para el análisis, o si mezclaste varias. Clarificar

no queda claro que principio. Si "el principio" es lo que viene después del dos puntos entonces lo correcto es "el siguiente principio". Ahora, si el "este" viene de antes en el texto no queda claro. Clarificar o reescribir

citar libro o fuente que hable de esto

conviene citar un libro o una fuente que hable de esta técnica

A distinção entre e-atividades síncronas e assíncronas é particularmente relevante, pois permite adequar a aprendizagem às necessidades e perfis dos estudantes. Considero que a flexibilidade das atividades assíncronas pode promover maior autonomia, mas concordo que exige atenção à interação, para evitar o isolamento.

(...) aprendizagem como processo (...)

Frase precisa conectar melhor com o objetivo do evento.

Podemos outra headline do Lucas que não foi usada no topo.

"Aprenda, de uma vez por todas, a usar Inteligência Artificial aplicada na prática à uma infraestrutura de redes e cibersegurança".

"Garanta seu ingresso antes que o lote vire"

Pode retirar também

Não tem necessidade.

• Copy pode estar mais alinhada a como o Analista de TI deve usar a IA de forma correta para ser produtivo e não correr o risco de ser substituído.
• Imagem das principais LLMs conectadas à uma topologia de rede.

Tanto para a copy, quanto para a imagem, usar referência dos criativo de vídeo 5 (Inclusive podemos usar essa imagem em criativos de imagem).

• Usar uma das headlines e sub-headline que o Lucas indicou
• Imagem do Lucas associada à IA

Was 16S more usable then?

O avanço tecnológico e a sua integração no ensino têm transformado a forma como se estrutura o processo de aprendizagem, exigindo novas posturas dos agentes educativos. Nesse contexto, as e-atividades – tarefas realizadas em ambientes digitais – ganham, de facto, destaque como mediadoras fundamentais do ensino e da aprendizagem. Baseadas em abordagens socio-construtivistas, promovem a participação ativa dos estudantes, valorizando os seus conhecimentos prévios, a autonomia e a interação social. Ao planear as e-atividades, os docentes devem considerar os objetivos de aprendizagem, a motivação dos alunos, as suas competências e as possíveis limitações tecnológicas. A flexibilidade dos ambientes digitais permite adaptar as e-atividades aos diferentes estilos e ritmos de aprendizagem, favorecendo uma formação mais personalizada e eficaz.

A minha reflexão sobre os princípios ilustrados na Figura 7 tem no caso do ensino de algumas áreas fundamentais da engenharia (por exemplo física, eletrónica, sistemas embebidos…), uma aplicação que considero um bom exemplo: a conceção e uso de laboratórios remotos (com experiências reais, através de equipamentos controlados à distância, e virtuais, através de simuladores). As e-atividades que este tipo de abordagem permite vão muito além de meros exercícios, materializando estes quatro princípios de forma exemplar. Propiciam a interação, não só entre os participantes, mas também a interação direta e prática com equipamentos e fenómenos reais. Estimulam a autonomia, ao permitir que os participantes conduzam experiências ao seu próprio ritmo, cometendo erros e aprendendo com eles, algo que é fundamental para a "aprendizagem profunda". Promovem a abertura, pelo acesso a equipamentos de laboratório dispendiosos e específicos, que de outra forma estariam inacessíveis à maioria dos participantes. Reconhecem a diversidade de aprendizagem, por permitirem um aprender pelo fazer, tanto a participantes com gosto pela prática, como aos que ficam inibidos na presença dos equipamentos (com medo de estragar pela “falta de jeito”), complementando a aprendizagem teórica. A pertinência deste modelo tem um exemplo paradigmático no que levou ao desenvolvimento dos ambientes (ferramentas, formas de uso, atores…) que tornaram possíveis os temas que abordámos neste curso. O trabalho de Sir Tim Berners-Lee no CERN, que deu origem à World Wide Web e aos primeiros web browsers, foi uma resposta a uma necessidade premente: permitir que milhares de cientistas, geograficamente dispersos, pudessem colaborar, aceder a dados e operar remotamente equipamentos nos projetos do maior laboratório de física de partículas do mundo. O artigo (https://iopscience.iop.org/article/10.1088/1748-0221/3/08/S08003) que apresenta a experiência ATLAS, uma das duas que comprovou em 2013 a existência do bosão de Higgs - a mítica partícula que se procurava sem sucesso há décadas - tem 2927 autores, ilustrando-o bem. A WWW nasceu, portanto, da necessidade de "laboratórios remotos" para uma comunidade científica global. Tendo vivido de perto essa experiência (um dos 2927), e sabendo que há assuntos que só se aprendem realmente fazendo, tenho a convicção, já com algum tempo (e também contacto com o assunto: https://www.physics.rutgers.edu/~eandrei/389/muon/1322-1326.pdf) de que estes laboratórios remotos, permitindo aos estudantes de cursos no formato de e-learning fazer trabalhos experimentais (e, como vimos, fazê-los de forma semelhante ao que se faz em algumas áreas da investigação fundamental), devem ser uma parte integrante destas e-atividades em determinados cursos. É certo que, como foi mencionado numa das sessões síncronas, o investimento inicial e a manutenção são exigentes, condicionando a oferta formativa. Parece-me, no entanto, que é um investimento com retorno seguro.

Paradoxalmente, também existe fenómeno inverso, há estudantes (particularmente de áreas tecnológicas) que dominam ferramentas como Discord, onde o ambiente de colaboração, através da comunicação instantânea e da partilha de conteúdos é sofisticado. Até mesmo plataformas como a Twitch, dedicadas à transmissão e interação em tempo real, influenciam a forma com os estudantes interagem com conteúdos síncronos. Numa resposta pedagógica construtiva, é possível converter a disparidade digital numa oportunidade de aprendizagem colaborativa: estudantes experientes nestas ferramentas podem funcionar como “mentores digitais” em atividades peer-to-peer, deixando ao docente o papel de facilitador e orientador do processo de aprendizagem.

DOI: 10.1242/jcs.263644

Resource: RRID:BDSC_27503

Curator: @maulamb

SciCrunch record: RRID:BDSC_27503

What is this?

DOI: 10.1016/j.phanu.2025.100446

Resource: (Thermo Fisher Scientific Cat# MA1-91399, RRID:AB_2273656)

Curator: @scibot

SciCrunch record: RRID:AB_2273656

What is this?

Prueba de anotación

El título actúa como una "tarjeta de presentación" del proyecto, condensando la esencia de la investigación. Incluir especificidad, espacialidad y temporalidad asegura que el título sea claro y delimite el alcance del estudio. Por ejemplo, un título como "Conocimientos sobre COVID-19 en estudiantes de la UVG, 2022" define claramente qué, dónde y cuándo, ayudando a los lectores a comprender inmediatamente el enfoque y contexto del trabajo.

Este punto destaca la importancia de contextualizar el problema para darle relevancia. Describir quiénes se ven afectados y las implicaciones (causas y consecuencias) permite al investigador justificar la pertinencia del estudio y conectar con las necesidades reales de una población o situación. Esto refuerza que un buen enunciado no solo describe el problema, sino que lo sitúa en un marco social, cultural o práctico significativo.

Este principio resalta la importancia de la reproducibilidad en la investigación científica. Detallar los recursos materiales (como software, equipos o documentos) asegura que el estudio sea transparente y verificable. Este aprendizaje refuerza que una investigación bien planificada considera no solo la ejecución, sino también la posibilidad de que otros puedan replicarla para validar los resultados.

Las preguntas auxiliares son esenciales para desglosar el problema en aspectos manejables. Esta pregunta específica guía la investigación hacia la evaluación de la efectividad de una herramienta, promoviendo un análisis crítico de sus fortalezas y limitaciones. Aprender a formular preguntas claras y enfocadas, como esta, ayuda a estructurar la investigación y a mantener el rumbo hacia el objetivo general.

La justificación es el "corazón" persuasivo de la investigación, ya que conecta el problema con su relevancia práctica o teórica. Al explicar por qué el estudio es necesario, el investigador no solo motiva su realización, sino que también convence a otros (como financiadores o académicos) de su valor. Este aprendizaje enfatiza la necesidad de alinear el proyecto con necesidades reales o vacíos de conocimiento.

El título actúa como una "tarjeta de presentación" del proyecto, condensando la esencia de la investigación. Incluir especificidad, espacialidad y temporalidad asegura que el título sea claro y delimite el alcance del estudio. Por ejemplo, un título como "Conocimientos sobre COVID-19 en estudiantes de la UVG, 2022" define claramente qué, dónde y cuándo, ayudando a los lectores a comprender inmediatamente el enfoque y contexto del trabajo.

Las preguntas auxiliares son esenciales para desglosar el problema en aspectos manejables. Esta pregunta específica guía la investigación hacia la evaluación de la efectividad de una herramienta, promoviendo un análisis crítico de sus fortalezas y limitaciones. Aprender a formular preguntas claras y enfocadas, como esta, ayuda a estructurar la investigación y a mantener el rumbo hacia el objetivo general.

Este punto destaca la importancia de contextualizar el problema para darle relevancia. Describir quiénes se ven afectados y las implicaciones (causas y consecuencias) permite al investigador justificar la pertinencia del estudio y conectar con las necesidades reales de una población o situación. Esto refuerza que un buen enunciado no solo describe el problema, sino que lo sitúa en un marco social, cultural o práctico significativo.

Este principio resalta la importancia de la reproducibilidad en la investigación científica. Detallar los recursos materiales (como software, equipos o documentos) asegura que el estudio sea transparente y verificable. Este aprendizaje refuerza que una investigación bien planificada considera no solo la ejecución, sino también la posibilidad de que otros puedan replicarla para validar los resultados.

La justificación es el "corazón" persuasivo de la investigación, ya que conecta el problema con su relevancia práctica o teórica. Al explicar por qué el estudio es necesario, el investigador no solo motiva su realización, sino que también convence a otros (como financiadores o académicos) de su valor. Este aprendizaje enfatiza la necesidad de alinear el proyecto con necesidades reales o vacíos de conocimiento.

Prezados autores, Maria Isabel e Gilberto

Considerações gerais:

O título está em sintonia com a proposta temática, o texto atende o objetivo proposto, o problema é olhado do ponto de vista de classe, a metodologia está coerente a perspectiva teórica do materialismo. Sugiro algumas correções pontuais, que seguem no decorrer do texto.

Author response:

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

Reviewer #1 (Public review):

Munday, Rosello, and colleagues compared predictions from a group of experts in epidemiology with predictions from two mathematical models on the question of how many Ebola cases would be reported in different geographical zones over the next month. Their study ran from November 2019 to March 2020 during the Ebola virus outbreak in the Democratic Republic of the Congo. Their key result concerned predicted numbers of cases in a defined set of zones. They found that neither the ensemble of models nor the group of experts produced consistently better predictions. Similarly, neither model performed consistently better than the other, and no expert's predictions were consistently better than the others. Experts were also able to specify other zones in which they expected to see cases in the next month. For this part of the analysis, experts consistently outperformed the models. In March, the final month of the analysis, the models' accuracy was lower than in other months and consistently poorer than the experts' predictions. 

A strength of the analysis is the use of consistent methodology to elicit predictions from experts during an outbreak that can be compared to observations, and that are comparable to predictions from the models. Results were elicited for a specified group of zones, and experts were also able to suggest other zones that were expected to have diagnosed cases. This likely replicates the type of advice being sought by policymakers during an outbreak. 

A potential weakness is that the authors included only two models in their ensemble. Ensembles of greater numbers of models might tend to produce better predictions. The authors do not address whether a greater number of models could outperform the experts. 

The elicitation was performed in four months near the end of the outbreak. The authors address some of the implications of this. A potential challenge to the transferability of this result is that the experts' understanding of local idiosyncrasies in transmission may have improved over the course of the outbreak. The model did not have this improvement over time. The comparison of models to experts may therefore not be applicable to the early stages of an outbreak when expert opinions may be less welltuned. 

This research has important implications for both researchers and policy-makers. Mathematical models produce clearly-described predictions that will later be compared to observed outcomes. When model predictions differ greatly from observations, this harms trust in the models, but alternative forms of prediction are seldom so clearly articulated or accurately assessed. If models are discredited without proper assessment of alternatives then we risk losing a valuable source of information that can help guide public health responses. From an academic perspective, this research can help to guide methods for combining expert opinion with model outputs, such as considering how experts can inform models' prior distributions and how model outputs can inform experts' opinions. 

Reviewer #2 (Public review):

Summary: 

The manuscript by Munday et al. presents real-time predictions of geographic spread during an Ebola epidemic in north-eastern DRC. Predictions were elicited from individual experts engaged in outbreak response and from two mathematical models. The authors found comparable performance between experts and models overall, although the models outperformed experts in a few dimensions. 

Strengths: 

Both individual experts and mathematical models are commonly used to support outbreak response but rarely used together. The manuscript presents an in-depth analysis of the accuracy and decision-relevance of the information provided by each source individually and in combination. 

Weaknesses: 

A few minor methodological details are currently missing.

We thank the reviewers for taking the time to consider our paper and for their positive reflections and suggestions for our study. We recognise and endorse their characterisation of the study in the public reviews and are greatful for their interest and support for this work. 

Reviewer #1 (Recommendations For The Authors): 

I initially found Table 1 difficult to interpret. In the final two columns, the rows relate to each other but in the other columns, rows within months don't relate to each other. Could this be made clearer? 

Thank you for your helpful suggestion. We agree that this is a little confusing and have now added vertical dividers to the table to indicate which parts of the table relate to each other.

In Figure 1A, the colours are the same as in the colour-bar for Figure 1B but don't have the same meaning. Could different colours be used or could Figure 1A have its own colour-bar to aid clarity? 

Thank you for your query. The colours are not the same pallette, but we appreciate that they look very similar. To help the reader we have changed the colour palette of panel A and added a legend to the left.  

In Figure 3, can labels for each expert be aligned horizontally, rather than moving above and below the timeline each month? 

Thank you for your perspective on this. We made the concious dicision to desplay the experts in this way as it allows the timeline to be presented in a shorter horizontal space. We appreciate that others may prefer a different design, but we are happy with this one. 

On lines 292 and 293, the authors state that experts were less confident that case numbers would cross higher thresholds. It seems that this would be inevitable given the number of cases is cumulative. Could this be clarified, please? 

Thank you for raising this point. We agree that this wording is confusing. We have now reworked the entire section in response to another reviewer. The equivalent section now reads: 

Experts correctly identified Mabalako as the highest-risk HZ in December. They attributed an average 82% probability of exceeding 2 cases; Mabalako reported 38 cases that month, exceeding all thresholds, although the probability assigned to exceeding the higher thresholds was similar to that of Beni (3 cases)

Reviewer #2 (Recommendations For The Authors): 

(1) Some methodological details seem to be missing. Most importantly, the results present multiple ensembles (experts, models, and both), but I can't seem to find anywhere in the Methods that details how these ensembles are calculated. Also, I think it would be useful to define the variables in each equation. It would have been easier to connect the equations to the description if the variables were cited explicitly in the text. 

Thank you for pointing out these omissions. We have included the following paragraph to detail how ensemble forecasts were calculated. 

“Enslemble forecasts

Ensemble forecasts were calculated as an average of the probabilities attributed by the members of the ensemble. For the expert ensemble the arithmetic mean was calculated across all experts with equal weighting. Similarly the model ensemble used the unweighted mean of the model forecasts. For the mixed (model and expert) ensemble, the mean was weighted such that the combined weight of the experts forecasts and the combined weight of the models forecasts were equal.”

(2) Overall, I think the results provide a strong analysis of model vs. expert performance. However, some sections were highly detailed (e.g., the text usually discusses results for every month and all health zones), which clouded my ability to see the salient points. For example, I found it difficult to follow all the details about expert/model predictions vs. observations in the "Expert panel and health zones..." subsection; instead, the graphical illustration of predictions vs. observations in Figure 4 was much easier to interpret. Perhaps some of these details could be trimmed or moved to the supplementary material. 

Thank you for your honest feedback on this point. We have shortened this section to highlight the key points that we feel are the most important. We have also simplified the text where we discuss the health zones nominated by experts. 

(3) Figure 5C is a nice visualization of the fallibility of relying on a single individual expert (or model). I wonder if it would be useful to summarize these results into the probability that a randomly selected expert outperforms a single model. Is it the case that a single expert is more unreliable than a single model? The discussion emphasizes the importance of ensembles and compares a single model to an ensemble of experts, but eliciting predictions from multiple experts may not always be possible. 

Thank you for raising this. We agree that this is an important point that eliciting expert opinions is not a trivial task and should not be taken for granted. We agree with the principle of your suggestion that it would be useful to understand how the models compare to indevidual experts. We don’t however believe that an additional analysis would add sufficiently more information than already shown in Figure 5, which already displays the full distribution of indevidual experts for each month and threshold. If you would like to try this analysis yourself, the relevant data (the indevidual score for each combination of expert, threshold, heal zone and month) is included in the github repo (https://github.com/epiforecasts/Ebola-Expert-Elicitation/blob/main/outputs/indevidual_results_with_scores.csv).

Minor comments: 

(1) Figure 2: the color scales in each panel are meant to represent different places, correct? The figure might be easier to interpret if the colors used were different.  

Thank you for bringing this to our attention. We have now changed the palette of panel A to differ from panel B.  

(2) Equation 7: is o(c>c_thresh) meant to be the indicator function (i.e. 1 if c>c_thresh) and 0 otherwise)? 

Thanks for raising this. The function o is the same as in the previous equation – an observation count function. We appreciate that this is not immediately clear so have added a sentence to explain the notation after the equation.

(3) Table 1: a brief description of the column headers would be useful.  

Thank you for the suggestion. We have now extended the table caption to include more description of the columns. 

“Table 1: Experts and health zones included in each round of the survey. The left part of the table details the experts interviewed (highlighted in green) the health zones included in the main survey in each month. In addition, the right part of the table details the health zones nominated by experts and the number of experts that nominated each one.”

DOI: 10.3390/biom15030413

Resource: (Abcam Cat# ab32760, RRID:AB_2145529)

Curator: @areedewitt04

SciCrunch record: RRID:AB_2145529

What is this?

DOI: 10.3389/fcell.2024.1394031

Resource: BioVoxxel Toolbox (RRID:SCR_015825)

Curator: @areedewitt04

SciCrunch record: RRID:SCR_015825

What is this?

DOI: 10.1016/j.isci.2025.113232

Resource: Flycircuit (RRID:SCR_006375)

Curator: @scibot

SciCrunch record: RRID:SCR_006375

What is this?

DOI: 10.1016/j.immuni.2025.07.001

Resource: (BioLegend Cat# 350506, RRID:AB_2563860)

Curator: @scibot

SciCrunch record: RRID:AB_2563860

What is this?

...momento tus ingresos, recibos o pagos.

Author response:

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

Reviewer #1 (Public Review): 

The manuscript by Feng et al. reported that the Endothelin B receptor (ETBR) expressed by the satellite glial cells (SGCs) in the dorsal root ganglions (DRG) acted to inhibit sensory axon regeneration in both adult and aged mice. Thus, pharmacological inhibition of ETBR with specific inhibitors resulted in enhanced sensory axon regeneration in vitro and in vivo. In addition, sensory axon regeneration significantly reduces in aged mice and inhibition of ETBR could restore such defect in aged mice. Moreover, the study provided some evidence that the reduced level of gap junction protein connexin 43 might act downstream of ETBR to suppress axon regeneration in aged mice. Overall, the study revealed an interesting SGC-derived signal in the DRG microenvironment to regulate sensory axon regeneration. It provided additional evidence that non-neuronal cell types in the microenvironment function to regulate axon regeneration via cell-cell interaction. 

However, the molecular mechanisms by which ETBR regulates axon regeneration are unclear, and the manuscript's structure is not well organized, especially in the last section. Some discussion and explanation about the data interpretation are needed to improve the manuscript. 

We thank the reviewer for the positive comments. We agree that the mechanisms by which ETBR signaling functions as a brake on axon growth and regeneration remain to be elucidated. We believe that unraveling the detailed molecular pathways downstream of ETBR signaling in SGCs that promote axon regeneration is beyond the scope of this manuscript. Answering these questions would first require cell specific KO of ETBR and Cx43 to confirm that this pathway is operating in SGCs to control axon regeneration. We would also need to identify how SGCs communicate with neurons to regulate axon regeneration, which is a large area of ongoing research that remains poorly understood. Our data showing that pharmacological inhibition of ETBR with specific FDA-approved inhibitors enhances sensory axon regeneration provide not only new evidence for non-neuronal mechanisms in nerve repair, but also a new potential clinical avenue for therapeutic intervention.

As suggested by the reviewer, we have extensively revised the organization of the manuscript, especially the last section of results. We have performed additional snRNAseq experiments to establish the impact of aging in DRG. We have also performed additional experiments to determine if blocking ETBR improves target tissue reinnervation. Following the reviewer’s suggestion, we have also expanded the Discussion section to discuss alternative mechanisms and o]er additional interpretation of our data. Below we describe how we address each point in detail.

(1) The result showed that the level of ETBR did not change after the peripheral nerve injury. Does this mean that its endogenous function is to limit spontaneous sensory axon regeneration? In other words, the results suggest that SGCs expressing ETBR or vascular endothelial cells expressing its ligand ET-1 act to suppress sensory axon regeneration. Some explanation or discussion about this is necessary. Moreover, does the protein level of ETBR or its ligand change during aging?  

We thank the reviewer for this point. Our results indeed indicate that one endogenous function of ETBR is to limit the extent of sensory axon regeneration. This may be a part of a mechanism to limit spontaneous sensory axon growth or plasticity and maladaptive neural rewiring after nerve injury. While the increased growth capacity of damaged peripheral axons can lead to reconnection with their targets and functional recovery, the increased growth capacity can also lead to axonal sprouting of the central axon terminals of injured neurons in the spinal cord, and to pain (see for example Costigan et al 2010, PMID: 19400724).  In the context of aging that we describe here, this protective mechanism may hinder beneficial recovery. Other mechanisms that slow axon regeneration have been reported, and include, for example, axonally synthesized proteins, which typically support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this process. One such RBP, the RNA binding protein KHSRP is locally translated following nerve injury. Rather than promoting axon regeneration, KHSRP promotes decay of other axonal mRNAs and slows axon regeneration.  Another example includes the Rho signaling pathway, which was shown to function as an inhibitory mechanism that slows the growth of spiral ganglion neurites in culture. We have now included these examples in the Discussion section.

To address the reviewer’s second question, we have checked protein levels of ETBR and ET-1 in adult and aged DRG tissue. We observed a robust increase in ET-1 in aged DRG, while the levels of ETBR did not appear to change significantly. These results are now presented in Figure 4- Figure Supplement 1, and further support the notion that in aging, activation of the ETBR signaling hinders axon regeneration.

(2) In ex vivo experiments, NGF was added to the culture medium. Previous studies have shown that adult sensory neurons could initiate fast axon growth in response to NGF within 24 hours. In addition, dissociated sensory neurons could also initiate spontaneous regenerative axon growth without NGF after 48 hours. Some discussion or rationale is needed to explain the di]erence between NGF-induced or spontaneous axon growth of culture adult sensory neurons and the roles of ETBR and SGCs. 

We appreciate the reviewer’s suggestion. In adult DRG explant or dissociated cultures, NGF is not typically required for survival or axon outgrowth. However, in dissociated culture, the addition of NGF to the medium stimulates growth from more neurons compared to controls (Smith and Skene 1997). In the DRG explant, NGF does not promote significant e]ects on axon growth, but stimulates glial cell migration (Klimovich et al 2020). We opted to included NGF in our explant assay to increase the potential of stimulating axon regeneration with pharmacological manipulations of ETBR. We have now clarified these considerations in the Method section.

(3) In cultured dissociated sensory neurons, inhibiting ETBR also enhanced axon growth, which meant the presence of SGCs surrounding the sensory neurons. Some direct evidence is needed to show the cellular relationship between them in culture.  

We thank the reviewer for raising this point and have added new data, now presented in Figure 2B, to show that in mixed DRG cultures, SGCs labeled with Fabp7 are present in the culture in proximity to neurons labeled with TUJ1, but they do not fully wrap the neuronal soma. These results are consistent with prior findings reporting that as time in culture progresses, SGCs lose their adhesive contacts with neuronal soma and adhere to the coverslip (PMID: 22032231, PMID: 27606776).  While in some cases SGCs can maintain their association with neuronal soma in the first day in culture after plating, in our hands, most SGCs have left the soma at the 24h time point we examined. 

(4) In Figure 3, the in vivo regeneration experiments first showed enhanced axon regeneration either 1 day or 3 days after the nerve injury. The study then showed that inhibiting ETBR could enhance sensory axon growth in vitro from uninjured naïve neurons or conditioning lesioned neurons. To my knowledge, in vivo sensory axon regeneration is relatively slow during the first 2 days after the nerve injury and then enters the fast regeneration mode on the 3rd day, representing the conditioning lesion e]ect in vivo. Some discussion is needed to compare the in vitro and the in vivo model of axon regeneration. 

We agree that axon growth is relatively slow the first 2 days and enters a fast growth mode on day 3. This has been elegantly demonstrated in Shin et al Neuron 2012 (PMID: 22726832), where an in vivo conditioning injury 3 days prior increases axon growth one day after injury. In vitro, similar e]ects have been described: a prior in vivo injury accelerates growth capacity within the first day in culture, but a similar growth mode occurs in naive adult neurons after 2-3 days in vitro (Smith and Skene 1996). We also know that the neurite growth in culture is stimulated by higher cell density, likely because non-neuronal cells can secrete trophic factors (Smith and Skene 1996). Our in vitro results thus suggest that blocking ETBR in SGCs in these mixed cultures may alter the media towards a more growth promoting state. In vivo, our data show that Bosentan treatment for 3 days partially mimics the conditioning injury and potentiate the e]ect of the conditioning injury. One possible interpretation is that inhibition of ETBR alters the release of trophic factors from SGCs. Future studies will be required to unravel how ETBR signaling influence the SGCs secretome and its influence on axon growth. We have now included these discussions points in the Results and Discussion Section.

(5) In Figure 5, the study showed that the level of connexin 43 increased after ETBR inhibition in either adult or aged mice, proposing an important role of connexin 43 in mediating the enhancing e]ect of ETBR inhibition on axon regeneration. However, in the study, there was no direct evidence supporting that ETBR directly regulates connexin 43 expression in SGCs. Moreover, there was no functional evidence that connexin 43 acted downstream of ETBR to regulate axon regeneration.  

We thank the reviewer for this point and agree that we do not provide direct evidence that connexin 43 acts downstream of ETBR to regulate axon regeneration. To obtain such functional evidence would require selective KO of ETBR and Cx43 in SGCs, which we believe is beyond the scope of the current study. We have revised the Results and Discussion sections to emphasize that while we observe that ETBR inhibition increases Cx43 levels and Cx43 levels correlates with axon regeneration, whether Cx43 directly mediates the e]ect on axon regeneration remains to be established.  We also discuss potential alternative mechanisms downstream of ETBR in SGCs that could contribute to the observed e]ects on axon regeneration. Specifically, we discuss the possibility that  ETBR signaling may limit axon regeneration via regulating SGCs glutamate reuptake functions, because of the following reasons: 1) Similarly to astrocytes, glutamate uptake by SGCs is important to regulate neuronal function, 2) exposure of cultured cortical astrocytes to endothelin results in a decrease in glutamate uptake that correlates with a major loss of basal glutamate transporter expression (GLT-1 and1), 3) Both glutamate transporters are expressed in SGCs in sensory ganglia 4) GLAST and glutamate reuptake function is important for lesion-induced plasticity in the developing somatosensory cortex. 

Reviewer #2 (Public Review): 

Summary: 

In this interesting and original study, Feng and colleagues set out to address the e]ect of manipulating endothelin signaling on nerve regeneration, focusing on the crosstalk between endothelial cells (ECs) in dorsal root ganglia (DRG), which secrete ET-1 and satellite glial cells (SGCs) expressing ETBR receptor. The main finding is that ETBR signaling is a default brake on axon growth, and inhibiting this pathway promotes axon regeneration after nerve injury and counters the decline in regenerative capacity that occurs during aging. ET-1 and ETBR are mapped in ECs and SGCs, respectively, using scRNA-seq of DRGs from adult or aged mice. Although their expression does not change upon injury, it is modulated during aging, with a reported increase in plasma levels of ET-1 (a potent vasoconstrictive signal). Using in vitro explant assays coupled with pharmacological inhibition in mouse models of nerve injury, the authors demonstrate that ET-1/ETBR curbs axonal growth, and the ETAR/ETBR antagonist Bosentan boosts regrowth during the early phase of repair. In addition, Bosentan restores the ability of aged DRG neurons to regrow after nerve lesions. Despite Bosentan inhibiting both endothelin receptors A and B, comparison with an ETAR-specific antagonist indicates that the e]ects can be attributed to the ET-1/ETBR pathway. In the DRGs, ETBR is mostly expressed by SGCs (and a subset of Schwann cells) a cell type that previous studies, including work from this group, have implicated in nerve regeneration. SGCs ensheath and couple with DRG neurons through gap junctions formed by Cx43. Based on their own findings and evidence from the literature, the pro-regenerative e]ects of ETBR inhibition are in part attributed to an increase in Cx43 levels, which are expected to enhance neuron-SGC coupling. Finally, gene expression analysis in adult vs aged DRGs predicts a decrease in fatty acid and cholesterol metabolism, for which previous work by the authors has shown a requirement in SGCs to promote axon regeneration. 

Strengths: 

The study is well-executed and the main conclusion that "ETBR signaling inhibits axon regeneration after nerve injury and plays a role in age-related decline in regenerative capacity" (line 77) is supported by the data. Given that Bosentan is an FDA-approved drug, the findings may have therapeutic value in clinical settings where peripheral nerve regeneration is suboptimal or largely impaired, as it often happens in aged individuals. In addition, the study highlights the importance of vascular signals in nerve regeneration, a topic that has gained traction in recent years. Importantly, these results further emphasize the contribution of longneglected SGCs to nerve tissue homeostasis and repair. Although the study does not reach a complete mechanistic understanding, the results are robust and are expected to attract the interest of a broader readership. 

We thank the reviewer for the positive comments, especially in regard to the rigor and originality of our study.

Weaknesses: 

Despite these positive comments provided above, the following points should be considered: 

(1) This study examines the contribution of the ET-1 pathway in the ganglia, and in vitro assays are consistent with the idea that important signaling events take place there. Nevertheless, it remains to be determined whether the accelerated axon regrowth observed in vivo depends also on cellular crosstalk mediated by ET-1 at the lesion site. Are ECs along the nerve secreting ET-1? What cells are present in the nerve stroma that could respond and participate in the repair process? Would these interactions be sensitive to Bosentan? It may be di]icult to dissect this contribution, but it should at least be discussed.  

We thank the reviewer for this important point and agree that the in vivo e]ects observed cannot rule out the contribution of ECs or SCs at the lesion site in the nerve. Dissecting the contribution of ETBR expressing cells in the nerve would require cell-specific manipulations that go beyond the scope of this manuscript. We have revised the Discussion section to highlight the potential contribution of ECs, fibroblast and SCs in the nerve.  

(2) It is suggested that the permeability of DRG vessels may facilitate the release of "vascularderived signals" (lines 82-84). Is it possible that the ET-1/ETBR pathway modulates vascular permeability, and that this, in turn, contributes to the observed e]ects on regeneration?  

We thank the reviewer for raising this interesting point. ET-1 can have an impact on vascular permeability. It was indeed shown that in high glucose conditions, increased trans-endothelial permeability is associated with increased Edn1, Ednra and Ednrb expression and augmented ET1 immunoreactivity (PMID: 10950122). It is thus possible that part of the e]ects observed results from altered vascular permeability. We have included this point in the Discussion section. Future experiments will be required to test how injury and age a]ects vascular permeability in the DRG.

(3) Is the a]inity of ET-3 for ETBR similar to that of ET-1? Can it be excluded that ET-3 expressed by fibroblasts is relevant for controlling SGC responses upon injury/aging?  

We thank the reviewer for raising this point. ET-1 binds to ETAR and ETBR with the same a]inity, but ET3 shows a higher a]inity to ETBR than to ETAR (Davenport et al. Pharmacol. Rev 2016 PMID: 26956245). We attempted to examine ET-3 level in adult and aged DRG by western blot, but in our hands the antibody did not work well enough, and we could not obtain clear results. We thus cannot exclude the possibility that ET-3 released by fibroblasts contribute to the e]ects we observe on axon regeneration. Indeed, in cultured cortical astrocytes, application of either ET-1 or ET-3 leads to inhibition of Cx43 expression. We have revised the text in the Discussion section to highlight the possibility that both ET-1 and ET-3 could participate on the ETBRdependent e]ect on axon regeneration.

(4) ETBR inhibition in dissociated (mixed) cultures uncovers the restraining activity of endothelin signaling on axon growth (Figure 2C). Since neurons do not express ET-1 receptors, based on scRNA-seq analysis, these results are interpreted as an indication that basal ETBR signaling in SGC curbs the axon growth potential of sensory neurons. For this to occur in dissociated cultures, however, one should assume that SGC-neuron association is present, similar to in vivo, or to whole DRG cultures (Figure 2C). Has this been tested?

We thank the reviewer for this point. In dissociated DRG culture, neurons, SGCs and other nonneuronal cells are present, but SGCs do not retain the surrounding morphology as they do in vivo. Within 24 hours in culture, SGCs lose their adhesive contacts with neuronal soma and adhere to the coverslip (PMID: 22032231, PMID: 27606776).  We have included new data in Figure 2B to show that in our culture conditions, SGCs are present, but do not wrap neurons soma as they do in vivo. We also know from prior studies that the density of the culture a]ects axon growth, an e]ect that was attributed to trophic factors released from non-neuronal cells (Smith and Skene 1997). Therefore, although SGCs do not surround neurons, the signaling pathway downstream of ETBR may be present in culture and contribute to the release of trophic factors that influence axon growth. We have revised the Results section to better explain our in vitro results and their interpretation.

In both in vitro experimental settings (dissociated and whole DRG cultures) how is ETBR stimulated over up to 7 days of culture? In other words, where does endothelin come from in these cultures (which are unlikely to support EC/blood vessel growth)? Is it possible that the relevant ligand here derives from fibroblasts (see point #6)? Or does it suggest that ETBR can be constitutively active (i.e., endothelin-independent signaling)? Is there any chance that endothelin is present in the culture media or Matrigel? 

We thank the reviewer for raising this point.  Our single-cell data indicate that ET-1 is expressed by endothelial cells and ET-3 by fibroblasts. In dissociated DRG culture at 24h time point, all DRGs cells are present, including endothelial cells and fibroblasts, and could represent the source of ET-1 or ET-3. In the explant setting, it is also possible that both ET-1 and ET-3 are released by endothelial cells and fibroblasts during the 7 days in culture. According to information for the suppliers, endothelin is not present neither in the culture media nor in the Matrigel. While mutations can facilitate the constitutive activity of the ETBR receptor, we are not aware of data showing that endogenous ETBR can be constitutively active.  Because the molecular mechanisms governing ETBR -mediated signaling remain incompletely understood (see for example PMID: 39043181, PMID: 39414992) future studies will be required to elucidate the detailed mechanisms activating ETBR in SGCs and its downstream signaling mechanisms.  We have now expanded the Results and discussion sections to clarify these points. 

(5) The discovery that ET-1/ETBR signaling in SGC curtails the growth capacity of axons at baseline raises questions about the physiological role of this pathway. What happens when ETBR signaling is prevented over a longer period of time? This could be addressed with pharmacological inhibitors, or better, with cell-specific knock-out mice. The experiments would certainly be of general interest, although not within the scope of this story. Nevertheless, it could be worth discussing the possibilities. 

We agree that this is an interesting point. As mentioned above in response to point #1 of reviewer 1, the physiological role of this pathway could be to limit plasticity and prevent maladaptive neural rewiring that can happen after injury (Costigan et al 2009, PMID: 19400724), but can also hinder beneficial recovery after injury. Other mechanisms that limit axon regeneration capacity have been described and involve local mRNA translation and Rho signaling. We have revised the Discussion section to include these points. We agree that understanding the consequence of blocking ETBR over longer time periods is beyond the scope of the current study, but we now discuss the possibility that blocking ETBR with a cell specific KO approach could unravel its physiological function on target innervation and behavior. 

(6) Assessing Cx43 levels by measuring the immunofluorescence signal (Figure 5E-F) is acceptable, particularly when the aim is to restrict the analysis to SGCs. The modulation of Cx43 expression by ET-1/ETBR plays an important part in the proposed model. Therefore, a complementary analysis of Cx43 expression by quantitative RT-PCR on sorted SGCs would be a valuable addition to the immunofluorescence data. Is this attainable? 

We agree and have attempted to perform these types of experiments but encountered technical di]iculties. We attempted to sorting SGCs from transgenic mice in which SGCs are fluorescently labeled. However, the cells did not survive the sorting process and died in culture.  We think that increasing the viability of cells after sorting would require capillary- free fluorescent sorting approaches. However, we do not currently have access to such technology. We attempted this experiment with cultured SGCs, following a previously published protocol (Tonello et al. 2023 PMID: 38156033). In these experiments, SGCs are cultured for 8 days to obtain purity. We did not observe any di]erence in Cx43 protein or mRNA level upon treatment with ET-1 with or without BQ788. However, in these SGCs cultures, Cx43 displayed a di]use localization, rather than puncta as observed in vivo. Therefore, despite our multiple attempts, quantifying Cx43 on sorted or purified SGCs was not attainable.

(7) The conclusions "We thus hypothesize that ETBR inhibition in SGCs contributes to axonal regeneration by increasing Cx43 levels, gap junction coupling or hemichannels and facilitating SGC-neuron communication" (lines 303-305) are consistent with the findings but seem in contrast with the e]ect of aging on gap junction coupling reported by others and cited in line 210: "the number of gap junctions and the dye coupling between these cells increases (Huang et al., 2006)". I am confused by what distinguishes a potential, and supposedly beneficial, increase in coupling after ETBR inhibition, from what is observed in aging. 

We agree that the aging impact of Cx43 level and gap junction number appears contradictory. Procacci et al 2008 reported that Cx43 expression in SGCs decreases in the aged mice. Huang et al 2006 report that both the number of gap junctions and the dye coupling between these cells were found to increase with aging. Procacci et al suggested as a possible explanation for this apparent discrepancy that additional connexin types other than Cx43 may contribute to the gap junctions between SGCs in aged mice. Our snRNAseq data did not allow us to verify this hypothesis, because there were less SGCs in aged mice compared to adult, and connexin genes were detected in only 20% or less of SGCs.  Furthermore, our quantification did not look specifically at gap junctions, but just at Cx43 puncta. Cx43 can also form hemichannels in addition to gap junctions, and can also perform non-channel functions, such as protein interaction, cell adhesion, and intracellular signaling. Thus, more research examining the role of Cx43 in SGCs is necessary to address this discrepancy in the literature. We have expanded the Discussion section to include these points. 

(8) I find it di]icult to reconcile the results in Figure 5F with the proposed model since (1) injury increases Cx43 levels in both adult and aged mice, (2) the injured aged/vehicle group has a similar level to the uninjured adult group, (3) upon injury, aged+Bosentan is much lower than adult+Bosentan (significance not tested). It seems hard to explain the e]ect of Bosentan only through the modulation of Cx43 levels. Whether the increase in Cx43 levels following ETBR inhibition actually results in higher SGC-neuron coupling has not been assessed experimentally. 

We thank the reviewer for this point and agree that the e]ect of Bosentan is likely not exclusively through the modulation of Cx43 levels in SGCs, and that Cx43 levels may simply correlate with axon regenerative capacity. We have revised the manuscript to clarify this point.  We have also added the missing significance test in Figure 5F.

Cell specific KO of Cx43 and ETBR would allow to test this hypothesis directly but is beyond the scope of the current study. We have not tested SGCs-neuron coupling, as these experiments are currently beyond our area of expertise. Cx43 has also other functions beyond gap junction coupling, such as protein interaction, cell adhesion, and intracellular signaling. Investigating the precise function of Cx43 would require in depth biochemical and cell specific experiments that are beyond the scope of this study. Furthermore, as we now mentioned in response to reviewer #2 point 5, ETBR signaling may also have other downstream e]ects in SGCs, such as glutamate transporters expression, or a]ect other cells in the nerve during the regeneration process. We have revised the Discussion section to include these alternative mechanisms.

Reviewer #3(Public Review): 

Summary: 

This manuscript suggests that inhibiting ETBR via the FDA-approved compound Bosentan can disrupt ET-1-ETBR signalling that they found detrimental to nerve regeneration, thus promoting repair after nerve injury in adult and aged mice. 

Strengths: 

(1) The clinical need to identify molecular and cellular mechanisms that can be targeted to improve repair after nerve injury. 

(2) The proposed mechanism is interesting. 

(3) The methodology is sound. 

We thank the reviewer for highlighting the strengths of our study

Weaknesses: 

(1) The data appear preliminary and the story appears incomplete. 

We appreciate the reviewer’s point. We would like to emphasize that our results provide compelling evidence that ETBR signaling is a default brake on axon growth, and inhibiting this pathway promotes axon regeneration after nerve injury and counters the decline in regenerative capacity that occurs during aging. We also provide evidence that ETBR signaling regulates the levels of Cx43 in SGCs. Furthermore, our results document the use of an FDA approved compound to increase axon regeneration may be of interest to the broader readership, as there is currently no therapies to improve or accelerate nerve repair after injury. We agree that the detailed mechanisms operating downstream of ETBR will need to be elucidated. Answering these questions would first require cell specific KO of ETBR and Cx43 to confirm that this pathway is operating in SGCs to control axon regeneration. We would also need to identify how SGCs communicate with neurons to regulate axon regeneration, which is a large area of ongoing research that remains poorly understood. This extensive and highly complex set of experiments is beyond the scope of the current study. As we discussed in our response to reviewer #1 and #2 we attempted to perform numerous additional experiments to better define the role of ETBR signaling in SGCs in aging and have included additional results in Fig. 2B, Fig 3G-H,  Fig 5A-E, and Figure 4- Figure Supplement 1and Figure 5- Figure Supplement 1. We have expanded the

Discussion to acknowledge the limitation of our study and to discuss possible mechanisms.  

(2) Lack of causality and clear cellular and molecular mechanism. There are also some loose ends such as the role of connexin 43 in SGCs: how is it related to ET-1- ETBR signalling?  

We thank the reviewer for this point and agree that the molecular mechanisms downstream of ETBR remain to be elucidated. However, we believe that our manuscript reports an interesting potential of an FDA-approved compound in promoting nerve repair. We focused on Cx43 downstream of ETBR signaling because decreased Cx43 expression in SGCs in ageing was previously established, but the mechanisms were not elucidated. Furthermore, it was reported that ET1 signaling in cultured astrocytes, which share functional similarities with SGCs, leads to the closure of gap junctions and reduction in Cx43 expression. Our study thus provides a mechanism by which ETBR signaling in SGCs regulates Cx43 expression. Whether Cx43 directly impact axon regeneration remains to be tested. Cell specific KO of Cx43 and ETBR would be required to answer this question. We have revised the Introduction and Discussion section extensively to provide a link between ETBR and Cx43 and to acknowledge the lack of causality in Cx43 in SGCs, as well as to provide additional potential mechanisms by which ETBR inhibition may promote nerve repair.

Reviewer #2 (Recommendations For The Authors): 

In addition to the points listed in the Public Review section, please consider the following comments: 

(1) ETAR, which is high in mural cells, does not seem to be implicated in the reported proregenerative e]ects. Even so, can vasoconstriction be ruled out as an underlying cause of the age-dependent decline in axon regrowth potential and, more generally, in the e]ects of ET-1 inhibition on regeneration? This could be discussed. 

We agree that we can’t exclude a role in vasoconstriction or e]ect on vascular permeability in the age-dependent decline in axon regrowth potential. However, our in vitro and ex vivo experiments, in which vascular related mechanisms are unlikely, suggest that vasoconstriction may not be a major contributor to the e]ects we observed.

(2) The manuscript (e.g. line 287-288) would benefit from a discussion of the role that blood vessels play in the peripheral nervous system, and possibly CNS, repair. Vessels were shown to accompany regenerating fibers and instruct the reorganization of the nerve tissue to favor repair potentially through the release of pro-regenerative signals acting on stromal cells, glia, and other cellular components. Highlighting these processes will help put the current findings into perspective. 

We agree and have revised the Discussion section to better explain the role of blood vessels in orientating Schwann cells migration and guiding axon regeneration.

(3) The vast majority of the cells that are sequenced and shown in the UMAP in Figure 1C are from adult (3-month-old) mice [16,923 out of 18,098]. It would be useful to include the UMAP split (or color-coded) by timepoint to appreciate changes in cell clustering that may occur with aging.  

We apologize for this misunderstanding, Figure 1C had all cells from all ages. However, the number of cells we obtained from the age group was insu]icient to perform in depth analysis of each cell type. We have thus revised this section and Figure 1, now only presenting the data from adult mice.  

It is not discussed why fewer cells were sequenced at later stages. Additionally, I do not know how to interpret the double asterisks next to the labeling "18,098 samples" in Figure 1C. 

Since our original sequencing of adult and aged mice using 10x yielded so few cells from the aged DRG, we tested and optimized a new technology for single cell preparation of DRG using Illumina Single Cell 3’ RNA Prep. This preparation creates templated emulsions using a vortex mixer to capture and barcode single-cell mRNA instead of a microfluidics system. This method yielded much better results for nuclei recovery from aged DRG, with more nuclei and better quality of nuclei. Thus, we now present in Figure 5 and Figure 5- Figure Supplement 1 the results from snRNA-sequencing of aged and adult DRG using the Illumina single cell kit. The results of the snRNA-sequencing show a decreased abundance of SGCs in aged mice, consistent with the results from our morphology analysis with EM. We were also able to perform SGCs-specific pathway analysis because of the increased number of nuclei captured in the aged SGCs, which we included in the manuscript.

(4) The in vivo studies are designed to examine the e]ects of ETBR inhibition during the first phase of axon regrowth after nerve injury (1-3 days post-injury, dpi). Is there a reason why later stages have not been studied? It would be interesting to understand whether ETBR inhibition improves long-term recovery or is only e]ective at boosting the initial growth of axons through the lesion. It is possible that early inhibition will be enough for long-term recovery. If so, these experiments would define a sensitivity window with therapeutic value. 

We agree that assessing functional recovery requires proper behavioral tests or morphological evaluations of reinnervation. To determine if Bosentan treatment has long-term e]ects on recovery, we administered Bosentan or vehicle for 3 weeks (daily for 1 week, and then once a week for the subsequent 2 weeks) after sciatic nerve crush. At 24 days after SNC, we assessed intraepidermal nerve fiber density (IENFD) in the injured paw and saw a trend towards increased fibers/mm in the treated animals (new Figure 3G,H). Future studies will examine how long-term Bosentan treatment a]ects functional recovery and innervation at later time points. Additionally, behavior assays will be needed to determine if these morphological changes relate to behavioral improvements using IENFD and behavior assays.

(5) I am unsure if the gene expression analysis shown in Figure 6 fits well into this story. It is interesting per se and in line with previous work from this group showing the relevance of fatty acid metabolism in SGCs for axon regeneration. Nevertheless, without a mechanistic link to endothelin signaling and Cx43/gap junction modulation, the observations derived from DEG analysis are not well integrated with the rest and may be more distracting than helpful. One limitation is that there is no cell-type information for the DEGs due to the small number of cells recovered from aged mice. For instance, if ETBR inhibition rescued gene downregulation associated with fatty acid/cholesterol metabolism, then the DGE results would become more relevant for understanding the cellular basis of the pro-regenerative e]ect, which at this point remains quite speculative (lines 264-265; lines 318-319).  

We agree and have added new snRNA sequencing data to replace these findings (see above response to point #4, new Figure 5 and Figure 5- Figure Supplement 1. The new data shows a decreased abundance of SGCs in aged mice, consistent with our TEM results. Pathway analysis revealed that aging triggers extensive transcriptional reprogramming in SGCs, reflecting heightened demands for structural integrity, cell junction remodeling, and glia–neuron interactions within the aged DRG microenvironment.  

(6) It would be interesting to determine whether Bosentan increases SGC coverage of neuronal cell bodies in aged mice (Figures 6A-C). 

We agree that this would be very interesting, but will require extensive EM analysis at di]erent time points and is beyond the scope of the current manuscript.

(7) Finally, adding a summary model would help the readers. 

We agree and have made a summary model, now presented in Figure 6F.

Reviewer #3 (Recommendations For The Authors): 

Longer time points post-injury and assessment of functional recovery after Bosentan would be of great value here. 

We agree that assessing functional recovery requires proper behavioral tests or morphological evaluations of reinnervation. To determine if Bosentan treatment has long-term e]ects on recovery, we administered Bosentan or vehicle for 3 weeks (daily for 1 week, and then once a week for the subsequent 2 weeks) after sciatic nerve crush. At 24 days after SNC, we assessed intraepidermal nerve fiber density in the injured paw and saw a trend towards increased fibers/mm in the treated animals (Fig 3). While the results do not reach significance, we decided to include this new data as it provides evidence that Bosentan treatment may also improves long term recovery. Future studies will be required examine how long-term Bosentan treatment a]ects functional recovery and innervation at later time points. Additionally, behavior assays will be needed to determine if these morphological changes relate to behavioral improvements.

It would be important to know how ET-1- ETBR signalling axis promotes the regeneration of axons:this remains unaddressed. What are the cells that are specifically involved? Endothelial cellsSGC- neurons- SC? There are no experiments addressing the role of any of these? 

We agree that the molecular and cellular mechanisms by which ETBR signaling in SGCs promote axon regeneration remains to be elucidated.  Answering these questions would first require cell specific KO of ETBR and Cx43 to confirm that this pathway is operating in SGCs to control axon regeneration. We would also need to identify how SGCs communicate with neurons to regulate axon regeneration, which is a large area of ongoing research that remains poorly understood. While these are important experiments, because of numerous technical and temporal constrains, we believe they are beyond the scope of the current manuscript. 

How does connexin 43 in SGCs related to ET-1- ETBR signalling? 

The relation between connexin 43 and ETBR signaling stems from observations made in astrocytes. ET1 signaling in cultured astrocytes, which share functional similarities with SGCs, was shown to lead to the closure of gap junctions and the reduction in Cx43 expression. Because Cx43 expression, a major connexin expressed in SGCs as in astrocytes, was previously shown to be reduced at the protein level in SGCs from aged mice, we decided to explore it this ETBR-Cx43 mechanism also operates in SGCs. We have revised the Introduction and Discussion section extensively to acknowledge the lack of causality in Cx43 expression SGCs and to provide additional potential mechanisms by which ETBR inhibition may promote nerve repair.

DOI: 10.1038/s41467-019-09943-y

Resource: RRID:BDSC_35789

Curator: @bdscstockkeepers

SciCrunch record: RRID:BDSC_35789

What is this?

I find it interesting that words like "wanton" and "harlot" were used to describe this woman. Although Shamash himself states how important of a woman she is. they will always see her as a "mistress" or "harlot." Those words in and of itself have negative connotations. It is as if they degrade her almost.

Art. 914. O executado, independentemente de penhora, depósito ou caução, poderá se opor à execução por meio de embargos.

(...)

§ 2º Na execução por carta, os embargos serão oferecidos no juízo deprecante ou no juízo deprecado, mas a competência para julgá-los é do juízo deprecante, salvo se versarem unicamente sobre vícios ou defeitos da penhora, da avaliação ou da alienação dos bens efetuadas no juízo deprecado.

Reviewer #1 (Public review):

This is a theoretical study addressing the problem of constructing integrator networks for which the activity state and integrated variables display non-trivial topologies. Historically, researchers in theoretical neuroscience have focused on models with simple underlying geometries (e.g., circle, torus), for which analytical models could be more easily constructed. How these models can be generalised to complex scenarios is, however, a non-trivial question. This is furthermore a time-sensitive issue, as population recordings from the brain in complex tasks and environments increasingly require the ability to construct such models.

I believe the authors do a good job of explaining the challenges related to this problem. They also propose a class of models that, although not fully general, overcome many of these difficulties while appearing solid and well-functioning. This requires some non-trivial mathematics, which is nevertheless conveyed in a reasonably accessible form. The manuscript is well written, and both the methodology and the code are well documented.

That said, I believe the manuscript has two major limitations, which could be addressed in a revision. First, some of the assumptions underlying this class of models are somewhat restrictive but are not sufficiently discussed. Second, although the stated goal of the manuscript is to provide practical recipes for constructing integrator networks, the methods section is not very explicit about the specific steps required for different geometries. I elaborate on these limitations below.


(1) The authors repeatedly describe MADE as a technique for constructing integrators of specified "topologies and geometries." What do they mean by "geometries"? Intuitively, I would associate geometry with properties beyond topology, such as embedding dimensionality or curvature. However, it is unclear to me to what extent these aspects are explicitly specified or controlled in MADE. It seems that geometry is only indirectly defined via the connectivity kernel, which itself obeys certain constraints (e.g., limited spatial scale; see below). I believe it is important for the authors to clarify what they mean by "geometry." They should also specify which aspects are under their control, and whether, in fact, all geometries can be realized.


(2) The authors make two key assumptions: that connectivity is purely inhibitory and that the connectivity kernel has a small spatial scale. They state that under these conditions, the homogeneous fixed point becomes unstable, leading to a non-periodic state. However, it seems to me that they do not demonstrate that this emergent state is necessarily a bump localized in all manifold dimensions -- although this is assumed throughout the manuscript. Are other solutions possible or observed? For example, might the network converge to states that are localized in one dimension but extended in another, yielding e.g., stripe-like activity in the plane rather than bumps? In other words, does the proposed recipe guarantee convergence to bumps? This is a critical point and should be clarified.


(3) Related to the question above: What are the failure modes when these two assumptions are violated? Does the network always exhibit runaway activity (as suggested in the text), or can other types of solutions emerge? It would be useful if the authors could briefly discuss this.


(4) Again, related to the question above: can this formalism be extended to activity profiles beyond bumps? For example, periodic fields as seen in grid cells, or irregular fields as observed in many biological datasets -- particularly in naturalistic environments? These activity profiles are of key importance to neuroscientists, so I believe this is an important point that should at least be addressed in the Discussion. Can MADE be naturally extended to these scenarios? What are the challenges involved?


(5) Line 119: "Since σ is the only spatial scale being introduced in the dynamics, we qualitatively expect that a localized bump state within the ball will have a spatial scale of O(σ)."
Is this statement always true? I understand that the spatial scale of the synaptic inputs exchanged via recurrent interactions (i.e., the argument of the function f in Equation 1) is characterised by the spatial scale σ. But the non-linear function f could modify that spatial scale -- for example, by "cutting" the bump close to its tip. Where am I wrong? Could the authors clarify?


(6) The authors provide beautiful intuition about the problem of constructing integrators on non-trivial topologies and propose a mathematically grounded solution using Killing vectors. Of course, solutions based on Killing vectors are more complex than those with constant offsets, which raises the question: Is the brain capable of learning and handling such complex structures? Perhaps the authors could speculate in the Discussion about the biological plausibility of these mechanisms.


(7) A great merit of this paper is that it provides mathematical tools for neuroscience researchers to build integrators on non-trivial geometries. I found that, although all the necessary information is present in the Methods, the authors could improve the presentation by schematizing the steps required to build each type of model. It would be extremely useful if, for each considered geometry, the authors provided a short list of required components: the manifold P, the choice of distance, and the connectivity offsets defined by the Killing vectors. Currently, this information is presented, but scattered (not grouped by geometry).

§ 9º Qualquer alteração na legislação federal que reduza ou eleve a arrecadação do imposto: (Incluído pela Emenda Constitucional nº 132, de 2023)

I - deverá ser compensada pela elevação ou redução, pelo Senado Federal, das alíquotas de referência de que trata o § 1º, XII, de modo a preservar a arrecadação das esferas federativas, nos termos de lei complementar; (Incluído pela Emenda Constitucional nº 132, de 2023)

II - somente entrará em vigor com o início da produção de efeitos do ajuste das alíquotas de referência de que trata o inciso I deste parágrafo. (Incluído pela Emenda Constitucional nº 132, de 2023)

§ 10. Os Estados, o Distrito Federal e os Municípios poderão optar por vincular suas alíquotas à alíquota de referência de que trata o § 1º, XII. (Incluído pela Emenda Constitucional nº 132, de 2023)

§ 11. Projeto de lei complementar em tramitação no Congresso Nacional que reduza ou aumente a arrecadação do imposto somente será apreciado se acompanhado de estimativa de impacto no valor das alíquotas de referência de que trata o § 1º, XII.

oportunidade pra SN que compra imobilizado aproveitar todo o crédito e depois fica livre pra trocar de regime.

The Writer is asking how he can express his love and grief for someone who has died. He wants to sing for them, but doesn’t know how to make it worthy. He wonders how to make his tribute beautiful enough for the kind soul who is gone. He’s also asking what gift or offering he can bring to their grave to show his love.

Carving Jack O Lanterns