Pergunta pessoal: imã é o mesmo que entendemos como aquilo que atrai, objetos imantados ou tem outro significado a mais nesse contexto?

Cerveira, Portugal. (?) Falta o ano

Aumentou ainda mais a sigla? Talvez seja bom colocar em nota. Nem eu sei o que é o PN....

O que é Gente diferenciada? Um coletivo? Do que? Saber local e ano

Saber se aqui também é em Salvador e o ano. Há acima mais 2/3 fotos sem o ano, o que destoa das outras. Apurar com Ana Dumas

DOI: 10.1371/journal.pgen.1010371

Resource: RRID:BDSC_7608

Curator: @bandrow

SciCrunch record: RRID:BDSC_7608

What is this?

DOI: 10.15252/embr.202153231

Resource: RRID:BDSC_55850

Curator: @scibot

SciCrunch record: RRID:BDSC_55850

What is this?

DOI: 10.3390/ijms222111613

Resource: RRID:BDSC_8760

Curator: @scibot

SciCrunch record: RRID:BDSC_8760

What is this?

A ABP facilita a promoção da IC ao proporcionar um ambiente onde os alunos podem trabalhar juntos, compartilhar conhecimentos e desenvolver soluções de forma colaborativa. Esse processo não apenas melhora o aprendizado individual, mas também potencializa o conhecimento coletivo do grupo.

O texto sugere que a IA traz grandes ganhos de produtividade e que a colaboração entre humanos e máquinas pode levar a realizações inimagináveis. No entanto, ele não aborda os desafios e limitações reais da IA, como questões éticas, viés nos dados, e a necessidade de regulamentação. Uma visão mais equilibrada seria mais informativa.

No relatório de avaliação de opções estratégicas para o aumento da capacidade aeroportuária da região de Lisboa, selecione aquelas partes que lhe parecem incluir técnicas de investigação operacional e comente.

Surtout : est-ce le rôle de la philosophie de terrain ? Peut-être préciser ici le niveau du discours : vous parlez des projets qui visent à régler les problèmes, ou bien vous parlez du philosophe ou de l'enquête philosophique qui prend pour terrain les acteurs de ces problèmes (ou de l'élaboration de leurs solutions) ? Ou bien le philosophe de terrain devient-il effectivement un des acteurs qui participe à l'élaboration de solutions ? O

Author response:

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

Editors’ recommendations for the authors

The reviewers recommend the following: 

(a) Digging deeper into the discussion of the density-dependent dispersal. 

(b) Clarifying the microfluidic setup.  

(c) Clarifying the description and interpretation of the transcriptomic evidence. 

(d) Toning down carbon cycle connections (some reviewers felt the evidence did not fully support the claims). 

We would like to thank the editors for their thoughtful evaluation of our manuscript and their clear suggestions. We have revised the manuscript in the light of these comments, as we outline below and address in detail in the point-by-point response to the reviewers’ comments that follows. 

(a) We have expanded the discussion of density-dependent dispersal and revised Figure 2C to improve clarity. 

(b) We have also added further information concerning the microfluidic setup in the results section and provide an illustration of the setup in a new figure panel, Figure 1A.

(c) Addressing the reviewers’ comments on the transcriptomic analysis, we have added more information in the description and interpretation of the results. 

(d) We have rephrased the text describing the role of degradation-dispersal cycles for carbon cycling to highlight it as the motivation of this study and emphasize the link to literature on foraging, without creating expectations of direct measurements of global carbon cycling.

Public Reviews:

Reviewer #1 (Public Review):

[...]

Weaknesses: 

Much of the genetic analysis, as it stands, is quite speculative and descriptive. I found myself confused about many of the genes (e.g., quorum sensing) that pop up enriched during dispersal quite in contrast to my expectations. While the authors do mention some of this in the text as worth following up on, I think the analysis as it stands adds little insight into the behaviors studied. However, I acknowledge that it might have the potential to generate hypotheses and thus aid future studies. Further, I found the connections to the carbon cycle and marine environments in the abstract weak --- the microfluidics setup by the authors is nice, but it provides limited insight into naturalistic environments where the spatial distribution and dimensionality of resources are expected to be qualitatively different. 

We thank the reviewer for their suggestions to improve our manuscript. We agree that the original manuscript would have benefitted from more detailed interpretation of the observed changes in gene expression. We have revised the manuscript to elaborate on the interpretation of the changes in expression of quorum sensing genes (see response to reviewer 1, comment 3), motility genes (see response to reviewer 1, comment 6), alginate lyase genes (see response to reviewer 1, comment 7 and reviewer 2, comment 2), and ribosomal and transporter genes (see response to reviewer 2, comment 2).

In general, we think that the gene expression study not only supports the phenotypic observations that we made in the microfluidic device, such as the increased swimming motility when exposed to digested alginate medium, but  also adds further insights. Our reasoning for studying the transcriptomes in well mixed-batch cultures was the inability to study gene expression dynamics to support the phenotypic observations about differential motility and chemotaxis in our microfluidics setup. The transcriptomic data clearly show that even in well-mixed environments, growth on digested alginate instead of alginate is sufficient to increase the expression of motility and chemotaxis genes. In addition, the finding that expression of alginate lyases and metabolic genes is increased during growth on digested alginate was revealed through the analysis of transcriptomes, something which would not have been possible in the microfluidic setup. We agree with the reviewer that our analyses implicate further, perhaps unexpected, mechanisms like quorum sensing in the cellular response to breakdown products, and that this represents an interesting avenue for further studies.

Finally, we  also agree with the reviewer that it would be good to be more explicit in the text that our microfluidic system cannot fully capture the complex dynamics of natural environments. Our approach does, however, allow the characterization of cellular behaviors at spatial and temporal scales that are relevant to the interactions of bacteria, and thus provides a better understanding of colonization and dispersal of marine bacteria in a manner that is not possible through in situ experiments. We have edited our manuscript to highlight this and modified our statements regarding carbon cycling towards emphasizing the role degradation-dispersal cycles in remineralization of polysaccharides (see response to reviewer 1, comment 2).  

Reviewer #2 (Public Review):

[...]

Weaknesses: 

The explanation of the microfluidics measurements is somewhat confusing but I think this could be easily remedied. The quantitative interpretation of the dispersal data could also be improved and I'm not clear if the data support the claim made. 

We thank the reviewer for their comments and helpful suggestions. We have revised the manuscript with these suggestions in mind and believe that the manuscript is improved by a more detailed explanation of the microfluidic setup. We have added more information in the text (detailed in response to reviewer 2, comments 1 and 2) and have added a depiction of the microfluidic setup (Fig. 1A). We have also modified the presentation and discussion of the dispersal data (Fig. 2C), as described in detail below in response to reviewer 2, comment 4, and argue that they clearly show density-dependent dispersal. We believe that this modification of how the results are presented provides a more convincing case for our main conclusion, namely that the presence of degradation products controls bacterial dispersal in a density-dependent manner.  

Reviewer #3 (Public Review):

[...]

Weaknesses: 

I find this paper very descriptive and speculative. The results of the genetic analyses are quite counterintuitive; therefore, I understand the difficulty of connecting them to the observations coming from experiments in the microfluidic device. However, they could be better placed in the literature of foraging - dispersal cycles, beyond bacteria. In addition, the interpretation of the results is sometimes confusing. 

We thank the reviewer for their suggestions to improve the manuscript. We have edited the manuscript to interpret the results of this study more clearly, in particular with regard to the fact that breakdown products of alginate cause cell dispersal (see response to reviewer 2, comment 1), gene expression changes of ribosomal proteins and transporters (see response to reviewer 2, comment 2), as well as genes relating to alginate catabolism (see response to reviewer 2, comment 3).

To provide more context for the interpretation of our results we now also embed our findings in more detail in the previous work on foraging strategies and dispersal tradeoffs.

Recommendations For The Authors:

Reviewer #1 (Recommendations For The Authors):

(1) The authors should clarify in more detail what they mean by density dependence in Figure 2. Usually density dependence refers to a per capita dependence, but here it seems that the per capita rate of dispersal might be roughly independent of density (Figure 2c; if you double the number of cells it doubles the number of cells leaving). Rather it seems the dispersal is such that the density of remaining cells falls below a threshold (~300 cells). 

We thank the reviewer for raising this important point. To analyze the data more explicitly in terms of per capita dependence and so make the density dependence in the dispersal from the microfluidic chambers more clear, we have modified Figure 2C and edited the text. 

In the modified Figure 2C, we computed the fraction of dispersed cells for each chamber (i.e the change in cell number divided by the cell number at the time of the nutrient switch). This quantity directly reveals the per-capita dependence, as mentioned by reviewer 1, and is now represented on the y-axis of Figure 2C instead of the absolute change in cell number. 

These data demonstrate that the fraction of dispersed cells increases with increasing numbers of cells present in the chamber at the time of switching, with more highly populated chambers showing a higher fraction of dispersed cells. These findings indicate that there is a strong density dependence in the dispersal process.

As pointed out by reviewer 1, another interesting aspect of the data is the transition at low cell number. The fraction of dispersed cells is negative in the case of the chamber with approximately 70 cells, consistent with no dispersal at this low density, and a moderate density increase as a function of continued growth.  

In addition to the new analysis presented in Figure 2C, we have modified the paragraph that discusses this result as follows (line 208):

“We indeed found that the nutrient switch caused a few or no cells to disperse from small cell groups (Fig. 2B), whereas a large fraction of cells from large cell groups dispersed (Fig. 2C). In fact, the e fraction of cells that dispersed upon imposition of the nutrient switch showed a strong positive relationship with the number of cells present, meaning that cells in chambers with many cells were more likely to disperse than cells in chambers with fewer cells (Fig. 2C).”

(2) The authors should tone down their claims about the carbon cycle in the abstract. I do not believe the results as they stand could be used to understand degradation-dispersal cycles in marine environments relevant to the carbon cycle, since these behaviors have been studied in microfluidic environments which in my understanding are quite different. As such, statements such as "degradation-dispersal cycles are an integral part in the global carbon cycle, we know little about how cells alternate between degradation and motility" and "Overall, our findings reveal the cellular mechanisms underlying bacterial degradation-dispersal cycles that drive remineralization in natural environments" are overstated in the abstract. 

We appreciate the reviewer’s comments regarding the connections of our work with the carbon cycle. We have now rephrased these statements in our manuscript to describe a potential connection between our work and the marine carbon cycle. The colonization of polysaccharides particles by bacteria and subsequent degradation has been widely acknowledged to play a significant role in controlling the carbon flow in marine ecosystems. (Fenchel, 2002; Preheim et al., 2011; Yawata et al., 2014, 2020). We still refer to carbon flow in the revised manuscript, though cautiously, as microbial remineralization of biomass, which is recognized as an important factor in the marine biological carbon pump (e.g., (Chisholm, 2000; Jiao et al., 2024). As stated in the previous version of the manuscript, the main motivation of our work was to study the growth behaviors of marine heterotrophic bacteria during polysaccharide degradation, especially to understand when bacteria depart already colonized and degraded particles and find novel patches to grow and degrade, a process that is poorly understood. Therefore, it is conceivable that degradation-dispersal cycles do play a role in the flow of carbon in marine ecosystems. However, we acknowledge that the carbon cycle is influenced by a multitude of biological and chemical processes, and the bacterial degradation-dispersal cycle might not be the sole mechanism at play. 

We also appreciate the reviewer’s comments highlighting that the complexity of natural environments is not fully captured in our microfluidics system. However, our microfluidics setup does allow us to quantify responses and behaviors of microbial groups at high spatial and temporal resolution, especially in the context of environmental fluctuations. Microbes in nature interact at small spatial scales and have to respond to changes in the environment, and the microfluidics setup enables the quantification of these responses. Moreover, dispersal of the bacterium V. cyclitrophicus that we use in our study, has been previously observed even during growth on particulate alginate (Alcolombri et al., 2021), but the cues and regulation controlling dispersal behaviors have been unclear.  Microfluidic experiments have now allowed us to study this process in a highly quantitative manner, and align well with observations from experiments from more nature-like settings. These quantitative experiments on bacterial strains isolated from marine particles are expected to constrain quantitative models of carbon degradation in the ocean (Nguyen et al., 2022).

We have now adjusted our statements throughout our manuscript to reflect the knowledge gaps in understanding the triggers of degradation-dispersal cycles and their links with carbon flow in marine ecosystems. The revised manuscript, especially, contains the following statements (line 47 and line 60):

“Even though many studies indicate that these degradation-dispersal cycles contribute to the carbon flow in marine systems, we know little about how cells alternate between polysaccharide degradation and motility, and which environmental factors trigger this behavioral switch.”

“Overall, our findings reveal cellular mechanisms that might also underlie bacterial degradation-dispersal cycles, which influence the remineralization of biomass in marine environments.”

(3) The authors should clarify why they think quorum-sensing genes are increased in expression on digested alginate. The authors currently mention that QS could be used to trigger dispersal, but given the timescales of dispersal in Figure 2 (~half an hour), I find it hard to believe that these genes are expressed and have the suggested effect on those timescales. As such I would have expected the other way round - for QS genes to be expressed highly during alginate growth, so that density could be sensed and responded to. Please clarify. 

We have now clarified this point in the revised manuscript. While the triggering of dispersal by quorum-sensing genes may indeed appear counterintuitive, and the response is rapid (we see dispersal of cells within 30-40 minutes), both observations are in line with previous studies in another model organism Vibrio cholerae. The dispersal time is similar to the dispersal time of V. cholerae cells from biofilms, as described by Singh and colleagues, (Figure 1E of Ref. Singh et al., 2017). In that case, induction of the quorum sensing dispersal regulator HapR was observed during biofilm dispersal within one hour after switch of condition (Fig. 2, middle panel of Ref. Singh et al., 2017). Even though the specific quorum sensing signaling molecules are probably different in our strain (there is no annotated homolog of the hapR gene in V. cyclitrophicus), we observed that the full set of quorum sensing genes was enriched in cells growing on digested alginate (as reported in line 314 and Fig. 4A).

We have added this information in the manuscript (line 317): 

“The set of quorum sensing genes was also positively enriched in cells growing on digested alginate (Fig. 4A and S4F, Table S13). This role in dispersal is in agreement with a previous study that showed induction of the quorum sensing master regulator in V. cholerae cells during dispersal from biofilms on a similar time scale as here (less than an hour) [28].”

Reviewer #2 (Recommendations For The Authors):

(1) Around line 144 - I don't really understand how you flow alginate through the microfluidic platform. It seems if the particles are transiently going through the microfluidic chamber then the flow rate and hence residence time of the alginate particles will matter a lot by controlling the time the cells have to colonize and excrete enzymes for alginate breakdown. Or perhaps the alginate is not particulate but is instead a large but soluble polymer? I think maybe a schematic of the microfluidic device would help -- there is an implicit assumption that we are familiar with the Dal Co et al device, but I don't recall its details and maybe a graphic added to Figure 1 would help. 

a. In reviewing the Dal Co paper I see that cells are trapped and the medium flows through channels and the plane where the cells are held. I am still a little confused about the size of the polymeric alginate -- large scale (>1um) particles or very small polymers? 

We have now provided a detailed description of our microfluidic experimental system. At the start of the experiments, cells are in fact not trapped within the microfluidic device, but grow and can move freely within a chamber designed with dimensions (sub-micron heights) so that growth occurs only as a monolayer. Cells were exposed to nutrients, either alginate or alginate digestion products, both in soluble form (not particles). These compounds were flowed into the device through a main channel, but entered the flowfree growth chambers by diffusion. To make these aspects of our experiments clearer, we have added further information on this in the Materials & Methods section (line 556), added this information in the abstract (line 51), and in the results (line123).

To make our microfluidic setup clearer, we have followed this advice and added a schematic as Figure 1A and have added more information on the setup to the main text (line 153):

“In brief, the microfluidic chips are made of an inert polymer (polydimethylsiloxane) bound to a glass coverslip. The PDMS layer contains flow channels through which the culture medium is pumped continuously. Each channel is connected to several growth chambers that are laterally positioned. The dimensions of these growth chambers (height: 0.85 µm, length: 60 µm, width: 90-120 µm) allow cells to freely move and grow as monolayers. The culture medium, containing either alginate or digested alginate in their soluble form, is constantly pumped through the flow channel and enters the growth chambers primarily through diffusion [15,16,4,17,8]. Therefore, the number of cells and their positioning within microfluidic chambers is determined by the cellular growth rate as well as by cell movement4. This setup combined with time-lapse microscopy allowed us to follow the development of cell communities over time.”

(2) What makes this confusing is the difference between Figure 1C and Figure S2A -- the authors state that the difference in Figure 1C is due to dispersal, but is there flow through the microfluidic device? So what role does that flow through the device have in dispersal? Is the adhesion of the cell groups driven at all by a physical interaction with high molecular weight polymers in the microfluidic devices or is this purely a biological effect? Could this also be explained by different real concentrations of nutrients in the two cases? 

We realize from this comment that the role of flow of the medium in the microfluidic setup was not clearly addressed in our manuscript. In fact, cells were not exposed to flow, and nutrients were provided to the growth chambers by diffusion. We have added a clearer explanation of this point on line 158:

“The culture medium, containing either alginate or digested alginate in their soluble form, is constantly pumped through the flow channel and enters the growth chambers primarily through diffusion [15,16,4,17,8]. Therefore, the number of cells and their positioning within microfluidic chambers is determined by the cellular growth rate as well as by cell movement4.“

One purely physical effect that we anticipate is that a high viscosity of the medium could immobilize cells. To address this point, we measured the viscosity of both alginate and digested alginate and conclude that the increase in viscosity is not strong enough to immobilize cells. We added a statement in the text (line 170)

“To test the role of increased viscosity of polymeric alginate in causing the increased aggregation of cells, we measured the viscosity of 0.1% (w/v) alginate or digested alginate dissolved in TR media. For alginate, the viscosity was 1.03±0.01 mPa·s (mean and standard deviation of three technical replicates) whereas the viscosity of digested alginate in TR media was found to be 0.74±0.01 mPa·s. Both these values are relatively close to the viscosity of water at this temperature (0.89 mPa·s18) and, while they may affect swimming behavior [19], they are insufficient to physically restrain cell movement [20].”

as well as a section in the Materials and Methods (line 594):

“Viscosity of the alginate and digested alginate solution

We measured the viscosity of alginate solutions using shear rheology measurements. We use a 40 mm cone-plate geometry (4° cone) in a Netzsch Kinexus Pro+ rheometer. 1200 uL of sample was placed on the bottom plate, the gap was set at 150 um and the sample trimmed. We used a solvent trap to avoid sample evaporation during measurement. The temperature was set to 25°C using a Peltier element. We measure the dynamic viscosity over a range of shear rates  = 0.1 – 100 s-1. We report the viscosity of each solution as the average viscosity measured over the shear rates 10 – 100 s-1, where the shear-dependence of the viscosity was low.

We measured the viscosity of 0.1% (w/V) alginate dissolved in TR media, which was 1.03 +/- 0.01 mPa·s (reporting the mean and standard deviation of three technical replicates.). The viscosity of 0.1% digested alginate in TR media was found to be 0.74+/-0.01 mPa·s. This means that the viscosity of alginate in our microfluidic experiments is 36% higher than of digested alginate, but the viscosities are close to those expected of water (0.89 mPa·s at 25 degree Celsius according to Berstad and colleagues [18]).”

While our microfluidic setup allows us to track the position and movement of cells in a spatially structured setting, these observations do not allow us to distinguish directly whether the differences in dispersal are a result of purely physical effects of polymers on cells or are a result of them triggering a biological response in cells that causes them to become sessile. It is known that bacterial appendages like pili interact with polysaccharide residues (Li et al., 2003). Therefore, it is quite plausible that cross-linking by polysaccharides can contribute growth behaviors on alginate. However, our analysis of gene expression demonstrates that flagellum-driven motility is decreased in the presence of alginate compared to digested alginate, alongside other major changes in gene expression. In addition, our measures of dispersal show that dispersal of cells when exposed to digested alginate is density dependent. Both observations suggest that the patterns in dispersal are governed by decision-making processes by cells resulting in changes in cell motility, rather than being a product of purely physical interactions with the polymer. 

The finding that viscosities of both alginate and digested alginate are similar to that of water, suggests that diffusion of nutrients in the growth chambers should be similar. Therefore, we think that the differences in real concentrations of nutrients is likely not contributing to the observed differences in behavior. 

(3) Why is Figure S1 arbitrary units? Does this have to do with the calibration of LC-MS? It would be better, it seems, to know the concentrations in real units of the monomer at least. 

We agree with the reviewer that it would have been better to have absolute concentrations for these compounds. However, to calibrate the mass spectrometer signals (ion counts) to absolute concentrations for the different alginate compounds, we would need an analytical standard of known concentration. We are not aware of such a standard and thus report only relative concentrations. We agree that the y-axis label of Figure S1 should not contain ‘arbitrary’ units, as it shows a ratio (of measurements in the same arbitrary units). We have edited the labels of Figure S1 accordingly and the figure legend in line 26 of the Supplemental Material (“Relative concentrations…”).

(4) Line 188 - density-dependent dispersal. The claim here is that "cells in chambers with many cells were more likely to disperse than cells in chambers with less cells." (my emphasis). Looking at the data in Figure 2C it appears that about 40% of the cells disperse irrespective of the density, before the switch to digested alginate. So it would seem that there is not a higher likelihood of dispersal at higher cell densities. For the very highest cell density, it does appear that this fraction is larger, but I'd be concerned about making this claim from what I understand to be a single experiment. To support the claim made should the authors plot Change in Cell number/Starting Cell number on the y-axis of Fig. 2C to show that the fraction is increasing? It would seem some additional data at higher starting cell densities would help support this claim more strongly. 

We thank the reviewer for this comment, which is in line with a remark made by reviewer 1 in their comment 1. In response to these two comments (and as described above), we have edited Figure 2C and now have plotted the change in cell number relative to starting cell number at the y axis to directly show the density dependence. We observe a positive (approximately linear) relationship between the fraction of dispersed cells with the number of cells present in the chamber at the time of switching. This indicates that there is a density dependence in the dispersal process, with highly populated chambers showing a higher fraction of dispersed cells. 

In addition to the change in Figure 2C, we have modified the paragraph around line 208: “We indeed found that the nutrient switch caused a few or no cells to disperse from small cell groups (Fig. 2B), whereas a large fraction of cells from large cell groups dispersed (Fig. 2C). In fact, the e fraction of cells that dispersed upon imposition of the nutrient switch showed a strong positive relationship with the number of cells present, meaning that cells in chambers with many cells were more likely to disperse than cells in chambers with fewer cells (Fig. 2C).”

The highest cell number at the start of the switch that we include is about 800 cells. The maximum number of cells that can fit into a chamber are ca. 1000 cells. Thus, 800 resident cells are close to the maximal density.

(5) A comment -- I find the result of significant chemotaxis towards alginate but not the monomers of alginate to be quite surprising. The ecological relevance of this (line 219) seems like an important result that is worth expanding on a bit at least in the discussion. For now, my question is whether the authors know of any mechanism by which chemotaxis receptors could respond to alginate but not the monomer. How can a receptor distinguish between the two? 

We agree that this result is surprising, given that oligomers can be more easily transported into the periplasm where sensing takes place, and they also provide an easier accessible nutrient source. Indeed, in case of the insoluble polymer chitin it has been shown that chemotaxis towards chitin is mediated by chitin oligomers (Bassler et al., 1991), which was suggested as a general motif to locate polysaccharide nutrient sources (Keegstra et al., 2022). However, a recent study has changed this perspective by showing widespread chemotaxis of marine bacteria towards the glucose-based marine polysaccharide laminarin, but not towards laminarin oligomers or glucose (Clerc et al., 2023). Together with our results on chemotaxis towards alginate (but not significantly toward alginate oligomers) this suggests that chemotaxis towards soluble polysaccharides can be mediated by direct sensing of the polysaccharide molecules.

As recommended, we expanded the discussion of the ecological relevance and also added more information on possible mechanisms of selective sensing of alginate and its breakdown products (around line 479).:

“Direct chemotaxis towards polysaccharides may facilitate the search for new polysaccharide sources after dispersal. We found that the presence of degradation products not only induces cell dispersal but also increases the expression of chemotaxis genes. Interestingly, we found that V. cyclitrophicus ZF270 cells show chemotaxis towards polymeric alginate but not digested alginate. This contrasts with previous findings for bacterial strains degrading the insoluble marine polysaccharide chitin, where chemotaxis was strongest towards chitin oligomers53, suggesting that oligomers may act as an environmental cue for polysaccharide nutrient sources55. However, recent work has shown that certain marine bacteria are attracted to the marine polysaccharide laminarin, and not laminarin oligomers56. Together with our results, this indicates that chemotaxis towards soluble polysaccharides may be mediated by the polysaccharide molecules themselves. The mechanism of this behavior is yet to be identified, but could be mediated by polysaccharide-binding proteins as have been found in Sphingomonas sp. A1 facilitating chemotaxis towards pectin57. Direct polysaccharide sensing adds complexity to chemosensing as polysaccharides cannot freely diffuse into the periplasm, which can lead to a trade-off between chemosensing and uptake58. Furthermore, most polysaccharides are not immediately metabolically accessible as they require degradation. But direct polysaccharide sensing can also provide certain benefits compared to using oligomers as sensory cues. First, it could enable bacterial strains to preferably navigate to polysaccharide nutrients sources that are relatively uncolonized and hence show little degradation activity. Second, strong chemotaxis towards degradation products could hinder a timely dispersal process as the dispersal then requires cells to travel against a strong attractant gradient formed by the degradation products. Overall, this strategy allows cells to alternate between degradation and dispersal to acquire carbon and energy in a heterogeneous world with nutrient hotspots [44,59–61].”

(6) Comment on lines 287-8 -- that the "positive enrichment of the gene set containing bacterial motility proteins matched the increase in motile cells that we observe in Fig 3E." I'm confused about what is meant by the word "matched" here. Is the implication that there is some quantitative correspondence between increased motility in Figure 3 and the change in expression in Figure 4? Or is the statement a qualitative one -- that motility genes are upregulated in the presence of digested alginate? Table S12 didn't help me answer this question. 

We thank the reviewer for their helpful comment. Our original statement was a qualitative one - observing that gene expression enrichment in genes associated with bacterial motility aligned with our expectations based on the previous observation of an increase in motile cells. We have now changed the wording to highlight the qualitative nature of this statement (line 315):

“The positive enrichment of the gene set containing bacterial motility proteins aligned with our expectations based on the increase in motile cells that we observed in Figure 3E (Fig. 4A, Table S12).”

(7) Line 326 - what is the explanation for the production of public enzymes in the presence of digest? How does this square with the previous narrative about cells growing on alginate digest expressing motility genes and chemotaxing towards alginate? It seems like the story is a bit tenuous here in the sense that digested alginates stimulate both motility - which is hypothesized to drive the discovery of new alginate particles - and lyase enzymes which are used to degrade alginate. So do the high motility cells that are chemotaxing towards alginate also express lyases en route? I'm of the opinion that constructing narratives like these in the absence of a more quantitative understanding of the colonization and degradation dynamics of alginate particles presents a major challenge and may be asking more of the data than the data can provide. 

a. I noted later that this is addressed later around lines 393 in the Discussion section.

Indeed, the notion that the presence of breakdown products triggers motility and also increases the expression of alginate lyases and other metabolic genes for alginate catabolism seems counterintuitive. We have now expanded our discussion of these results to contextualize these findings (around line 443):

"One reason for this observation may be that cells primarily rely on intracellular monosaccharide levels to trigger the upregulation of genes associated with polysaccharide degradation and catabolism, as has previously been observed for E. coli across various carbon sources [50,51]. In fact, the majority of carbon sources are sensed by prokaryotes through one‑component sensors inside the cell50. In the one‑component internal sensing scheme, the enzymes and transporters for the use of various carbon sources are expressed at basal levels, which leads to an increase in pathway intermediates upon nutrient availability. The pathway intermediates are sensed by an internal sensor, usually a transcription factor, and lead to the upregulation of transporter and enzyme expression [50,51]. This results in a positive feedback loop, which enables small changes in substrate abundance to trigger large transcriptional responses [50,52]. Thus, the presence of alginate breakdown products may likely result in increased expression of all components of the alginate degradation pathway, including the expression of degrading enzymes. As the gene expression analysis was performed on well-mixed cultures in culture medium containing alginate breakdown products, we therefore expect a strong stimulation of alginate catabolism. In a natural scenario, where cells disperse from a polysaccharide hotspot before its exhaustion, the expression of alginate catabolism genes may likely decrease again once the local concentration of breakdown products decreases. However, continued production of alginate lyases could also provide an advantage when encountering a new alginate source and continued production of alginate lyases may thus help cells to prepare for likely future environments. Further investigations of bacterial enzyme secretion in changing nutrient environments and at relevant spatial scales are required to improve our understanding of the regulation of enzyme secretion along nutrient gradients."

(8) I like Figure 6, and I think this hypothesis is a good result from this paper, but I think it would be important to emphasize this as a proposal that needs further quantitative analysis to be supported. 

We have now edited the manuscript to make this point more clear. While both degradation and dispersal are well-appreciated parts of microbial ecology, the transitions and underlying mechanisms are unclear. We have edited the discussion to improve the clarity (line 419): 

“This cycle of biomass degradation and dispersal has long been discussed in the context of foraging e.g., [44,45,13,46,47], but the cellular mechanisms that drive the cell dispersal remain unclear.”

Also, we have updated Figure 6 to indicate more clearly which new findings this work proposes (now bold font) and which previous findings that were made in different bacterial taxa and carbon sources that aligns with our  work (now light font). We edited the figure legend accordingly (line 503):

"By integrating our results with previous studies on cooperative growth on the same system, as well as results on dispersal cycles in other systems, we highlight where the specific results of this work add to this framework (bold font)."

Minor comments 

(1) Is there any growth on the enzyme used for alginate digestion? E.g. is the enzyme used to digest the alginate at sufficiently high concentrations that cells could utilize it for a carbon/nitrogen source? 

We thank the reviewer for raising this point. We added the following paragraph as Supplemental Text to address it (line 179):

“Protein amount of the alginate lyases added to create digested alginate

Based on the following calculation, we conclude that the amount of protein added to the growth medium by the addition of alginate lyases is so small that we consider it negligible. In our experiment we used 1 unit/ml of alginate lyases in a 4.5 ml solution to digest the alginate. As the commercially purchased alginate lyases are 10,000 units/g, our 4.5 ml solution contains 0.45 mg of alginate lyase protein. The digested alginate solution diluted 45x when added to culture medium. This means that we added 0.18 µg alginate lyase protein to 1 ml of culture medium.

As a comparison, for 1ml of alginate medium, 1000µg of alginate is added or for 1 ml of Lysogeny broth (LB) culture medium, 3,500 µg of LB are added.  Thus, the amount of alginate lyase protein that we added is ca. 5000 - 20,000 times smaller than the amount of alginate or LB that one would add to support cell growth. Therefore, we expect the growth that the digestion of the added alginate lyases would allow to be negligible.”

(2) The lines in Figure 2B are very hard to see. 

We have addressed this comment by using thicker lines in Figure 2B.

(3) The black background and images in Figure 3A and B are hard to see as well. 

We have now replaced Figure 3A and B, now using a white background.

(4) Typo at the beginning of line 251? 

Unfortunately we failed to find the typo referred to. We are happy to address it if it still exists in the revised manuscript.

Reviewer #3 (Recommendations For The Authors):

(1) I think there is not enough experimental evidence to conclude that the underlying cause of increased motility is the accumulation of digested alginate products. To conclusively show that this is the cause and not just some signal linked to cell density, perhaps the experiment should be repeated with a different carbon source. 

We thank the reviewer for their comment, which made us realize that we did not make the nature of the dispersal cue clear. The gene expression data was obtained from batch cultures and measured at the same approximate bacterial densities in batch, which indeed shows that the digested alginate is a sufficient signal for an increase in motility gene expression. This agrees very well with our observation that cells growing on digested alginate in microfluidic chambers have an increased fraction of motile cells in comparison with cells exposed to alginate (Fig 3E). However, we did not mean to suggest that the observed dispersal by bacterial motility is not influenced by cell density, in fact, we see that dispersal (and hence the increase in cell motility) in microfluidic chambers that are switched from polymeric to digested alginate depends on the bacterial density in the chamber, with higher bacterial densities showing increased dispersal. This shows that the presence of alginate oligomers does trigger dispersal through motility, but this signal affects bacterial groups in a cell density dependent manner.

Similar observations have been made in Caulobacter crescentus, which was found to form cell groups on the polymer xylan while cells disperse when the corresponding monomer xylose becomes available (D’Souza et al., 2021). We reference the additional work in lines 179 and 230. Taken together, these observations indicate a more general phenomenon in dispersal from polysaccharide substrates.

(2) About the expression data: 

• Ribosomal proteins and ABC transporters are enriched in cells grown on digested alginate and the authors discuss that this explains the difference in max growth rate between alginate and digested alginate. However, in Figure S2E the authors report no statistical difference between growth rates. 

We have now edited the manuscript to clarify this point. We found that cells grown on degradation products reached their maximal growth rate around 7.5 hours earlier (Fig. S2D) and showed increased expression of ribosomal biosynthesis and ABC transporters in late-exponential phase (Fig. 4A). We consider this shorter lag time as a sign of a different growth state and therefore a possible reason for the difference in ribosomal protein expression.

As the reviewer correctly points out, the maximum growth rates that were computed from the two growth curves were not significantly different (Fig. S2E). However, for our gene expression analysis, we harvested the transcriptome of cells that reached OD 0.39-0.41 (mid- to late-exponential phase). At this time point, the cell cultures may have differed in their momentary growth rate.

We edited the manuscript to make this clearer (line 287):

“Both observations likely relate to the different growth dynamics of V. cyclitrophicus ZF270 on digested alginate compared to alginate (Fig. S2A), where cells in digested alginate medium reached their maximal growth rate 7.5 hours earlier and thus showed a shorter lag time (Fig. S2D). As a consequence, the growth rate at the time of RNA extraction (mid-to-late exponential phase) may have differed, even though the maximum growth rate of cells grown in alginate medium and digested alginate medium were not found to be significantly different (Fig. S2E).”

• The increased expression of transporters for lyases in cells grown on digested alginate (lines 273-274 and 325-328) is very confusing and the explanation provided in lines 412-420 is not very convincing. My two cents on this: Expression of more enzymes and induction of motility might be a strategy to be prepared for more likely future environments (after dispersal, alginate is the most likely carbon source they will find). This would be in line with observed increased chemotaxis towards the polymer rather than the monomer (Similar to C. elegans). 

This comment is in line with reviewer 2, comment 7. In response to these two comments (and as described above), we expanded our discussion of these results to contextualize these findings (around line 443):

“One reason for this observation may be that cells primarily rely on intracellular monosaccharide levels to trigger the upregulation of genes associated with polysaccharide degradation and catabolism, as has previously been observed for E. coli across various carbon sources [50,51]. In fact, the majority of carbon sources are sensed by prokaryotes through one‑component sensors inside the cell [50]. In the one‑component internal sensing scheme, the enzymes and transporters for the use of various carbon sources are expressed at basal levels, which leads to an increase in pathway intermediates upon nutrient availability. The pathway intermediates are sensed by an internal sensor, usually a transcription factor, and lead to the upregulation of transporter and enzyme expression [50,51]. This results in a positive feedback loop, which enables small changes in substrate abundance to trigger large transcriptional responses [50,52]. Thus, the presence of alginate breakdown products may likely result in increased expression of all components of the alginate degradation pathway, including the expression of degrading enzymes. As the gene expression analysis was performed on well-mixed cultures in culture medium containing alginate breakdown products, we therefore expect a strong stimulation of alginate catabolism. In a natural scenario, where cells disperse from a polysaccharide hotspot before its exhaustion, the expression of alginate catabolism genes may likely decrease again once the local concentration of breakdown products decreases. However, continued production of alginate lyases could also provide an advantage when encountering a new alginate source and continued production of alginate lyases may thus help cells to prepare for likely future environments. Further investigations of bacterial enzyme secretion in changing nutrient environments and at relevant spatial scales are required to improve our understanding of the regulation of enzyme secretion along nutrient gradients.”

Additionally, we agree with the intriguing comment that continued expression of alginate lyases may also prepare cells for likely future environments. Further studies that aim to answer whether marine bacteria are primed by their growth on one carbon source towards faster re-initiation of degradation on a new particle will be an interesting research question. We now address this point in our manuscript (line 458):

“However, continued production of alginate lyases could also provide an advantage when encountering a new alginate source and continued production of alginate lyases may thus help cells to prepare for likely future environments. Further investigations of bacterial enzyme secretion in changing nutrient environments and at relevant spatial scales are required to improve our understanding of the regulation of enzyme secretion along nutrient gradients.“

(3) The yield reached by Vibrio on alginate is significantly higher than the yield in digested alginate, not similar, as stated in lines 133-134. Only cell counts are similar. Perhaps the author can correct this statement and speculate on the reason leading to this discrepancy: perhaps cells tend to aggregate in alginate despite the fact that these are well-mixed cultures. 

We have edited the description of the OD measurements accordingly and agree with the reviewer that aggregation is indeed a possible reason for the discrepancy (line 141):

“We also observed that the optical density at stationary phase was higher when cells were grown on alginate (Fig. S2B and C). However, colony counts did not show a significant difference in cell numbers (Fig. S3), suggesting that the increased optical density may stem from aggregation of cells in the alginate medium, as observed for other Vibrio species [7].”

(4) I suggest toning down the importance of the results presented in this study for understanding global carbon cycling. There is a link but at present it is too much emphasized. 

We have edited our statements regarding the carbon cycle. In the revised manuscript we stress the lack of direct quantifications of carbon cycling. . We still refer to carbon flow in the revised manuscript, as we would argue that microbial remineralization of biomass is recognized as an important factor in the marine biological carbon pump (e.g., Chisholm, 2000) and research on marine bacterial foraging investigates how bacterial cells manage to find and utilize this biomass.

Our revised manuscript contains the following modified statements (line 47 and line 60): “Even though many studies indicate that these degradation-dispersal cycles contribute to the carbon flow in marine systems, we know little about how cells alternate between polysaccharide degradation and motility, and which environmental factors trigger this behavioral switch.”

“Overall, our findings reveal cellular mechanisms that might also underlie bacterial degradation-dispersal cycles, which influence the remineralization of biomass in marine environments.”

References

• Alcolombri, U., Peaudecerf, F. J., Fernandez, V. I., Behrendt, L., Lee, K. S., & Stocker, R. (2021). Sinking enhances the degradation of organic particles by marine bacteria. Nature Geoscience, 14(10), 775–780. https://doi.org/10.1038/s41561-021-00817-x
• Bassler, B. L., Gibbons, P. J., Yu, C., & Roseman, S. (1991). Chitin utilization by marine bacteria. Chemotaxis to chitin oligosaccharides by Vibrio furnissii. Journal of Biological Chemistry, 266(36), 24268–24275. https://doi.org/10.1016/S0021-9258(18)54224-1
• Chisholm, S. W. (2000). Stirring times in the Southern Ocean. Nature, 407(6805), 685–686. https://doi.org/10.1038/35037696
• Chubukov, V., Gerosa, L., Kochanowski, K., & Sauer, U. (2014). Coordination of microbial metabolism. Nature Reviews. Microbiology, 12(5), 327–340. https://doi.org/10.1038/nrmicro3238
• Clerc, E. E., Raina, J.-B., Keegstra, J. M., Landry, Z., Pontrelli, S., Alcolombri, U., Lambert, B. S., Anelli, V., Vincent, F., Masdeu-Navarro, M., Sichert, A., De Schaetzen, F., Sauer, U., Simó, R., Hehemann, J.-H., Vardi, A., Seymour, J. R., & Stocker, R. (2023). Strong chemotaxis by marine bacteria towards polysaccharides is enhanced by the abundant organosulfur compound DMSP. Nature Communications, 14(1), 8080. https://doi.org/10.1038/s41467-023-43143z
• Dal Co, A., van Vliet, S., Kiviet, D. J., Schlegel, S., & Ackermann, M. (2020). Shortrange interactions govern the dynamics and functions of microbial communities. Nature Ecology and Evolution, 4(3), 366–375. https://doi.org/10.1038/s41559-019-1080-2
• D’Souza, G., Ebrahimi, A., Stubbusch, A., Daniels, M., Keegstra, J., Stocker, R., Cordero, O., & Ackermann, M. (2023). Cell aggregation is associated with enzyme secretion strategies in marine polysaccharide-degrading bacteria. The ISME Journal. https://doi.org/10.1038/s41396-023-01385-1
• D’Souza, G. G., Povolo, V. R., Keegstra, J. M., Stocker, R., & Ackermann, M. (2021). Nutrient complexity triggers transitions between solitary and colonial growth in bacterial populations. The ISME Journal, 15(9), 2614–2626. https://doi.org/10.1038/s41396-021-00953-7
• D’Souza, G., Schwartzman, J., Keegstra, J., Schreier, J. E., Daniels, M., Cordero, O. X., Stocker, R., & Ackermann, M. (2023). Interspecies interactions determine growth dynamics of biopolymer-degrading populations in microbial communities. Proceedings of the National Academy of Sciences of the United States of America, 120(44), e2305198120. https://doi.org/10.1073/pnas.2305198120
• Fenchel, T. (2002). Microbial Behavior in a Heterogeneous World. Science, 296(5570), 1068–1071. https://doi.org/10.1126/science.1070118
• Jiao, N., Luo, T., Chen, Q., Zhao, Z., Xiao, X., Liu, J., Jian, Z., Xie, S., Thomas, H., Herndl, G. J., Benner, R., Gonsior, M., Chen, F., Cai, W.-J., & Robinson, C. (2024). The microbial carbon pump and climate change. Nature Reviews Microbiology. https://doi.org/10.1038/s41579-024-01018-0
• Keegstra, J. M., Carrara, F., & Stocker, R. (2022). The ecological roles of bacterial chemotaxis. Nature Reviews Microbiology, 20(8), 491–504. https://doi.org/10.1038/s41579-022-00709-w
• Konishi, H., Hio, M., Kobayashi, M., Takase, R., & Hashimoto, W. (2020). Bacterial chemotaxis towards polysaccharide pectin by pectin-binding protein. Scientific Reports, 10(1), 3977. https://doi.org/10.1038/s41598-020-60274-1
• Li, Y., Sun, H., Ma, X., Lu, A., Lux, R., Zusman, D., & Shi, W. (2003). Extracellular polysaccharides mediate pilus retraction during social motility of Myxococcus xanthus. Proceedings of the National Academy of Sciences, 100(9), 5443–5448. https://doi.org/10.1073/pnas.0836639100
• Martínez-Antonio, A., Janga, S. C., Salgado, H., & Collado-Vides, J. (2006). Internal sensing machinery directs the activity of the regulatory network in Escherichia coli. Trends in Microbiology, 14(1), 22–27. https://doi.org/10.1016/j.tim.2005.11.002
• McDougald, D., Rice, S. A., Barraud, N., Steinberg, P. D., & Kjelleberg, S. (2012). Should we stay or should we go: Mechanisms and ecological consequences for biofilm dispersal. Nature Reviews Microbiology, 10(1), 39–50. https://doi.org/10.1038/nrmicro2695
• Nguyen, T. T. H., Zakem, E. J., Ebrahimi, A., Schwartzman, J., Caglar, T., Amarnath, K., Alcolombri, U., Peaudecerf, F. J., Hwa, T., Stocker, R., Cordero, O. X., & Levine, N. M. (2022). Microbes contribute to setting the ocean carbon flux by altering the fate of sinking particulates. Nature Communications, 13(1), 1657. https://doi.org/10.1038/s41467-022-29297-2
• Norris, N., Alcolombri, U., Keegstra, J. M., Yawata, Y., Menolascina, F., Frazzoli, E., Levine, N. M., Fernandez, V. I., & Stocker, R. (2022). Bacterial chemotaxis to saccharides is governed by a trade-off between sensing and uptake. Biophysical Journal, 121(11), 2046–2059. https://doi.org/10.1016/j.bpj.2022.05.003
• Povolo, V. R., D’Souza, G. G., Kaczmarczyk, A., Stubbusch, A. K., Jenal, U., & Ackermann, M. (2022). Extracellular appendages govern spatial dynamics and growth of Caulobacter crescentus on a prevalent biopolymer. bioRxiv, 2022.06.13.495907. https://doi.org/10.1101/2022.06.13.495907
• Preheim, S. P., Boucher, Y., Wildschutte, H., David, L. A., Veneziano, D., Alm, E. J., & Polz, M. F. (2011). Metapopulation structure of Vibrionaceae among coastal marine invertebrates. Environmental Microbiology, 13(1), 265–275. https://doi.org/10.1111/j.1462-2920.2010.02328.x
• Schwartzman, J. A., Ebrahimi, A., Chadwick, G., Sato, Y., Orphan, V., & Cordero, O. X. (2021). Bacterial growth in multicellular aggregates leads to the emergence of complex lifecycles. bioRxiv, 2021.11.01.466752. https://doi.org/10.1101/2021.11.01.466752
• Singh, P. K., Bartalomej, S., Hartmann, R., Jeckel, H., Vidakovic, L., Nadell, C. D., & Drescher, K. (2017). Vibrio cholerae Combines Individual and Collective Sensing to Trigger Biofilm Dispersal. Current Biology, 27(21), 3359-3366.e7. https://doi.org/10.1016/j.cub.2017.09.041
• Ulrich, L. E., Koonin, E. V., & Zhulin, I. B. (2005). One-component systems dominate signal transduction in prokaryotes. Trends in Microbiology, 13(2), 52–56. https://doi.org/10.1016/j.tim.2004.12.006
• Wall, M. E., Hlavacek, W. S., & Savageau, M. A. (2004). Design of gene circuits: Lessons from bacteria. Nature Reviews Genetics, 5(1), 34–42. https://doi.org/10.1038/nrg1244
• Yawata, Y., Carrara, F., Menolascina, F., & Stocker, R. (2020). Constrained optimal foraging by marine bacterioplankton on particulate organic matter. Proceedings of the National Academy of Sciences, 117(41), 25571–25579. https://doi.org/10.1073/pnas.2012443117
• Yawata, Y., Cordero, O. X., Menolascina, F., Hehemann, J.-H., Polz, M. F., & Stocker, R. (2014). Competition–dispersal tradeoff ecologically differentiates recently speciated marine bacterioplankton populations. Proceedings of the National Academy of Sciences, 111(15), 5622–5627. https://doi.org/10.1073/pnas.1318943111
• Zöttl, A., & Yeomans, J. M. (2019). Enhanced bacterial swimming speeds in macromolecular polymer solutions. Nature Physics, 15(6), 554–558. https://doi.org/10.1038/s41567-019-0454-3

Author response:

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

Response to Reviewer 1

(Cys25)PTH(1-84) does not show efficacy surpassing that of the previously used rhPTH(1-34). This needs to be discussed biologically and clinically.

Thank you very much for your valuable comments for enhancing the manuscript. We appreciate your input and have noted that this aspect was not addressed in the discussion. The authors have included the following paragraph in discussion section.

“This biological difference is thought to be due to dimeric R25CPTH(1-34) exhibiting a more preferential binding affinity for the RG versus R0 PTH1R conformation, despite having a diminished affinity for either conformation. Additionally, the potency of cAMP production in cells was lower for dimeric R25CPTH compared to monomeric R25CPTH, consistent with its lower PTH1R-binding affinity.  (Noh et al., 2024) One of the potential clinical advantages of dimeric R25CPTH(1-34) is its partial agonistic effect in pharmacodynamics. This property may allow for a more fine-tuned regulation of bone metabolism, potentially reducing the risk of adverse effects associated with full agonism, such as hypercalcemia and bone resorption by osteolcast activity. Moreover, the dimeric form may offer a more sustained anabolic response, which could be beneficial in the context of long-term treatment strategies. (Noh et al., 2024) Also, the effects of dimer were prominent, as we mentioned better bone formation than the control group.” (2nd paragraph, Discussion section)

The terms (Cys25)PTH(1-84) and Dimeric R25CPTH(1-34) are being used interchangeably and incorrectly. A unification of these terms is necessary.

We totally agree with the reviewer’s notion. R25CPTH(1-84) represents mutated human PTH, rhPTH(1-34) and dimeric R25CPTH(1-34) are synthesized PTH analogs. To clarified the terminology, we thus have changeed the terminology in the manuscript appear in red.

The figure legend is incorrect. Not all figures are described, and even though there are figures from A to I, only up to E is explained, or the content is different.

We apologize for our negligence. As suggested by a reviewer, we've fixed the figure legends throughout before the list of references in the manuscript as follows.

“Figure legends

Figure 1. Micro-CT analysis (A-D) Experimental design for the controlled delivery of rhPTH(1-34) and dimeric R25CPTH(1-34) in ovariectomized beagle model. Representative images for injection and placement of titanium implant. (E) Micro-CT analysis. bone mineral density (BMD), bone volume (TV; mm3), trabecular number (Tb.N; 1/mm), trabecular thickness (Tb. Th; um), trabecular separation (Tb.sp; ㎛). Error bars indicate standard deviation. Data are shown as mean ± s.d. *p<0.05, **p<0.01, ***p<0.001, n.s., not significant.  P, posterior. R, right

Figure 2. (A-I) Histological analysis of the different groups stained in Goldner’s trichrome. The presence of bone is marked by the green color and soft tissue in red. Red arrows indicate the position with soft tissues without bone around the implant threads. The area of bone formed was the widest in the rhPTH(1-34)-treated group. In the dimeric R25CPTH(1-34)treated group, there is a greater amount of bone than vehicle-treated group. Green arrows represent the bone formed over the implant. blue dotted line, margin of bone and soft tissue; Scale bars: 1mm

Figure 3. Histological analysis using Masson trichrome staining results in the rhPTH(1-34) and dimeric R25CPTH(1-34)-treated group (A-L) Masson trichrome-stained sections of cancellous bone in the mandibular bone. The formed bone is marked by the color red. Collagen is stained blue. Black dotted box magnification region of trabecular bone in the mandible. Scale bars, A-C, G-I: 1mm; D-F, J-L: 200 ㎛

Figure 4. Immunohistochemical analysis using TRAP staining for bone remodeling activity (A-L) TRAP staining is used to evaluate bone remodeling by staining osteoclasts. Osteoclasts is presented by the purple color. Black dotted box magnification region of trabecular bone in the mandible. (M, N) The number of TRAP-positive cells in the mandible of the rhPTH(1-34) and dimeric R25CPTH(1-34)-treated beagles. Scale bars, A-C, G-I: 1mm; D-F, J-L: 200 ㎛. Error bars indicate standard deviation. Data are shown as mean ± s.d. *p<0.05, **p<0.01, n.s., not significant

Figure 5. Measurement of biochemical Marker Dynamics in serum. The serum levels of calcium, phosphorus, P1NP, and CTX across three time points (T0, T1, T2) following treatment with dimeric dimeric R25CPTH(1-34), rhPTH(1-34), or control. (A-B) Calcium and phosphorus levels exhibit an upward trend in response to both PTH treatments compared to control, suggesting enhanced bone mineralization. (C) P1NP levels, indicative of bone formation, remain relatively unchanged across time and treatments. (D) CTX levels, associated with bone resorption, show no significant differences between groups. Data points for the dimeric R25CPTH(1-34), rhPTH(1-34), and control are marked by squares, circles, and triangles, respectively, with error bars representing confidence intervals.

Supplementary Figure. Three-dimensional reconstructed image of the bone surrounding the implants. Three-dimensional reconstructed images of the peri-implant bone depicting the osseointegration after different therapeutic interventions. (A) Represents the bone response to recombinant human parathyroid hormone fragment (rhPTH 1-34) treatment, showing the most robust degree of bone formation around the implant in the three groups. (B) Shows the bone response to a modified PTH fragment (dimeric R25CPTH(1-34)), indicating a similar level of bone growth and integration as seen with rhPTH(1-34), although to a slightly lesser extent. (C) Serves as the control group, demonstrating the least amount of bone formation and osseointegration. The upper panel provides a top view of the bone-implant interface, while the lower panel offers a cross-sectional view highlighting the extent of bony ingrowth and integration with the implant surface.”

In Figure 5, although the descriptions of T0, T1, T2 are mentioned in the method section, it would be more clear if there was a timeline like in Figure 1.

Based on the reviewer’s advice, we have indicated the timing of T0, T1, and T2 in the materials & methods section describing the serum biochemical assay, and we have shown a timeline in figure 5.

In Figure 5, instead of having calcium, phosphorus, P1NP, CTX graphs all under Figure 5, it would be more convenient for referencing in the text to label them as Figure 5A, Figure 5B, Figure 5C, Figure 5D.

We totally understood the reviewer’s comment. As the reviewer’s suggested, we have corrected the labeling in the text for figure 5 as follows.

“The levels of calcium, phosphorus, CTX, and P1NP were analyzed over time using RM-ANOVA (Figure 5). There were no significant differences between the groups for calcium and phosphorus at time points T0 and T1 (Figure 5A). However, after the PTH analog was administered at T2 (Figure 5A), the levels were highest in the rhPTH(1-34) group, followed by the dimeric R25CPTH(1-34) group, and then, lowest in the control group, which was statistically significant (Figure 5B,C). (P < 0.05) The differences between the groups over time for CTX and P1NP were not statistically significant (Figure 5D, E).”

Significance should be indicated in the figure (no asterisk present).

As the reviewer’s comment, we put the asterisk in the figure 5.

Addition of Figures in Text:

Line 112: change from "figure 2" to "figure 1" / Line 115: mention "figure 1. E"

Line 120: refer to "figure 1. E" / Line 123: change from "figure 3" to "figure 2"

Line 128: refer to "figure 2.A-C" / Line 137: mention "figure 3"

Line 138: refer to "figure 3. A-L" / Line 143: mention "figure 3. A-L"

Line 144: refer to "figure 3. E,F,K,L" / Line 148: mention "figure 4"

Line 150: refer to "figure 4 M,N" / Line 152: mention "figure 4. M,N"

Line 155: refer to "figure 5" / Line 157: mention "figure 5"

Line 159: refer to "figure 5" / Line 171: mention "figure 1 E"

Line 175: refer to "figure 2 M, N"/ Line 194: mention "figure 3"

Above all, thank you for the reviewer’s notion. We corrected detailed figure labeling in text to red color.

Response to Reviewer 2

First, the authors should clarify why they compared the effects of rhPTH(1-34) and of dimeric R25C2 PTH(1-34)? In most of the parameters, rhPTH(1-34) seems to be superior to dimeric R25C2 PTH(1-34). Why did the authors insist that the anabolic effects of dimer were prominent? Even though implication of dimeric R25C2 PTH(1-34) was drawn from genetic mutation studies, the authors should describe more clearly in the discussion the potential clinical benefits of the dimeric R25C2 PTH(1-34) compared to rhPTH(1-34), especially if dimeric R25C2 PTH(1-34) has just partial agonistic effect in pharmacodynamics.

Thank you for your insightful comments and questions regarding our results between rhPTH(1-34) and dimeric R25CPTH(1-34). rhPTH(1-34) is a well-characterized therapy for osteoporosis. In this study, rhPTH(1-34) generally showed superior outcomes in most parameters tested, the dimeric R25CPTH(1-34) exhibited specific anabolic effects that are not as pronounced with rhPTH(1-34). We recognized R25CPTH(1-34) as a anabolic effector. One of the potential advantages of dimeric R25CPTH(1-34) is its partial agonistic effect in pharmacodynamics. This property may allow for a more fine-tuned regulation of bone metabolism, potentially reducing the risk of adverse effects associated with full agonism, such as hypercalcemia and bone resorption by osteolast activity. Moreover, the dimeric form may offer a more sustained anabolic response, which could be beneficial in the context of long-term treatment strategies. Also, based on our results, we notes that the effects of dimer were prominent, as we mentioned better bone formation than the control group. We appreciate your input and have noted that this aspect was not addressed in the discussion. As a result, we have included the following paragraph in discussion section.

“This biological difference is thought to be due to dimeric R25CPTH(1-34) exhibiting a more preferential binding affinity for the RG versus R0 PTH1R conformation, despite having a diminished affinity for either conformation. Additionally, the potency of cAMP production in cells was lower for dimeric R25CPTH compared to monomeric R25CPTH, consistent with its lower PTH1R-binding affinity.  (Noh et al., 2024) One of the potential clinical advantages of dimeric R25CPTH(1-34) is its partial agonistic effect in pharmacodynamics. This property may allow for a more fine-tuned regulation of bone metabolism, potentially reducing the risk of adverse effects associated with full agonism, such as hypercalcemia and bone resorption by osteolcast activity. Moreover, the dimeric form may offer a more sustained anabolic response, which could be beneficial in the context of long-term treatment strategies. (Noh et al., 2024) Also, the effects of dimer were prominent, as we mentioned better bone formation than the control group.” (2nd paragraph, Discussion section)

Second, please describe the intermittent and continuous application of PTH analogues. Many of the readers may misunderstand that the authors' daily injection of PTHs were actually to mimic the clinical intermittent application or continuous one. Incorporation of the author's intention for experimental design would be more helpful for readers.

Thank you for your insightful comments regarding the need for clearer differentiation between intermittent and continuous applications of PTH analogs in this study. We appreciate your concern that the readers may not fully grasp whether our daily injection protocol was intended to mimic clinical intermittent or continuous PTH administration. To address this, we have revised the manuscript to explicitly clarify that the daily injections of rhPTH(1-34) and dimeric R25CPTH(1-34) were designed to simulate the intermittent dosing regimen commonly used in clinical practice. This regimen is known to maximize the anabolic effects on bone while minimizing potential catabolic actions associated with more frequent or continuous hormone exposure. We have added detailed explanations in the Introduction, Methods, and Discussion sections to help readers understand our experimental design and its relevance to clinical settings.

Introduction section

“Administration of prathyroid hormone (PTH) analogs can be categorized into two distinct protocols: intermittent and continuous. Intermittent rhPTH(1-34) therapy, typically characterized by daily injections, is clinically used to enhance bone formation and strength. This method leverages the anabolic effects of rhPTH(1-34) without significant bone resorption, which can occur with more frequent or continuous exposure. On the other hand, continuous rhPTH(1-34) exposure, often modeled in research as constant infusion, tends to accelerate bone resorption activities, potentially leading to bone loss (Silva and Bilezikian, 2015; Jilka, 2007). Understanding these differences is crucial for interpreting the therapeutic implications of rhPTH(1-34) in bone health.”

Silva, B. C., & Bilezikian, J. P. (2015). Parathyroid hormone: anabolic and catabolic actions on the skeleton. Current Opinion in Pharmacology, 22, 41-50.

Jilka, R. L. (2007). Molecular and cellular mechanisms of the anabolic effect of intermittent PTH. Bone, 40(6), 1434-1446.

Materials and Methods section

“Each animal received one injection per day, aimed at replicating the intermittent rhPTH(1-34) exposure proven beneficial for bone regeneration and overall skeletal health in clinical settings (Neer et al., 2001; Kendler et al., 2018). This regimen was chosen to investigate the potential anabolic effects of these specific PTH analogs under conditions closely resembling therapeutic use.”

Neer, R. M., Arnaud, C. D., Zanchetta, J. R., Prince, R., Gaich, G. A., Reginster, J. Y., Hodsman, A. B., Eriksen, E. F., Ish-Shalom, S., Genant, H. K., Wang, O., and Mitlak, B. H. (2001). Effect of Parathyroid Hormone (1-34) on Fractures and Bone Mineral Density in Postmenopausal Women with Osteoporosis. The New England Journal of Medicine, 344(19), 1434-1441.

Kendler, D. L., Marin, F., Zerbini, C. A. F., Russo, L. A., Greenspan, S. L., Zikan, V., Bagur, A., Malouf-Sierra, J., Lakatos, P., Fahrleitner-Pammer, A., Lespessailles, E., Minisola, S., Body, J. J., Geusens, P., Moricke, R., & Lopez-Romero, P. (2018). Effects of Teriparatide and Risedronate on New Fractures in Post-Menopausal Women with Severe Osteoporosis (VERO): A Multicenter, Double-Blind, Double-Dummy, Randomized Controlled Trial. The Lancet, 391(10117), 230-240.

Discussion section

“The use of daily injections in this study was intended to simulate intermittent PTH therapy, a well-established clinical approach for managing osteoporosis and enhancing bone regeneration. Intermittent administration of PTH, as opposed to continuous exposure, is critical for maximizing the anabolic response while minimizing the catabolic effects that are associated with higher frequency or continuous hormone levels. Our findings support the notion that even with daily administration, both rhPTH(1-34) and dimeric dimeric R25CPTH(1-34) promote bone formation and osseointegration, consistent with the outcomes expected from intermittent therapy. It’s important for future research to consider the dosage and timing of administration to further optimize the therapeutic benefits of PTH analogs (Dempster et al., 2001; Hodsman et al., 2005).”

Dempster, D. W., Cosman, F., Kurland, E. S., Zhou, H., Nieves, J., Woelfert, L., Shane, E., Plavetic, K., Müller, R., Bilezikian, J., & Lindsay, R. (2001). Effects of Daily Treatment with Parathyroid Hormone on Bone Microarchitecture and Turnover in Patients with Osteoporosis: A Paired Biopsy Study. Journal of Bone and Mineral Research, 16(10), 1846-1853.

Hodsman, A. B., Bauer, D. C., Dempster, D. W., Dian, L., Hanley, D. A., Harris, S. T., Kendler, D. L., McClung, M. R., Miller, P. D., Olszynski, W. P., Orwoll, E., Yuen, C. K. (2005). Parathyroid Hormone and Teriparatide for the Treatment of Osteoporosis: A Review of the Evidence and Suggested Guidelines for Its Use. Endocrine Reviews, 26(5), 688-703.

Third, please unify the nomenclature. Ensure consistency in the nomenclature throughout the article. Unify the naming conventions for PTH analogues, such as rhPTH(1-34) vs teriparatide and (Cys25)PTH(1-84) vs R25CPTH(1-34) vs R25CPTH(1-34) vs (1-84). Choose one nomenclature for each analogue and use it consistently throughout the article.

We totally agree with the reviewer’s notion. R25CPTH(1-84) represents mutated human PTH, rhPTH(1-34) and dimeric R25CPTH(1-34) are synthesized PTH analogs. To clarified the terminology, we thus have changed the terminology in the manuscript appear in red.

Response to Reviewer 3

I would recommend to rewrite the manuscript in a form that is more understandable to the readers. In fact, it appears to me that this work was originally formatted in a way that would need the Materials and Methods to precede the results. As presented (and as requested by the eLife formatting) the Materials and Methods are available only at the end of the reading and, as a consequence, the readers needs to refer to the Materials and Methods to have a general and initial understanding of the study design (i.e. type of treatment for each group, etc are not well specified in the Results section).

Thank you for you constructive comments and suggestions regarding the manuscript. We appreciate your feedback on the organization of the manuscript entirely. As reviewer mentioned, Materials and methods were placed after the discussion section in accordance with the format of the elife journal. For a better and initial understanding, a description of each experimental group has been added to the Results section as follow. Thank you again for your valuable comments.

“To investigate evaluating and comparing the efficacy of rhPTH(1-34) and the dimeric R25CPTH(1-34) in promoting bone regeneration and healing in a clinically relevant animal model. In our study, beagle dogs were selected as the model due to their anatomical similarity to human oral structures, suitable size for surgeries, human-like bone turnover rates, and established oral health profiles, ensuring comparable and ethically sound research outcomes. The normal saline injected-control group, injected with 40ug/day PTH (Forsteo, Eli Lilly) group, and 40ug/day PTH analog-injected group. Animals in each group were injected subcutaneously for 10 weeks.”

DOI: 10.1038/s41467-023-42306-2

Resource: RRID:BDSC_39209

Curator: @scibot

SciCrunch record: RRID:BDSC_39209

What is this?

DOI: 10.1038/s41467-023-42306-2

Resource: RRID:BDSC_33413

Curator: @scibot

SciCrunch record: RRID:BDSC_33413

What is this?

DOI: 10.1126/sciadv.adp3756

Resource: Northwestern University Biological Imaging Core Facility (RRID:SCR_017767)

Curator: @scibot

SciCrunch record: RRID:SCR_017767

What is this?

DOI: 10.1111/head.14752

Resource: Connectivity Toolbox (RRID:SCR_009550)

Curator: @scibot

SciCrunch record: RRID:SCR_009550

What is this?

DOI: 10.1016/j.celrep.2024.114401

Resource: (BD Biosciences Cat# 742390, RRID:AB_2740746)

Curator: @scibot

SciCrunch record: RRID:AB_2740746

What is this?

El glosario debe estar en estricto orden alfabético. No puede seguir, después de la "S", términos que empiezan por "A".

La relación entre investigador y participantes es fundamental para el desarrollo de la investigación social, creando un nuevo espacio social que permite una comprensión más profunda de los fenómenos estudiados. Objetividad: Uso de metodologías sistemáticas y herramientas que permitan replicar y verificar resultados. Recurso para estimular y profundizar el diálogo de manera genuina y estructurada. Subjetividad: La interacción y la relación del investigador con los participantes afectan la interpretación de los datos. La necesidad de ganar la confianza y el interés de los participantes, adaptándose a sus contextos y respuestas emocionales.

Respondiendo a la pregunta ¿Qué aspectos determinan la objetividad y la subjetividad en la investigación de fenómenos sociales? Fernando González sostiene que la objetividad y la subjetividad no son opuestas sino complementarias en la investigación social. Mientras que la objetividad tradicional puede simplificar y limitar la comprensión de fenómenos complejos a datos cuantificables, la subjetividad permite explorar la profundidad y riqueza de las experiencias humanas y contextos culturales. En lugar de reducir la complejidad a simples categorías, integrar la subjetividad en la investigación ofrece una perspectiva más creativa, lo cual es necesario para desafiar sistemas autoritarios y de control que imponen visiones más conservadoras

Aquí Fernando González argumenta a través de Foucault que, en lugar de simplemente etiquetar lo que no entendemos como una enfermedad mental, deberíamos tratar de comprender el contexto y la perspectiva del individuo. Esto subraya que nuestra manera de interpretar y entender el comportamiento de las personas, basada en sus propias experiencias y sentimientos, es crucial para la investigación social.

Objetividad y Subjetividad en la Investigación Social Objetividad: Tradicionalmente se entiende como la capacidad de observar y analizar fenómenos de manera imparcial, sin influencias personales o emocionales. Sin embargo, el texto sugiere que esta forma de objetividad es limitada, ya que no puede existir un conocimiento completamente independiente de los sentidos humanos.

Subjetividad: Reconoce que los individuos interpretan el mundo a través de sus propios sentidos, experiencias y emociones. Este enfoque valora las perspectivas individuales y contextuales, proporcionando una comprensión más rica y matizada de los fenómenos sociales.

Cuestionamientos sobre la Objetividad y Subjetividad: Definición de Objetividad:

Lenin: Define la materia como todo lo que existe independientemente de los sentidos. Crítica de FGR: FGR discrepa de esta definición, argumentando que el conocimiento no puede construirse independientemente de los sentidos, ya que estos participan en la construcción del saber. Implicación: La crítica sugiere que la objetividad absoluta, definida como una separación total de los sentidos, es inalcanzable e irrealista. Subjetividad como Objeto de las Ciencias Sociales:

Debate con Martin Packer: Packer plantea que es problemático considerar la subjetividad como objeto de estudio en las ciencias sociales debido a su oposición a la objetividad. Respuesta de FGR: FGR argumenta que no hay una oposición real entre subjetividad y objetividad en el pensamiento científico, y cuestiona cuál es la definición de objetividad que se está utilizando. Concreción de la Materialidad:

Lenin: Incluye lo subjetivo dentro de la materialidad, lo que sugiere que todo lo que existe, incluyendo las percepciones subjetivas, es material. FGR: Señala que, aunque las emociones y el mundo psicológico son tan reales como el mundo físico, la subjetividad no puede ser completamente objetivada o materializada. Influencia de Vygotsky:

Vygotsky: Reconoce que las emociones y el mundo psicológico son reales y se expresan en nuestras acciones y fantasías. FGR: Utiliza la afirmación de Vygotsky para subrayar que la subjetividad es una parte esencial de la realidad humana y que no puede ser ignorada en la investigación social. Relación entre Subjetividad y Cultura:

FGR: Argumenta que la cultura es una producción subjetiva y que la subjetividad y la cultura se configuran recíprocamente. Las producciones subjetivas definen nuevas formas de cultura y viceversa. Implicación: Este punto resalta que la subjetividad humana es fundamental para la creación y recreación de la cultura, lo que sugiere que cualquier estudio cultural debe considerar la subjetividad. Potencial Creativo de la Subjetividad:

FGR: La subjetividad humana permite un potencial creativo que no está definido únicamente por las condiciones objetivas en las que surge el creador. Implicación: Este argumento sugiere que la creatividad y la innovación están intrínsecamente ligadas a la subjetividad, desafiando la noción de que las condiciones objetivas son las únicas determinantes.

Objetividad y Subjetividad en la Investigación Social Objetividad: Tradicionalmente se entiende como la capacidad de observar y analizar fenómenos de manera imparcial, sin influencias personales o emocionales. Sin embargo, el texto sugiere que esta forma de objetividad es limitada, ya que no puede existir un conocimiento completamente independiente de los sentidos humanos.

Subjetividad: Reconoce que los individuos interpretan el mundo a través de sus propios sentidos, experiencias y emociones. Este enfoque valora las perspectivas individuales y contextuales, proporcionando una comprensión más rica y matizada de los fenómenos sociales.

A la pregunta, ¿De qué forma los sujetos modifican la manera en la que se establece un fenómeno social? El texto señala que los sujetos modifican la manera en que se establece un fenómeno social a través de la interacción activa con el investigador. Este proceso transforma el fenómeno, ya que el investigador ajusta los instrumentos de investigación para fomentar un diálogo auténtico y dinámico. Así, la investigación se convierte en una co-creación entre el investigador y los sujetos, afectando cómo se configura y comprende el fenómeno social.

La falta de comprensión sobre comportamientos o fenómenos puede llevar a etiquetarlos como patológicos, lo cual refleja una falta de objetividad y una tendencia a simplificar la realidad para ajustarla a esquemas preexistentes.

La subjetividad: Aquí se destaca la ambigüedad del concepto de subjetividad, que puede ser visto desde la perspectiva del sujeto racional cartesiano o como una amenaza para la objetividad científica, este conflicto subraya la dificultad de integrar la subjetividad en paradigmas que priorizan la objetividad y el conocimiento empírico.

En la psicología soviética, la subjetividad estaba restringida debido a las implicaciones ideológicas del materialismo frente al idealismo, esto nos muestra cómo los contextos políticos y filosóficos pueden limitar o influir en el enfoque de las ciencias sociales. La ausencia de una discusión abierta sobre la subjetividad refleja una resistencia a integrar aspectos del sujeto que no encajaban con las doctrinas materialistas predominantes.

La personalidad fue una forma de abordar la subjetividad dentro de un marco aceptable en la psicología soviética, esto lleva a una adaptación dentro de las limitaciones ideológicas para explorar aspectos del sujeto que estaban más cerca de lo que se consideraba aceptable, como la motivación humana.

Modificación de Fenómenos Sociales por los Sujetos: Construcción Social de la Realidad: Los sujetos, a través de sus interacciones y prácticas cotidianas, construyen y modifican continuamente la realidad social. La forma en que interpretan y actúan en el mundo influye en la definición y percepción de los fenómenos sociales. Ejemplo: Las normas sociales y valores culturales son construidos y reforzados mediante la interacción social. Participación y Agencia: Los individuos tienen agencia, es decir, capacidad de actuar y tomar decisiones que afectan los fenómenos sociales. Sus acciones pueden desafiar, reproducir o transformar las estructuras sociales existentes. Ejemplo: Los movimientos sociales y políticos pueden surgir de la agencia de individuos y grupos que buscan cambiar las condiciones sociales. Subjetividad y Experiencia Personal: La subjetividad y las experiencias personales de los individuos influyen en cómo perciben y reaccionan ante los fenómenos sociales. Sus interpretaciones y respuestas pueden variar significativamente según su contexto y biografía. Ejemplo: La percepción de la justicia o la injusticia puede variar entre individuos según sus experiencias personales con el sistema legal. Innovación y Cambio Cultural: Los sujetos pueden introducir nuevas ideas, prácticas y tecnologías que transforman los fenómenos sociales. La innovación cultural y tecnológica es un proceso constante que redefine la realidad social. Ejemplo: El impacto de las redes sociales en la comunicación y la organización social ha cambiado profundamente las formas en que las personas interactúan y participan en la sociedad. Retroalimentación y Reflexividad: Los fenómenos sociales son reflexivos, es decir, los sujetos no solo participan en ellos, sino que también reflexionan sobre ellos y modifican sus comportamientos en respuesta a nuevos entendimientos y conocimientos. Ejemplo: La adopción de nuevas políticas públicas puede ser influenciada por la retroalimentación y el activismo de los ciudadanos.

Definición de Objetividad: La objetividad se refiere a la capacidad de observar y analizar fenómenos sin la influencia de las emociones o prejuicios del investigador. En el contexto del texto, se menciona que la objetividad puede ser entendida de diferentes maneras, incluyendo la definición de Lenin sobre materia, que considera todo lo que existe independientemente de los sentidos

Definición de Objetividad: La objetividad se refiere a la capacidad de observar y analizar fenómenos sin la influencia de las emociones o prejuicios del investigador. En el contexto del texto, se menciona que la objetividad puede ser entendida de diferentes maneras, incluyendo la definición de Lenin sobre materia, que considera todo lo que existe independientemente de los sentidos

Este concepto es altamente interesante, puesto que, se lleva el concepto de objetividad, hacia una percepción general y bastante profunda. Así pues, muchas veces se cree que la subjetividad logra tener participación dentro de nuestros conceptos o criterios propios, pero, a raíz del dominio de ideales establecidos por los prejuicios o conductas sociales, se crea un tipo de cultura objetiva, en la cual, las emciones, justamente, no obtienen mucha participación.

Asimismo, en una cultura dominada por la objetividad y el sometimiento, como la que describen, la subjetividad tiende entonces, a ser marginada y desvalorizada; esta predominancia de la objetividad en las prácticas culturales y sociales, puede limitar la capacidad de las personas para explorar y expresar sus experiencias y perspectivas personales de manera auténtica, lo que a su vez afecta la comprensión completa y matizada de los fenómenos sociales.

La capacidad del investigador en obtener el interés o, como indica el texto, "ganarse" a los participantes, dentro de una investigación, resulta ser entonces, un punto clave, en cuestión del reconocimiento de un fenómeno social respectivo. Sin dejar de lado los hechos (negativos o no) de los tiempos investigativos de dicho fenómeno, el hecho de generar un espacio social para lograr relacionarse, dentro de ese concepto, si logra facilitar la cooperación de los participantes y puede revelar nuevas perspectivas que podrían no ser evidentes en una investigación más distante.

Específicamente, este enfoque puede reconocer la complejidad y autonomía del individuo, sugiriendo, tal vés, una perspectiva donde los procesos subjetivos pueden trascender y ser independientes de los factores sociales y culturales que contribuyeron a su formación inicial. Aquí es donde se puede desatacar esa participación del criterio propio, dentro de un concepto totalmente analítico del uso de la subjetividad, entendiendo así, cómo este tipo de percepciones hacia dicho concepto, resaltan la manera en que la subjetividad puede trascender y ser entendida como un recurso donde la personalidad si obtiene un papel participativo.

1. Objetividad y subjetividad: Son dos conceptos que se contraponen, pero a la vez pueden complementarse. Para entenderlo mejor, González Rey pone como ejemplo el estudio de la cultura. Si es objetivo, se trata de un monumento simbólico o un lenguaje milenario, algo totalmente estático, mientras que lo subjetivo pretende ir más allá y explorar las emociones humanas desde, en este caso, la cultura. Permite romper con moldes o esquemas e indagar en la forma del saber humano. Agrega que desde la subjetividad se configura un gran potencial creativo.

El conocimiento de obras literarias clásicas como las de Dostoievski, Tolstoi, Milan Kundera y Sándor Márai enriquece profundamente la comprensión de los psicólogos sobre las realidades humanas subjetivas, ya que estos textos aportan perspectivas valiosas que influyen en la interpretación personal y profesional de los lectores.

El investigador entonces debe librarse de normas y modelos, esto le permitirá realizar investigaciones de una forma inmersiva, planteando de esta forma nuevos recursos de lectura de una problemática o temática de estudio. El autor plantea que la estadística es un significado, pero que no es en su totalidad un criterio de legitimidad.

Para mi, esta es la hipótesis del texto. Ya que es el punto de partida para que el autor se replanteé, precisamente a través de sus reflexiones una nueva forma de generar conocimiento o de hacer investigación, teniendo en cuenta la subjetividad del investigador, que se convierte en una variable a considerar en los resultados del estudio.

Leyendo el texto, a mí opinión, el siguiente párrafo es sacado del documento para hacer referencia a cuál es el objetivo general: Yo creo que el punto más impor- tante y diferenciador de la epistemología cualitativa es, como te decía, el carácter constructivo-interpretativo del conocimiento que orienta la investigación cuali- tativa concreta. ¿En qué sentido esto ocurre? En la construcción de indicadores que nos permiten ir avanzando, no por expresiones explícitas de las personas estudiadas, sino por elementos indirectos que van tomando un valor en la construcción del inves- tigador, y que nos permiten generar hipótesis para producir un saber sobre la subjetividad, para construir un saber sobre el cual esta epistemología se desarrolló, y que nos permite construcciones que están más allá de la conciencia, de la intención y del lenguaje intencional de las personas estudiadas. Esto es un gran desafío, estos son caminos difíciles de inteligibilidad. Sin embargo, nos permiten saberes para explicar problemas que las otras teorías y epistemologías no nos posibilitaron.

Esta epistemología busca comprender y aprovecharla para obtener un conocimiento más profundo y matizado de la realidad social. La subjetividad no se ve como un obstáculo, sino como una fuente valiosa de información que puede revelar aspectos de la realidad social que no son accesibles a través de métodos puramente objetivos.

Argumentos que Soportan el Texto

1. Carácter Constructivo-Interpretativo: El argumento central del texto es que la epistemología cualitativa tiene un carácter constructivo-interpretativo. Esto significa que el conocimiento se construye a través de la interpretación de los datos, en lugar de simplemente recolectarlos y analizarlos de manera objetiva. Esta interpretación permite al investigador generar hipótesis y construir un saber que va más allá de lo explícitamente expresado por los sujetos estudiados.

2. Indicadores Indirectos: Otro argumento clave es la importancia de los indicadores indirectos en la investigación cualitativa. A diferencia de los enfoques tradicionales que se centran en las expresiones directas y explícitas de los sujetos, la epistemología cualitativa se enfoca en elementos indirectos que pueden no ser evidentes a simple vista. Estos indicadores indirectos son esenciales para comprender la subjetividad de los sujetos y para construir un conocimiento más profundo y complejo.

3. Más Allá de la Conciencia y el Lenguaje Intencional: El texto argumenta que el conocimiento sobre la subjetividad no puede limitarse a lo que los sujetos son conscientes o a lo que expresan intencionalmente. La epistemología cualitativa permite acceder a niveles más profundos de la subjetividad que están más allá de la conciencia y el lenguaje intencional de los sujetos. Esto amplía las posibilidades de comprensión y explicación de los fenómenos sociales.

4. Desafíos y Caminos de Inteligibilidad: Finalmente, el texto reconoce que esta aproximación presenta grandes desafíos y que los caminos para alcanzar esta comprensión profunda son difíciles de inteligibilidad. Sin embargo, sostiene que estos caminos permiten obtener saberes que son cruciales para explicar problemas que las teorías y epistemologías tradicionales no han podido resolver.

ID: 851661

Appears as of SW 01-07-02: Indication in case of bus signal problems. Main pcb cannot communicate with the following parts:

Combination of faults possible i.e.: 10 = 2+8 - 1: I/O PCB - 2: Motor bottom - 4: Motor top - 8: Ignition module top - 16: Ignition module bottom - Check bus cable plug and cable for connection and damage

Appears for 30 sec. after switch ON - Display can be cancelled by touch - Water level o. k., - Level electrode is working - Too many pulses measured by CDS sensor - Check electrode or water leakage through check valve

• ADI 2260 MC
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. MOREIRA ALVES
• Julgamento: 14/02/2001
• Publicação: 02/08/2002

Ementa EMENTA: Ação direta de inconstitucionalidade com pedido de liminar. Artigo 1º da Medida Provisória 2.027-40, de 29 de junho de 2000, na parte que acrescenta parágrafo único ao artigo 10 do Decreto-Lei nº 3.365, de 11 de junho de 1941. - De há muito, a jurisprudência desta Corte afirmou que a ação de desapropriação indireta tem caráter real e não pessoal, traduzindo-se numa verdadeira expropriação às avessas, tendo o direito à indenização que daí nasce o mesmo fundamento da garantia constitucional da justa indenização nos casos de desapropriação regular. - Não tendo o dispositivo ora impugnado sequer criado uma modalidade de usucapião por ato ilícito com o prazo de cinco anos para, através dele, transcorrido esse prazo, atribuir o direito de propriedade ao Poder Público sobre a coisa de que ele se apossou administrativamente, é relevante o fundamento jurídico da presente argüição de inconstitucionalidade no sentido de que a prescrição extintiva, ora criada, da ação de indenização por desapropriação indireta fere a garantia constitucional da justa e prévia indenização, a qual se aplica tanto à desapropriação direta como à indireta. - Ocorrência, no caso, do requisito da conveniência para a concessão da liminar requerida. - Já com referência à parte final do dispositivo impugnado no que tange à "ação que vise a indenização por restrições decorrentes de atos do Poder Público", não se configura a plausibilidade jurídica de sua argüição de inconstitucionalidade. Liminar que se defere em parte, para suspender, com eficácia "ex nunc" e até o julgamento final desta ação, as expressões "ação de indenização por apossamento administrativo ou desapropriação indireta, bem como" contidas no parágrafo único do artigo 10 do Decreto-Lei nº 3.365/1941, a ele acrescentado pelo artigo 1º da Medida Provisória nº 2.027-40, de 29 de junho de 2000, e suas subseqüentes reedições.

Contestação na ação de desapropriação somente poderá versar sobre vício processual e valor do imóvel.

1. Direito de extensão é o que assiste ao proprietário de exigir que se inclua no plano de desapropriação a parte remanescente do bem, que se tornou inútil ou de difícil utilização.

2. "(...) o pedido de extensão é formulado na via administrativa, quando há a perspectiva de acordo, ou na via judicial, neste caso por ocasião da contestação. O réu, impugnando o valor ofertado pelo expropriante, apresenta outra avaliação do bem, considerando a sua integralidade, e não a sua parcialidade, como pretendia o autor. O juiz, se reconhecer presentes os elementos do direito, fixará a indenização correspondente à integralidade do bem. Resulta daí que é o bem, da mesma forma em sua integralidade, que se transferirá ao patrimônio do expropriante" (José dos Santos Carvalho Filho, Manual de Direito Administrativo, 11ª edição, Editora Lumen Juris, Rio de Janeiro, 2004, p. 723).

3. O direito de extensão nada mais é do que a impugnação do preço ofertado pelo expropriante. O réu, quando impugna na contestação o valor ofertado, apresenta outra avaliação do bem, abrangendo a integralidade do imóvel, e não apenas a parte incluída no plano de desapropriação. Assim, o pedido de extensão formulado na contestação em nada ofende o art. 20 do Decreto-Lei 3.365/41, segundo o qual a contestação somente pode versar sobre "vício do processo judicial ou impugnação do preço". Precedentes de ambas as Turmas de Direito Público. (...)

(REsp 986.386/SP, Rel. Ministro CASTRO MEIRA, SEGUNDA TURMA, julgado em 04/03/2008, DJe 17/03/2008)

OJ-SDI2-152 AÇÃO RESCISÓRIA E MANDADO DE SEGU-RANÇA. RECURSO DE REVISTA DE ACÓRDÃO REGIO-NAL QUE JULGA AÇÃO RESCISÓRIA OU MANDADO DE SEGURANÇA. PRINCÍPIO DA FUNGIBILIDADE. INAPLICABILIDADE. ERRO GROSSEIRO NA INTERPOSIÇÃO DO RECURSO (DEJT divulgado em 03, 04 e 05.12.2008) - A interposição de recurso de revista de decisão definitiva de Tribunal Regional do Trabalho em ação rescisória ou em mandado de segurança, com fundamento em violação legal e divergência jurisprudencial e remissão expressa ao art. 896 da CLT, configura erro grosseiro, insuscetível de autorizar o seu recebimento como recurso ordinário, em face do disposto no art. 895, “b”, da CLT.

SUM-303 FAZENDA PÚBLICA. REEXAME NECESSÁRIO (nova redação em decorrência do CPC de 2015) - Res. 211/2016, DEJT divulgado em 24, 25 e 26.08.2016

I - Em dissídio individual, está sujeita ao reexame necessário, mesmo na vigência da Constituição Federal de 1988, decisão contrária à Fazenda Pública, salvo quando a condenação não ultrapassar o valor correspondente a: - a) 1.000 (mil) salários mínimos para a União e as respectivas autarquias e fundações de direito público; - b) 500 (quinhentos) salários mínimos para os Estados, o Distrito Federal, as respectivas autarquias e fundações de direito público e os Municípios que constituam capitais dos Estados; - c) 100 (cem) salários mínimos para todos os demais Municípios e respectivas autarquias e fundações de direito público.

II – Também não se sujeita ao duplo grau de jurisdição a decisão fundada em: - a) súmula ou orientação jurisprudencial do Tribunal Superior do Trabalho; - b) acórdão proferido pelo Supremo Tribunal Federal ou pelo Tribunal Superior do Trabalho em julgamento de recursos repetitivos; - c) entendimento firmado em incidente de resolução de demandas repetitivas ou de assunção de competência; - d) entendimento coincidente com orientação vinculante firmada no âmbito administrativo do próprio ente público, consolidada em manifestação, parecer ou súmula administrativa.

III - Em ação rescisória, a decisão proferida pelo Tribunal Regional do Trabalho está sujeita ao duplo grau de jurisdição obrigatório quando desfavorável ao ente público, exceto nas hipóteses dos incisos anteriores. (ex-OJ nº 71 da SBDI-1 - inserida em 03.06.1996)

IV - Em mandado de segurança, somente cabe reexame necessário se, na relação processual, figurar pessoa jurídica de direito público como parte prejudicada pela concessão da ordem. Tal situação não ocorre na hipótese de figurar no feito como impetrante e terceiro interessado pessoa de direito privado, ressalvada a hipótese de matéria administrativa. (ex-OJs nºs 72 e 73 da SBDI-1 – inseridas, respectivamente, em 25.11.1996 e 03.06.1996)

1. consumerism The paragraph A mentions that Provo believed the scheme "was an answer to the perceived threats of air pollution and consumerism." Dịch: Họ tin rằng chương trình, được gọi là Witte Fietsenplan, là câu trả lời cho các mối đe dọa được nhận thức là ô nhiễm không khí và "chủ nghĩa tiêu dùng."

1. activists In paragraph A: "Provo, the organisation that came up with the idea, was a group of Dutch activists who wanted to change society." Câu tóm tắt đang đề cập đến những người trong nhóm này, vì vậy "activists" (các nhà hoạt động) là từ chính xác cần sử dụng ở đây.

Để LLMs có thể thích nghi với các ngôn ngữ mới, các tokens từ các ngôn ngữ này có thể được thêm vào. Tuy nhiên, thay vì mở rộng bộ từ điển của mô hình, nghiên cứu sẽ thay thể một số lượng nhất định các token ít phổ biến nhất trong tập từ điển hiện tại bằng các token mới. Cách này giúp cho kích thước tập từ vựng được giữ nguyên. Cụ thể, 1 BPE encoder với một tập từ điển gồm k từ của một ngôn ngữ nhất định sẽ được huấn luyện trên 1 bộ dữ liệu của ngôn ngữ đó. Sau đó, bộ từ điển của 2 BPE encoder sẽ được so sánh với nhau với o là tập các từ có trong cả 2 bộ từ điển của 2 BPE encoder. Sau đó, các từ xuát hiện ít nhất trong BPE encoder gốc mà không có trong tập o sẽ được thay thế bởi các từ mới có trong tập k. Các embedding tương ứng với các token bị thay thế cũng được tái khởi tạo.

boltons???

lyanna

kinda what theon did in a way

uh well tywin is there...

DOI: 10.1371/journal.pgen.1008832

Resource: RRID:BDSC_9693

Curator: @AniH

SciCrunch record: RRID:BDSC_9693

What is this?

DOI: 10.1016/j.isci.2020.101335

Resource: (BDSC Cat# 34775,RRID:BDSC_34775)

Curator: @scibot

SciCrunch record: RRID:BDSC_34775

What is this?

A declaração de nulidade enseja sempre a indenização pelo que foi executado ou comprovado o prejuízo. Somente não haverá indenização caso se comprove que a declaração de nulidade foi imputável ao contratado.

Nesse sentido: - 5. A jurisprudência do STJ é de que, mesmo que seja nulo o contrato realizado com a Administração Pública, por ausência de prévia licitação, é devido o pagamento pelos serviços prestados, desde que comprovados, nos termos do art. 59, parágrafo único, da Lei 8.666/1993, sob pena de enriquecimento ilícito da Administração. - 6. O STJ reconhece que, ainda que ausente a boa fé do contratado e que tenha ele concorrido para nulidade, é devida a indenização pelo custo básico do serviço, sem qualquer margem de lucro. - 7. A inexistência de autorização da Administração para subcontratação é insuficiente para afastar o dever de indenização, no caso dos autos, porque a própria contratação foi irregular, haja vista que não houve licitação e o contrato foi verbal. Assim, desde que provada a existência de subcontratação e a efetiva prestação de serviços, ainda que por terceiros, e que tais serviços se reverteram em benefício da Administração, será devida a indenização dos respectivos valores. Na mesma linha: REsp n. 468.189/SP, Rel. Min. José Delgado, Primeira Turma, julgado em 18/3/2003, DJ de 12/5/2003, p. 221.

(REsp n. 2.045.450/RS, relator Ministro Herman Benjamin, Segunda Turma, julgado em 20/6/2023, DJe de 28/6/2023.)

Usa-se o pregão sempre que o desempenho e a qualidade poder ser objetivamente definido

for - quote - rise of populism

RHC 163.334:

O contribuinte que, de forma contumaz e com dolo de apropriação, deixa de recolher o ICMS cobrado do adquirente da mercadoria ou serviço incide no tipo penal do art. 2º, II, da Lei nº 8.137/1990.

• É considerado crime material, no entanto.

Observar Súmula Vinculante nº 24, que dispõe que os crimes previstos nos incisos de I a IV somente são típicos após o lançamento definitivo:

Súmula Vinculante 24 - Não se tipifica crime material contra a ordem tributária, previsto no art. 1º, incisos I a IV, da Lei 8.137/1990, antes do lançamento definitivo do tributo.

(...) "tratando-se de crime material contra a ordem tributária (art. 1º da Lei n. 8.137/1990), a competência para processar e julgar o delito é do local onde houver ocorrido a sua consumação, por meio da constituição definitiva do crédito tributário, sendo irrelevante a mudança de domicílio fiscal do contribuinte" (CC 120.850/BA, Rel. Ministro MARCO AURÉLIO BELLIZZE, TERCEIRA SEÇÃO, julgado em 08/08/2012, DJe 30/08/2012)

Teoria regulacji pobudzenia (integrucjąca)

DOI: 10.1371/journal.pgen.1008863

Resource: BDSC_41123

Curator: @scibot

SciCrunch record: RRID:BDSC_41123

What is this?

POCI = in Abstract

e.g. P: adults with type 2 diabetes

O response variables: glycated hemoglobin (AIC), subject's weight * two time periods (pre- and 6-month-post program)

C: 2 types of education being received (diabetes patient education OR augumanted by a community self-management program) = explanatory variables

I: 2 types of education being received *assigned

POCI ⇒ Inerventional RQ ⇒ experiment

via u/IrmaBecx at https://www.reddit.com/r/typewriters/comments/1e73576/comment/ldy6tjq/?utm_source=reddit&utm_medium=web2x&context=3

With Antares portable typewriters on can often use the top cover of the case upside down and the typewriter will sit snugly inside and can be used as a lap desk.

DOI: 10.1016/j.crphar.2024.100196

Resource: (Millipore Cat# SCC129, RRID:CVCL_0321)

Curator: @scibot

SciCrunch record: RRID:CVCL_0321

What is this?

DOI: 10.1016/j.isci.2024.109912

Resource: (Cell Signaling Technology Cat# 2956, RRID:AB_1196615)

Curator: @scibot

SciCrunch record: RRID:AB_1196615

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

1. General Statements

We are grateful for the valuable, constructive comments of the reviewers, which helped to substantially improve the quality of our manuscript. We particularly agree that the original structure of the manuscript was confusing and in parts misleading, since we followed the history of the project, which first identified the RBM39 mediated impact on IRF3 expression, whereas the -omics studies, identifying additional factors, were done at a far later point. Many discrepancies further arose from the low sensitivity of our initial proteomics analysis, which we now repeated, thereby obtaining far more sensitive detection of the key factors we also found in the transcriptomics data.

We have re-structured the entire manuscript by moving the -omics data from the end of the paper towards the middle and provide similar depth downstream analysis of all relevant key factors identified (RIG-I/MDA5, IFN receptors, STAT1/2), to reduce the focus on IRF3, as suggested. We further changed the title and abstract to reflect this major conceptual change. Thanks to this helpful comment, we think that our manuscript is now conceptually much clearer.

We further added new data to support the central claims of our manuscript, including a repetition of the proteomics study. Proteomics and transcriptomics now consistently demonstrate the impact of RMB39 knockdown as well as indisulam treatment on several key factors of innate immunity, including IRF3, STAT1/2, RIG-I and MDA5 (now in Fig. 5), with IFNAR2 and IL10RB additionally found in transcriptomics. We provide additional functional evidence that IRF3 is the key factor affected in the TLR3 pathway (IRF3 overexpression, Fig. 6B, C), whereas diminished abundance of RIG-I/MAD5 is equally important in the respective pathway, thereby also affecting NF-κB response (Fig. 6F-I). We further show the functional significance of IFN-receptor/STAT downregulation on type I and III IFN responses (Fig. 7E-G).

The reviewers also pointed to some datasets showing the expected trends, but in some cases lacking statistical significance, due to variability in knockdown efficiency. We repeated all mentioned datasets with new batches of siRNA with sufficient biological replicates (n=3). We thereby obtained consistent, statistically significant data in all cases. Importantly, all experiments implementing the RMB39.esc control now show consistent rescue (Fig2. A-E).

To generate a homogenous experimental design for virus infections, we further added new data showing a comparable impact of siRNA knockdown (Fig. 3F) and indisulam treatment (new Fig. 3G) on Sendai virus infection in A549 cells and took this as a rationale to consistently use indisulam for all other infections.

2. Point-by-point description of the revisions

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __ This manuscript by Li and colleagues examines the role of RBM39 in innate immune signaling. Splicing factor RBM39 was identified through a genome wide screen with a death reporter under control of the IFIT1 promoter that got stimulated with pIC in a TLR3-dependent manner. Besides IFIT1, further experiments showed that RBM39 is also involved in optimal expression of other innate immunity genes like IFNB, CXCL10, RIG-I or MDA5. While NFkB-dependent genes seem not to depend on RBM39, for IRF3 it was shown that protein levels decrease under conditions of RBM39 depletion, because IRF3 mRNAs are (slightly) reduced and spliced differently. The sulfonamid Indisulam could largely recapitulate the phenotype of RBM39 depletion. Further analyses using proteomics and transcriptomics showed that RBM39 is required for mRNA splicing and expression of a large set of other proteins. Altogether, this well designed and written study highlights the fundamental role played by RBM39 in in maintaining the pathways of immunity and metabolism. The key conclusions are convincing but some additional experiments would strengthen them further.

We are grateful for the very positive general comments of this reviewer.

Major comments: - For the statistics, authors seem not to have done multiple tests but rather tested individual datasets within larger graphs against each other. Please explain where this is the case and use corrections if multiple testing was done

We apologize for not have been clearer here, we indeed used multiple testing. In the proteomics, statistical significance was evaluated by "two-sample tests" (Student's T-test with permutation-based FDR 0.05 and 250 number of randomizations). For the analysis of RNAseq data, p values were calculated with the Wald test and corrected for multiple testing according to Benjamini-Hochberg. We have now included this information in the materials and methods section and in the respective figure legends.

• Fig. 4 shows that RBM39 depletion reduces IFIT expression in virus infected cells and slightly increases virus replication. RBM39 has a major effect on IRF3 levels, but also on other players in innate immunity. What happens if IRF3 is ectopically expressed as in figure 5? With this experiment one could measure how high the contribution of IRF3 miss-splicing is to innate immunity.

We thank this reviewer for the valuable suggestion. We restructured the entire manuscript, to address several reviewer comments regarding the focus on IRF3 and the lack of data on other factors in the pathway. We now clearly demonstrate that ectopic IRF3 expression entirely rescues the TLR3 response to poly(I:C) in PH5CH cells (Fig. 6B-C), which also explains the lack of impact on the NF-κB pathway (Fig. 2G-H). In contrast, overexpression of IRF3 does not rescue the RIG-I/MDA5 response in A549 cells (new data, Fig. 6F-I). Here, also the NF-κB pathway is affected by knockdown of RBM39, suggesting that reduced RIG-I/MDA5 abundance upon RMB39 knockdown substantially contributed to the diminished innate immune response.

• Fig. 4 A uses siRNAs but B, C and D only indisulam treatment. It would be better if siRNAs would also be used for the other viruses.

We agree that a homogenous setup for virus infection would be favorable, however, the use of different cell lines was authorative due to limited permissivess of the used cell types towards virus infection and it appeared challenging to achieve similar knockdown efficiencies. To generate a homogenous experimental design, we now added new data showing a comparable impact of siRNA knockdown (Fig. 3F) and indisulam treatment (new Fig. 3G) on Sendai virus infection in A549 cells and took this as a rationale to consistently use indisulam for all other infections.

• RBM39 depletion strongly reduces IRF3 levels in the WB, but not so much in RT-PCR and not at all in proteomics. Is the antibody used for WB perhaps recognizing a domain that is underrepresented in isoforms after disturbed splicing? Please clarify.

Our previous proteomics data suffered from a very low sensitivity, therefore we missed clear detection of many factors, including IRF3. We repeated the whole proteomics analysis with siRNA and indisulam treatment (new Fig. 5A, B) and now found significantly reduced IRF3 protein levels in both conditions (new Fig. S5C), in agreement with the WB data. The lower impact on IRF3 mRNA abundance is due to the additional contribution of alternative splicing (Fig. 6A, Fig. S6A-D), which both in combination affect protein abundance.

• Volcano plots in figure 7 show a lot of hits obtained after both RBM38 siRNA and indisulam (green dots), and some that are additionally identified in transcriptomes and in proteomes (red dots). Nonetheless only innate immunity and stress response genes are marked, although they do not belong to these highly conserved classes. Please elaborate more on the most RBM39-dependent genes, e.g. by presenting them in a heat map.

To our knowledge, our study is the first with a comprehensive comparison on the impact of RBM39 knockdown and indisulam treatment on the host cell proteome and transcriptome. However, several studies already did -omics studies on individual conditions/readouts (e.g. (Coomar et al, 2023; Dou et al, 2023; Mai et al, 2016; Nijhuis et al, 2022)). These studies already identified and described in detail key changes in transcriptome and proteome e.g. affecting genes involved in cell cycle control and metabolism, which we find as well. However, the novelty of our paper is the impact on innate immune response, we therefore rather decided to put an even stronger focus on these genes and to omit other factors, like stress response pathway components, etc.. This strategy is supported by the higher sensitivity of our new proteome analysis, which now generated a far better overlap with the transcriptomics, favoring a display setting on highlighting only those factors that were further analyzed in detail in the volcano blots (Fig. 5). Still, interested readers will find the comprehensive list of data in the supplementary Excel-datasheets as well as in our primary data in online depositories.

Minor comments: - Some abbreviations are not explained, like PGK, siNT, siVTN

We apologize and have added the missing explanation of abbreviations.

• Welsch should read Welch

Corrected.

• Fig. 2H: were cells also stimulated and if yes, how?

These were unstimulated conditions, to show the impact of RBM39 on basal expression of the IFNlambda receptor chains. However, we deleted this dataset due to the re-organisation of the manuscript. The analysis of the type I and type III receptor and STAT1/2 expression is now comprehensively shown in Fig. 7/S6E, F, solely based on the transcriptomic data for consistency reasons, along with the functional impact on the IFN response.

• Fig. 6E: I cannot see a difference between to IRF3-203 and 228 isoforms. And what are the white boxes?

• Also 6E: Location of the primers is barely visible

Due to the re-organization of the manuscript these data are now shown in Fig. S6D. Both isoforms are indeed very similar and only differ by a very small (16nt) additional exon in isoform 228. The white boxes are exons not translated in the respective isoforms. We have included this important information in the legend to Fig. S6 and increased the arrows indicating the positions of the primer.

• Some materials are not properly referenced, like the death reporter, the lentiviral system, or the Rift Valley fever luciferase virus

We are sorry for the missing information, which has now been added to the materials and methods section.

• Supplement has no page numbers

We have added page numbers to the supplementary information.

Reviewer #1 (Significance (Required)):

The study advances our knowledge about the regulation of innate immunity. Strengths are the discovery of a novel layer of innate immunity regulation by splicing and the in-depth analysis of the importance of RBM39 for cellular gene expression. A potential weakness might be the focus on innate immunity as other biological functions seem even more dependent on RBM39. However, this reviewer sees the necessity that covering all aspects of RBM39 finction would be beyond the scope of a single study. The relevant literature is appropriately cited (except for some materials, see minor comments). Results will be of interest not only to people doing basic research on innate immunity, but also to those interested in gene regulation in general or to cancer researchers using indisulam

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ The authors performed a CRISPR-based screen for genes required for TLR3-mediated signaling and gene expression in Hepatoma cells. Interferon-stimulated expression of an apoptosis inducer was used as a read-out system. A number of candidate genes were identified and one of these, RBM39, investigated in detail. The protein has previously been linked to both transcriptional control and RNA processing. Validation studies confirm that reduction of cellular RBM39 results in less TLR3-mediated IFN-beta synthesis and lower levels of ISG mRNA synthesis. Initial studies suggest a role of RBM39 in regulating of IRF3 levels, the transcription factor activated by TLR3 signaling to induce IFN-beta synthesis. However, the effect is variable and poorly supported by transcriptomic and proteomic data. Moreover, only one out of four cell-based viral infection models reports a substantial effect of the RBM39 knockdown.

We apologize for the lack of consistency among several datasets, which was mainly due to the low sensitivity of the proteomic analysis. This has been repeated and now fully confirms all other data. In part due to the comments of this reviewer, we further broadened the scope of the manuscript away from IRF3, including a change of the title.

Major comments:

1. The data do not support the claim that RBM39 is a broadly acting player in innate immune responses. In addition, they suggest that IRF3 may not be the only relevant RBM39 target. The most informative knockdown control in this regard would be IRF3 siRNA.

We have re-structured the entire manuscript and added new data to support the central claims of our manuscript, including a repetition of the proteomics study. Proteomics and transcriptomics now consistently demonstrate the impact of RMB39 knockdown as well as indisulam treatment on several key factors of innate immunity, including IRF3, STAT1/2, RIG-I and MDA5 (now in Fig. 5), with IFNAR2 and IL10RB additionally found in transcriptomics. We further provide functional evidence that IRF3 is the key factor affected in the TLR3 pathway (IRF3 overexpression, Fig. 6B, C), whereas diminished abundance of RIG-I/MAD5 is equally important in the respective pathway, thereby also affecting NF-κB response (Fig. 6F-I). We further show the functional significance of IFN-receptor/STAT downregulation on type I and III IFN responses (Fig. 7E-G). We hope this reviewer now agrees with our claim that RBM39 is a broadly acting player in innate immune responses.

1. The structure of the manuscript is rather confusing because IRF3 is presented as the main RBM39 target in figures 3-6, but the -omics data in figures 7 and 8 do not support this view. The authors argue different sensitivities of the experimental approaches, but I think few people would agree that western blots are more sensitive than MS. To my opinion a narrative with less focus on IRF3 and a broader integration of candidates of the -omics approaches would be preferable.

We are grateful for this valuable comment and fully agree that the original structure of the manuscript was confusing and in parts misleading, which was mainly due to the fact that we followed the history of the project, which first identified the RBM39 mediated impact on IRF3 expression, whereas the -omics studies, identifying additional factors, were done at a far later point. Many discrepancies further arose from the low sensitivity of our proteomics analysis, which we now repeated, thereby obtaining far more sensitive detection of the key factors we also found in the transcriptomics data. We now moved the -omics data from the end of the paper towards the middle and provide similar depth downstream analysis of all relevant key factors identified (RIG-I/MDA5, IFN receptors, STAT1/2, to reduce the focus on IRF3, as suggested. We further changed the title and abstract to reflect this major conceptual change. Thanks to this helpful comment, we think that our manuscript is now conceptually much clearer.

Investigating the role of RBM39 by RNA-seq in pIC-treated cells would further strengthen the manuscript. It will yield a broader view of the protein's role in induced innate immunity.

We did not add pIC treatment to the RNA-seq analysis, since, based on own experience and numerous papers, this will change the expression of literally thousands of genes. Based on the key factors of the pIC response modulated by RBM39 (RLRs and IRF3), this would very likely simply result in reduced induction of the whole ISG panel (as exemplified for IFIT1, ISG15, MxA and CXCL10 in Fig. 2B-E).

3.The results in figures 6A-C are confusing for two reasons. First, the siRNA-mediated knockdown should result in reduced RBM39 protein as well (as shown in Fig. 3A) and, therefore, in an increase in RBM39 levels. Second, why was this effect not noted in the experiments shown in figs. 1-5? To avoid this confusion it might be good to mention which IRF3 splice isoforms are detected by the primers and antibodies used in these figures.

Unfortunately, the reviewer seems to have conceptually misinterpreted Fig. 6A-C of the original paper, which did not show protein, but transcriptome data. We now added the corresponding data of the proteomic analysis in the new Fig. S5, for all detectable, relevant candidates, showing consistency to all previous data. The confusing point in previous Fig. 6B, which the reviewer appears to refer to, is the upregulation of RBM39 transcript levels upon indisulam treatment, which was not apparent in previous experiments, since we always used WB to show diminished RBM39 protein levels upon indisulam treatment. This increase in RBM39 mRNA is due to an autoregulation of RBM39 mRNA by protein abundance, which has been reported in literature (Campagne et al, 2023). Since this is rather confusing and not relevant for our study, we removed previous Fig. 6B and show this aspect only in the volcano blot in Fig. 5D, mentioning and citing the paper on autoregulation.

Minor comments.

1. Fig S1: the figure panels and legend are inconsistent. IFIT1 is labeled as ISG56 in panel S1A.

We apologie for this inconsistency and now use IFIT1 throughout the paper.

1. Data with the siRNA escape mutant of RBM39 are inconsistent. For example, why is its effect significantly different only in 1 out of 4 ISG in figures S2A-D?

We apologize for the inconsistency, which is due to variability of silencing efficiency. We repeated the entire set of experiments (n=3) with a new batch of siRNA and obtained comparable, significant differences for all ISGs analyzed (new Fig. 2B-E).

1. Line 164: the statement that TRIF and RBM39 siRNAs produce effects of similar magnitude is incorrect for the IFIT1 gene in figure S2A.

This experiment was repeated (see previous point), now obtaining significant, more homogenous data. We have modified the text accordingly.

4.Fig. 2H: In absence of additional evidence for functional implications, the data showing reduced IL10RB expression should be omitted.

We omitted the data, as suggested by the reviewer, however, we provide a more in depth analysis of the type I and III IFN response in Fig. 7, based on the transcriptomic data and a functional analysis.

5.Fig. 3: More datapoints would be needed in panel A to sustain the lack of significant difference between the untreated and escape mutant samples. Are the viability data in panels B and C normalized to untreated cells to control for Indisulam toxicity? In figure S3A the effect of the mutant is rather small. To allow for comparison, the Indisulam titration curves should be adapted to the concentrations used in Fig. 3.

Fig. 3 (now Fig. 4) was replaced by another representative experiment, now also containing the quantification of the shown western blots, however, the statistical analysis shown in the previous version was and is based on three independent biological replicates, as indicated in the figure legend. Viability data was normalized to controls and this information is now added to the figure lengend as well. The mutant analyzed in Fig. S3A (now S4A) confers only partial resistance, which explains the limited but clear rescue. We did not include higher indisulam concentrations here due to the increased cytotoxicity of concentration above 5 µM in PH5CH, in the absence of pronounced additional effects on RBM39 abundance (Fig. 4B).

6.RNA-seq measures steady-state RNA, not transcription.

This is of course correct, we changed all sentences, where our wording might have indicated that we are measuring transcription by RNAseq. However, we still need to differentiate between the role of RBM39 in transcriptional regulation and splicing, where changes in RNA abundance found in RNAseq rather point to transcriptional regulation.

Reviewer #2 (Significance (Required)):

The identification of RBM39 as a candidate player in innate immune responses is of interest to a large scientific community with interest in signalling by pattern recognition receptors. Its role should be strengthened with additional infection models. It is puzzling that three out of four viruses don't benefit from the reduced IFN-beta synthesis in the RBM39 knockdown. Moreover, the data are not convincing (or too diverse) to nail down IRF3 as a major, or the most relevant, RBM39 target.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ CRISPR Screen for factors that are required for dsRNA-dependent ISG production. Found a large number of hits but most did not validate in subsequent assays. The authors follow up the one candidate that did pass secondary screening criteria, RBM39, although re-expression of RBM39 only rescues the phenotype of the siRNAs against RBM39 (siRBM39) in one of the two cell lines tested. Additionally, siRBM39 impacts only a subset of polyIC-induced ISGs and does not regulate NFkB-driven gene expression. They go on to attempt to investigate the impact of siRBM39 on other key innate immune genes and proteins, although many key controls and appropriate methods are missing.

We thank this reviewer for pointing at inconsistencies and missing controls in our manuscript. We have critically re-evaluated the respective datasets.

Major comments: 1) The authors propose some rationale for the limited success of the screen, however, while RBM39 may have a role in dsRNA-induced innate immunity, in general the screen seems to have limited value.

The aim of our CRISPR/Cas9 death reporter screen was the identification of so far unknown contributors to innate immune response. This was achieved by identifying a critical role of RBM39, followed by an in depth validation focusing on RBM39. We further found known components of the TLR3 pathway in our candidate list (e.g. TRIF and UNC93B1), pointing to the overall quality of the experimental setup. At no point of the manuscript we claim that our screen aimed for or delivered a comprehensive overview on innate immunity pathways. Honestly, no comparable screen (e.g. on cytopathic viruses) has delivered such data.

2) Given that the siRBM39 clearly has off-target effects (since expression of a resistant RBM39 cDNA only gives limited rescue in many cases - Fig S2), each of the experiments in which siRBM39 is used (i.e. Fig 2) should have the RBM39.esc control - especially those that drive subsequent experiments such as the expression of IFNbeta and IFNLR1 (Fig 2a, h)

The inconsistency in some datasets, showing all the same trends, but in some cases lacking statistical significance was due to variability in knockdown efficiency. We repeated all mentioned datasets with new batches of siRNA with sufficient biological replicates (n=3) with now all of them revealing consistent, statistically significant data. Importantly, all experiments implementing the RMB39.esc control now show consistent rescue.

3) Since RBM39 reduction has an apparent impact even if IFNLR1-deficient cells (although need the rescue control to know if this is real) the authors conclude that RBM39 regulates the initial wave of dsRNA signaling-events, but this should be tested with the use of Ruxilitinib to block JAK-STAT signaling.

Due to the general major re-organization of the manuscript, aiming for a less confusing data presentation and consistency towards depth of candidate evaluation, we have removed the data on the IFNLR-deficient cell line. The claim that RBM39 affects the initial wave of ISG responses is based on reduced IFNb expression, which is exclusively induced by the initial wave of ISG response and by the general impact on ISG expression, which we measure at 6h after induction, too early for autocrine IFN stimulation (Burkart et al, 2023). However, we further demonstrate that downregulation of type I and type III IFN receptors in conjunction with STAT1/2 affect the type I and the type III IFN response as well (Fig. 7E-G, in part new data). Therefore, RBM39 affects both, the intial wave and the auto-/paracrine IFN response, and we therefore undertook no further efforts to separate these effects.

4) IRF3 expression in the Indisulam-treated cells more closely tracks cell viability than RBM39 expression. For example in Fig 3C 10 microM gives 50% IRF3 expression and 50% viability but still 95% RBB39 expression - arguing that the impact of siRBM39 on IRF3 might be very indirect (and error bars on rescue are large so unclear if the rescue really worked in Fig 3A).

Based on this reviewer comment we re-evaluated the quantification in previous Fig. 3C (now Fig. 4C), which combines data from three independent experiments. We deeply apologize, but the initial quantification proved to be wrong, due erroneous background subtraction, which was relatively high in one of the PHH-replicates (Replicate 1, see Reviewer Fig. 1 in uploaded file). The re-evaluated quantification revealed 55% for the RBM39 abundance at 10µM indisulam, which better reflects the data shown and is now in line with the impact on cytotoxicity and IRF3 abundance.

5) It is unclear in Fig 4 why some cell/virus combinations are tested with siRBM39 and others are tested with Indisulam. Also the conclusion that RBM39 "substantially contributes to the cell intrinsic innate immune response to viral infections" is greatly overstated given that the differences are between ~3 fold and non-significant.

We agree that a homogenous setup for virus infection would be favorable, however, the use of different cell lines was authoritave due to limited permissivess of the used cell types towards virus infection and it appeared challenging to achieve similar knockdown efficiencies. To generate a homogenous experimental design, we now added new data showing a comparable impact of siRNA knockdown (Fig. 3F) and indisulam treatment (new Fig. 3G) on Sendai virus infection in A549 cells and took this as a rationale to consistently use indisulam for all other infections. Overall, the aim of the virus infection experiments was using a variety of natural triggers of innate immunity beyond synthetic poly(I:C). Here we found indeed significant reductions of ISG induction for all viruses tested, similar to poly(I:C), this is the basis for the statement that RBM39 contributes the cell intrinsic innate immune response to viral infections. Our experimental design did not intend to see pronounced effects on viral replication, this was only measured to secure that reduced ISG induction was not due to inhibition of viral replication. We have explained this strategy now clearer and tuned down corresponding statements, to exclude potential overinterpretation of the data.

6) Neither DTU/DRIMseq or qPCR are valid methods to measure splice isoform differences. The authors need to use rMATS or MAJIQ and validate by gel-based RT-PCR.

Output generated by modern alignment algorithms like salmon is suitable for studies on an isoform level (Love et al, 2018) and has been used in a variety of studies (e.g.(Jabs et al, 2020; Xiong et al, 2023). MAJIQ and rMATS are only superior tools if the detection of so far unknown isoforms is of interest (Love et al., 2018), which is beyond the scope of this project. We have validated the data for IRF3 in RT-qPCR, showing close to identical results to the DTU analysis (compare Fig. 6A and S6D). We disagree that a gel-based RT-PCR analysis would be superior here, due to the lack of quantification.

7) The conclusions from the proteomic and transcriptomic analyses should be treated with extreme caution given the caveats of methodology and controls discussed above.

We are aware of the caveats of these technologies. The previous proteomic analysis indeed suffered from low sensitivity, failing to detect essential candidates like IRF3. The repetition of the experiment (new Fig. 5A, B, new Fig. S5) now revealed data very consistent with the transcriptomic data. Overall, the strength of our approach is the direct comparison of siRNA based RBM39 knockdown and RBM39 depletion by indisulam throughout transcriptomics and proteomics analyses. The wide overlap argues for the validity of our data and suggests that we thereby circumvented many caveats.

Reviewer #3 (Significance (Required)):

Innate immune signaling is a complex and essential pathway for maintaining health. While much is known about key components of this pathway, additional regulators are likely to exist. This manuscript describes an attempt to identify new regulators of dsRNA-mediated gene expression.

References

Burkart SS, Schweinoch D, Frankish J, Sparn C, Wust S, Urban C, Merlo M, Magalhaes VG, Piras A, Pichlmair A et al (2023) High-resolution kinetic characterization of the RIG-I-signaling pathway and the antiviral response. Life Sci Alliance 6

Campagne S, Jutzi D, Malard F, Matoga M, Romane K, Feldmuller M, Colombo M, Ruepp MD, Allain FH (2023) Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39. Nat Commun 14: 5366

Coomar S, Mota P, Penson A, Schwaller J, Abdel-Wahab O, Gillingham D (2023) Overlaid Transcriptional and Proteome Analyses Identify Mitotic Kinesins as Important Targets of Arylsulfonamide-Mediated RBM39 Degradation. Mol Cancer Res 21: 768-778

Dou Z, Zhang X, Su W, Zhang T, Ye F, Zhao D, Chen X, Li Q, Zhang H, Di C (2023) Indisulam exerts anticancer effects via modulation of transcription, translation and alternative splicing on human cervical cancer cells. Am J Cancer Res 13: 2922-2937

Jabs S, Biton A, Becavin C, Nahori MA, Ghozlane A, Pagliuso A, Spano G, Guerineau V, Touboul D, Giai Gianetto Q et al (2020) Impact of the gut microbiota on the m(6)A epitranscriptome of mouse cecum and liver. Nat Commun 11: 1344

Love MI, Soneson C, Patro R (2018) Swimming downstream: statistical analysis of differential transcript usage following Salmon quantification. F1000Res 7: 952

Mai S, Qu X, Li P, Ma Q, Cao C, Liu X (2016) Global regulation of alternative RNA splicing by the SR-rich protein RBM39. Biochim Biophys Acta 1859: 1014-1024

Nijhuis A, Sikka A, Yogev O, Herendi L, Balcells C, Ma Y, Poon E, Eckold C, Valbuena GN, Xu Y et al (2022) Indisulam targets RNA splicing and metabolism to serve as a therapeutic strategy for high-risk neuroblastoma. Nat Commun 13: 1380

Xiong L, Liu J, Han SY, Koppitch K, Guo JJ, Rommelfanger M, Miao Z, Gao F, Hallgrimsdottir IB, Pachter L et al (2023) Direct androgen receptor control of sexually dimorphic gene expression in the mammalian kidney. Dev Cell 58: 2338-2358 e2335

Reviewer #2 (Public Review):

Summary:

This manuscript from Lee-Odegard et al reports proteomic profiling of exercise plasma in humans, leading to the discovery of CD300LG as a secreted exercise-inducible plasma protein. Correlational studies show associations of CD300LG with glycemic traits. Lastly, the authors query available public data from CD300LG-KO mice to establish a causal role for CD300LG as a potential link between exercise and glucose metabolism. However, the strengths of this manuscript were balanced by the moderate to major weaknesses. Therefore in my opinion, while this is an interesting study, the conclusions remain preliminary and are not fully supported by the experiments shown so far.

Strengths:

(1) Data from a well-phenotyped human cohort showing exercise-inducible increases in CD300LG.

(2) Associations between CD300LG and glucose and other cardiometabolic traits in humans, that have not previously been reported.

(3) Correlation to CD300LG mRNA levels in adipose provides additional evidence for exercise-inducible increases in CD300LG.

Weaknesses:

(1) CD300LG is by sequence a single-pass transmembrane protein that is exclusively localized to the plasma membrane. How CD300LG can be secreted remains a mystery. More evidence should be provided to understand the molecular nature of circulating CD300LG. Is it full-length? Is there a cleaved fragment? Where is the epitope where the o-link is binding to CD300LG? Does transfection of CD300LG to cells in vitro result in secreted CD300LG?

(2) There is a growing recognition of specificity issues with both the O-link and somalogic platforms. Therefore it is critical that the authors use antibodies, targeted mass spectrometry, or some other methods to validate that CD300LG really is increased instead of just relying on the O-link data.

(3) It is insufficient simply to query the IMPC phenotyping data for CD300LG; the authors should obtain the animals and reproduce or determine the glucose phenotypes in their own hands. In addition, this would allow the investigators to answer key questions like the phenotype of these animals after a GTT, whether glucose production or glucose uptake is affected, whether insulin secretion in response to glucose is normal, effects of high-fat diet, and other standard mouse metabolic phenotyping assays.

(4) I was unable to find the time point at which plasma was collected at the 12-week time point. Was it immediately after the last bout of exercise (an acute response) or after some time after the training protocol (trained state)?

Author response:

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

Reviewer #1 (Public Review):

Summary:

In this paper, proteomics analysis of the plasma of human subjects that underwent an exercise training regime consisting of a combination of endurance and resistance exercise led to the identification of several proteins that were responsive to exercise training. Confirming previous studies, many exercise-responsive secreted proteins were found to be involved in the extra-cellular matrix. The protein CD300LG was singled out as a potential novel exercise biomarker and the subject of numerous follow-up analyses. The levels of CD300LG were correlated with insulin sensitivity. The analysis of various open-source datasets led to the tentative suggestion that CD300LG might be connected with angiogenesis, liver fat, and insulin sensitivity. CD300LG was found to be most highly expressed in subcutaneous adipose tissue and specifically in venular endothelial cells. In a subset of subjects from the UK Biobank, serum CD300LG levels were positively associated with several measures of physical activity - particularly vigorous activity. In addition, serum CD300LG levels were negatively associated with glucose levels and type 2 diabetes. Genetic studies hinted at these associations possibly being causal. Mice carrying alterations in the CD300LG gene displayed impaired glucose tolerance, but no change in fasting glucose and insulin. Whether the production of CD300LG is changed in the mutant mice is unclear.

Strengths:

The specific proteomics approach conducted to identify novel proteins impacted by exercise training is new. The authors are resourceful in the exploitation of existing datasets to gain additional information on CD300LG.

Weaknesses:

While the analyses of multiple open-source datasets are necessary and useful, they lead to relatively unspecific correlative data that collectively insufficiently advance our knowledge of CD300LG and merely represent the starting point for more detailed investigations. Additional more targeted experiments of CD300LG are necessary to gain a better understanding of the role of CD300LG and the mechanism by which exercise training may influence CD300LG levels. One should also be careful to rely on external data for such delicate experiments as mouse phenotyping. Can the authors vouch for the quality of the data collected. 

Thank you for the valuable feedback on our manuscript. We recognize concerns about the specificity of correlative data from open-source datasets and the limitations it presents for understanding CD300LG's role. To address this, we have expanded the manuscript with a paragraph in the discussion regarding the need of targeted experiments confirm CD300LG’s functions and relationship with glucose metabolism. We also emphazise caution regarding external data reliance and we acknowledge the need for generating primary data including direct phenotyping of mice with CD300LG gene alterations to better understand its regulatory mechanisms and effects on glucose tolerance. Please see lines 446-456.

Reviewer #2 (Public Review):

Summary:

This manuscript from Lee-Odegard et al reports proteomic profiling of exercise plasma in humans, leading to the discovery of CD300LG as a secreted exercise-inducible plasma protein. Correlational studies show associations of CD300LG with glycemic traits. Lastly, the authors query available public data from CD300LG-KO mice to establish a causal role for CD300LG as a potential link between exercise and glucose metabolism. However, the strengths of this manuscript were balanced by the moderate to major weaknesses. Therefore in my opinion, while this is an interesting study, the conclusions remain preliminary and are not fully supported by the experiments shown so far.

Strengths:

(1) Data from a well-phenotyped human cohort showing exercise-inducible increases in CD300LG.

(2) Associations between CD300LG and glucose and other cardiometabolic traits in humans, that have not previously been reported.

(3) Correlation to CD300LG mRNA levels in adipose provides additional evidence for exercise-inducible increases in CD300LG.

Weaknesses:

(1) CD300LG is by sequence a single-pass transmembrane protein that is exclusively localized to the plasma membrane. How CD300LG can be secreted remains a mystery. More evidence should be provided to understand the molecular nature of circulating CD300LG. Is it full-length? Is there a cleaved fragment? Where is the epitope where the o-link is binding to CD300LG? Does transfection of CD300LG to cells in vitro result in secreted CD300LG?

(2) There is a growing recognition of specificity issues with both the O-link and somalogic platforms. Therefore it is critical that the authors use antibodies, targeted mass spectrometry, or some other methods to validate that CD300LG really is increased instead of just relying on the O-link data.

(3) It is insufficient simply to query the IMPC phenotyping data for CD300LG; the authors should obtain the animals and reproduce or determine the glucose phenotypes in their own hands. In addition, this would allow the investigators to answer key questions like the phenotype of these animals after a GTT, whether glucose production or glucose uptake is affected, whether insulin secretion in response to glucose is normal, effects of high-fat diet, and other standard mouse metabolic phenotyping assays.

(4) I was unable to find the time point at which plasma was collected at the 12-week time point. Was it immediately after the last bout of exercise (an acute response) or after some time after the training protocol (trained state)?

We acknowledge the importance of understanding the molecular form of CD300LG in circulation. We have expanded the discussion with a paragraph regarding the need of follow-up experiments on whether circulating CD300LG is full-length or a cleaved fragment, to identify the epitope for O-link binding, and assess CD300LG secretion in vitro through transfection experiments. We also discuss the need of targeted mass spectrometry and antibody-based validation of O-link measurements of CD300LG, and the need for more validation experiments on CD300LG-deficient mice. Please see lines 446-456.

The plasma collected post-intervention is in a state that reflects the new baseline trained condition of the subjects, 3 days after the last exercise session during the intervention. We have clarified this in our manuscript. The information is updated in line 491-493.

Reviewer #1 (Recommendations For The Authors):

In the present form, the paper raises interest in the potential role of CD300LG in the response to exercise training but unfortunately does not provide clear answers. The authors should focus their efforts on firmly validating the status of CD300LG as an exercise biomarker in humans and carefully examine the function of CD300LG through mechanistic and animal-based studies.

The authors are encouraged to acquire CD300LG-deficient mice and perform specific experiments to validate hypotheses forthcoming from the analysis of the open-source datasets. In addition, it needs to be validated that the cd300lgtm1a(KOMP)Wtsi mice are actually deficient in CD300LG. It is not uncommon that Tm1a mice have (almost) normal expression of the targeted gene.

We have now revised the manuscript and added a new section to the discussion regarding the limitations with open-source data, cd300lgtm1a(KOMP)Wtsi mice and the need for more validation experiments on CD300LG-deficient mice. Please see lines 446-456.

The value of the correlative data presented in Figure 5 is rather limited. The same can be argued for the data presented in Supplementary Figure 2. If CD300LG is expressed in endothelial cells, it stands to reason that its expression is correlated with angiogenesis. Hence, this observation does not really carry any additional value.

We agree that correlations cannot imply causality. However, similar patterns were observed in several tissues and across different data sets, which at least suggest a role CD300LG related to angiogesis. We have included a section in the discussion were we clarify that our observations should only be regarded as indications and that follow-up studies are needed to confirm any causal role for CD300LG on angiogenesis/oxidativ capacity. Please see lines 446-456.

Figure 6 may be better accommodated in the supplement.

Figure 6 is now moved to the supplement.

Figure 3A and B are a bit awkward. The description "no overlap" is confusing. Isn't it more accurate to say "no enrichment" or "no over-representation"? There will always be some overlap with certain pathways. However, there may be no enrichment. Furthermore, the use of arrows to indicate No overlap is visually not very appealing. Maybe the numbers can be given a specific color?

We have now removed the arrows and text, and rather stated in the text that there were no enrichements other than for the proteins down-regulated in the overweight group.

The description of the figure legend of figure 5E-H is incomplete.

The description is now completed.

I don't see my preview test ;o

DOI: 10.1016/j.isci.2024.110461

Resource: (IZSLER Cat# BS TCL 156, RRID:CVCL_0033)

Curator: @vtello

SciCrunch record: RRID:CVCL_0033

What is this?

DOI: 10.1016/j.cmet.2020.08.017

Resource: Flybase_FBst0477680

Curator: @scibot

SciCrunch record: RRID:Flybase_FBst0477680

What is this?

DOI: 10.1016/j.cmet.2020.08.017

Resource: (BDSC Cat# 63762,RRID:BDSC_63762)

Curator: @scibot

SciCrunch record: RRID:BDSC_63762

What is this?

DOI: 10.1126/science.adk5382

Resource: (Jackson ImmunoResearch Labs Cat# 115-035-062, RRID:AB_2338504)

Curator: @scibot

SciCrunch record: RRID:AB_2338504

What is this?

DOI: 10.1016/j.ccell.2024.05.024

Resource: Statistical Analysis System (RRID:SCR_008567)

Curator: @scibot

SciCrunch record: RRID:SCR_008567

What is this?

DOI: 10.1016/j.ccell.2024.06.001

Resource: (BioLegend Cat# 108452, RRID:AB_2564249)

Curator: @scibot

SciCrunch record: RRID:AB_2564249

What is this?

DOI: 10.1016/j.cell.2024.06.012

Resource: (Thermo Fisher Scientific Cat# 12-5856-82, RRID:AB_1311270)

Curator: @scibot

SciCrunch record: RRID:AB_1311270

What is this?

Overall Rating (⭐⭐☆☆☆): This manuscript addresses a relevant topic in cardiovascular health, exploring the relationship between Metabolic Syndrome (MetS) components and risk of Heart Failure (HF). The focus on a relatively understudied population is a strength. However, there are significant methodological limitations and I have several concerns about the interpretation and presentation of the findings. - The case-control design with data collected at one time point severely limits causal inference and results in high risks for reverse causation. The small sample size results in limited power. - The rationale and specific research gap addressed by this study are not clear, and there are inconsistencies between the stated aims and the actual analyses performed and findings discussed. - The reporting of the study population, data collection methods, and statistical analyses is not sufficiently clear or complete for adequate interpretation and replication of the study. Also, I’m not a native English speaker, but I would recommend textual editing as some sentences seem a bit awkward and difficult to understand (e.g. ‘diabetes or insulin resistance had the higher risk of developing heart failure” – should this be ‘patients with diabetes or IR had a higher odds of HF as compared to…. ’?).

Additional minor comments: - The introduction is rather long and could be shortened and more focused on the specific research aims. - The first line of the abstract, "Metabolic dysfunction of metabolic syndromes," is unclear to me. - The statement that cardiovascular disease is the leading cause of death in the US (first line) is true, but I would suggest expanding this to that this is true globally.- Please check consistency in terminology and use of abbreviations (e.g., insulin resistance vs. IR) throughout the manuscript.

Impact (⭐⭐☆☆☆): While the study addresses a relevant topic, giving the high and rising prevalence of risk factors included in the metabolic syndrome and HF, its potential impact is limited by several factors, as outlined above.

The specific research gap addressed and the aims of the study need further clarification throughout the manuscript. The introduction suggests that many studies have already examined MetS as a whole in relation to HF, while also implying that individual components have been less studied. This seems contradictory to the existing literature, which includes numerous studies on individual risk factors (e.g., blood pressure, cholesterol levels, obesity) and their relationship to HF. Subsequently, the authors highlight that the study of different combinations of MetS components are not well studied, but in their analyses they do not study all combinations available or test e.g. interactions. In the discussion, the authors again only focus on single MetS components. This all seems rather contradictory.

With the available data, maybe the authors could maybe include: interaction testing (additive or multiplicative) across individual MetSyn components on HF risk and/or focus on whether single components are independently associated or may mediate each other. This may help guide prevention strategies among different subgroups of patients with particular metabolic risk factors and thereby increase potential impact.

Methods (⭐⭐☆☆☆): <br /> 1. Study population: More information is needed about the source population. Are there specific patient groups or professions represented in the Marshall Health Network? Is this a population-based or patient-based sample? 2. Data collection: More information is required on how data were collected, both for HF definitions and for exposures and covariates. The timing and methods of data collection are crucial for understanding potential biases. 3. Study design: in line with this; based on the current text I understand that data are all collected at a single time point. This cross-sectional nature o the study needs to be explicitly stated and integrated in the interpretation and discussion of findings. 4. MetSyn definition: the authors should state which definition they used for MetS and provide a rationale for their choice. 5. Analyses: The description of how MetS components were included in the models needs clarification. What does "simultaneously" mean in this context? Are all five factors included in one model, or are all different combinations tested? 6. Analyses: consider including analyses of interaction effects between different MetS components on HF (as hinted to by the authors in the introduction). 7. Minor: The statement about "combinations of previously identified significant components" (lines 168-169) is unclear to me.

Results (⭐⭐☆☆☆): 1. Please provide footnotes for each table indicating the models used, confounders adjusted for, abbreviations used, etc. 2. Table 2a: The analysis for obesity is based on a very small sample size (only 10 HF cases and 5 controls are not overweight or obese). I would not do this analyses with such a small sample. Based on the text in the methods (please provide info in the footnotes as well), there are also several confounders included in these model, which makes the degrees of freedom for this analysis very low. 3. Related to this: to enhance statistical power and retain more information from this limited sample size: consider analyzing all components also as continuous variables in addition to meeting the MetSyn cutoff yes/no. E.g. include blood pressure and not only hypertension yes/no. Especially because cutoffs for MetS components are somewhat arbitrary and vary across definitions and study populations. 4. Table 2b: The rationale for selecting only these specific combinations of MetSyn components is not clear. Why were not all possible combinations tested? 5. Table 3, major: In my opinion, the results presenting odds ratios (ORs) for the number of MetSyn components do not show a clear dose-response trend. While there is a large OR for having any single MetSyn component compared to none, the ORs for having at least 2, 3, or 4 components are generally similar with almost completely overlapping confidence intervals. This observation does not support the interpretation provided in lines 216-219 of the manuscript or in lines 230-231 of the conclusion.

Discussion(⭐⭐⭐☆☆): 1. The cross-sectional nature of the study, the risk for reverse causation, residual confounding, and the limited sample size should be discussed as major limitations. 2. Related to this: please be careful with using causal language throughout the manuscript. 3. In general, the discussion is not in line with the aims, the discussion should focus more on combinations and interactions of MetS components, rather than primarily on individual factors. 4. The findings in line with the obesity paradox (seemingly protective effect of obesity) in this study are very likely largely due to reverse causation. This should be explicitly discussed. 5. Given the limitations of this observational study, the authors should be very cautious in their interpretation and in drawing clinical implications. Any conclusions or suggestions for clinical practice should be framed as hypotheses for future research rather than definitive findings or recommendations.

Reviewer Information Dr. Trudy Voortman has a PhD in nutritional epidemiology, 15+ years of research experience, focusing on the role of nutrition and lifestyle in population health across the lifecourse.

Dr. Trudy Voortman on ResearchHub: https://www.researchhub.com/user/1791011/overview

ResearchHub Peer Reviewer Statement: This peer review has been uploaded from ResearchHub as part of a paid peer review initiative. ResearchHub aims to accelerate the pace of scientific research using novel incentive structures.

Reviewer #1 (Public Review):

Summary:

This manuscript describes the crystallographic screening of a number of small molecules derived from the natural substrates S-adenosyl methionine (SAM) and adenine, against the SARS-CoV-2 2'-O-methyltransferase NSP16 in complex with its partner NSP10. High-quality structures for several of these are presented together with efforts to evaluate their potential biophysical binding and antiviral activities. The structures are of high quality and the data are well presented but do not yet show potency in biophysical binding. They only offer limited insights into the design of inhibitors of NSP16/10.

Strengths:

The main strengths of the study are the high quality of the structural data, and associated electron density maps making the structural data highly accurate and informative for future structure-based design. These results are clearly presented and communicated in the manuscript. Another strength is the authors' attempts to probe the binding of the identified fragments using biophysical assays. Although in general the outcome of these experiments shows negative data or very weak binding affinities the authors should be commended for attempting several techniques and showing the data clearly. This study is also useful as an example of the complexities associated with drug discovery on a bi-substrate target such as a methyltransferase, several of the observed binding poises were unexpected with compounds that are relatively similar to substrates binding in different parts of the active site or other unexpected orientations. This serves as an example of how experimental structural information is still of crucial importance to structure-based drug design. In general, the claims in the manuscript are well supported by the data.

Weaknesses:

The main limitations of the study are that the new structures generated in the study are fairly limited in terms of chemical space being similar to either SAM or RNA-CAP analogues. It feels a little bit of a lost opportunity to expand this to more diverse ligands which may reveal potential inhibitors that are distinct from current methyltransferase inhibitors based on SAM analogues and truly allow a selective targeting of this important target.

Another limitation is the potentially misleading nature of the antiviral assays. It is not possible to say if these compounds display on-target activity in these assays or even if the inhibition of NSP16/10 would have any effect in these assays. Whilst the authors do mention these points I think this should be emphasized more strongly.

Minor critical points:

The authors state that their crystals and protein preps have co-purified SAM occupying the active site of the crystals. Presumably, this complicates the interpretation of electron density maps as many of the ligands share overlap with the existing SAM density making traditional analysis of difference maps challenging. The authors did not utilize the PanDDA analysis for this step, perhaps this is related to the presence of SAM in the ground state datasets? Also, occupancies are reported in the manuscript in some cases to two significant figures, this seems to be an overestimation of the ability of refinement to determine occupancy based on density alone and the authors should clarify how these figures were reached.

The molecular docking approach to pre-selection of library compounds to soak did not appear to be successful. Could the authors make any observations about the compounds selected by docking or the docking approach used that may explain this?

Author response:

eLife assessment

This important study describes the crystallographic screening of a number of small molecules against a viral enzyme critical for the 5' capping of SARS-CoV-2 RNA and viral replication. While the high-quality crystal structures and complementary biophysical assays in this study provide solid evidence to support the major claims regarding how these small molecule compounds bind to the viral enzyme, the mismatch between the antiviral activity and binding to the viral enzyme of several small molecule compounds could have been more thoroughly investigated or discussed. This paper would be of interest to the fields of coronavirus biology, structural biology, and drug discovery.

We do fully agree that the antiviral assay results could be brought better into context clarifying that the antiviral effects of tubercine and its derivates are due to off-target effects.

Reviewer #1 (Public Review):

Summary:

This manuscript describes the crystallographic screening of a number of small molecules derived from the natural substrates S-adenosyl methionine (SAM) and adenine, against the SARS-CoV-2 2'-O-methyltransferase NSP16 in complex with its partner NSP10. High-quality structures for several of these are presented together with efforts to evaluate their potential biophysical binding and antiviral activities. The structures are of high quality and the data are well presented but do not yet show potency in biophysical binding. They only offer limited insights into the design of inhibitors of NSP16/10.

Strengths:

The main strengths of the study are the high quality of the structural data, and associated electron density maps making the structural data highly accurate and informative for future structure-based design. These results are clearly presented and communicated in the manuscript. Another strength is the authors' attempts to probe the binding of the identified fragments using biophysical assays. Although in general the outcome of these experiments shows negative data or very weak binding affinities the authors should be commended for attempting several techniques and showing the data clearly. This study is also useful as an example of the complexities associated with drug discovery on a bi-substrate target such as a methyltransferase, several of the observed binding poises were unexpected with compounds that are relatively similar to substrates binding in different parts of the active site or other unexpected orientations. This serves as an example of how experimental structural information is still of crucial importance to structure-based drug design. In general, the claims in the manuscript are well supported by the data.

Weaknesses:

The main limitations of the study are that the new structures generated in the study are fairly limited in terms of chemical space being similar to either SAM or RNA-CAP analogues. It feels a little bit of a lost opportunity to expand this to more diverse ligands which may reveal potential inhibitors that are distinct from current methyltransferase inhibitors based on SAM analogues and truly allow a selective targeting of this important target.

It is true that it makes sense to screen for more diverse compounds to expand to a more diverse ligand set and we do hope our study motivates to do so. Given the limited number of crystal structures of nsp10-16 with potential drug molecules, the aim of this study was to upgrade the data base with new complex structures to have a pool of complex structures for future compound designs with increased selectivity. Furthermore, some of the hits are known inhibitors of similar enzymes and most prominent and potent methyltransferase inhibitors are structurally related to SAM, like sinefungin and tubercidine. We do think that knowing which SAM compounds or fragments of SAM are able to bind in the nsp10-16 active site is highly valuable for further specific and optimized inhibitor design.

Another limitation is the potentially misleading nature of the antiviral assays. It is not possible to say if these compounds display on-target activity in these assays or even if the inhibition of NSP16/10 would have any effect in these assays. Whilst the authors do mention these points I think this should be emphasized more strongly.

That is a very valid point and we do not believe that the antiviral activity is based on on-target effects. We do agree that the way it is currently presented can be considered misleading and we indeed clarify this point in the revised version.

Minor critical points:

The authors state that their crystals and protein preps have co-purified SAM occupying the active site of the crystals. Presumably, this complicates the interpretation of electron density maps as many of the ligands share overlap with the existing SAM density making traditional analysis of difference maps challenging. The authors did not utilize the PanDDA analysis for this step, perhaps this is related to the presence of SAM in the ground state datasets? Also, occupancies are reported in the manuscript in some cases to two significant figures, this seems to be an overestimation of the ability of refinement to determine occupancy based on density alone and the authors should clarify how these figures were reached.

We have used PanDDA in parallel for hit finding. We however did not see any advantages for this target over the hit finding results from the visual inspection. This is probably as mentioned because of SAM being present is the “ground state” which complicates the PanDDA map calculations.

Regarding the occupancies, we fully agree with this comment and change it to reasonable digits and clarify how the figures were reached.  

The molecular docking approach to pre-selection of library compounds to soak did not appear to be successful. Could the authors make any observations about the compounds selected by docking or the docking approach used that may explain this?

Yes, it is a good point to give possible explanations why the docking approach was not successful to facilitate similar approaches in future studies.

Reviewer #2 (Public Review):

Summary:

The study by Kremling et al. describes a study of the nsp16-nsp10 methyl transferase from SARS CoV-2 protein which is aimed at identifying inhibitors by x-ray crystallography-based compound screening.<br /> A set of 234 compounds were screened resulting in a set of adenosine-containing compounds or analogues thereof that bind in the SAM site of nsp16-nsp10. The compound selection was mainly based on similarity to SAM and docking of commercially available libraries. The resulting structures are of good quality and clearly show the binding mode of the compounds. It is not surprising to find that these compounds bind in the SAM pocket since they are structurally very similar to portions of SAM. Nevertheless, the result is novel and may be inspirational for the future design of inhibitors. Following up on the crystallographic screen the identified compounds were tested for antiviral activity and binding to np16-nsp10. In addition, an analysis of similar binding sites was presented.

Strengths:

The crystallography is solid and the structures are of good quality. The compound binding constitutes a novel finding.

Weaknesses:

The major weakness is the mismatch between antiviral activity and binding to the target protein. Only one of the compounds could be demonstrated to bind to the nsp16-nsp10 protein. By performing a displacement experiment using ITC Sangivamycin is concluded to bind with a Kd > 1mM. However, the same compound displays antiviral activity with an EC50 of 0.01 microM. Even though the authors do not make specific claims that the antiviral effect is due to inhibition of nsp16-nsp10, it is implicit. If the data is included, it should state specifically that the effect is not likely due to nsp16-nsp10 inhibition.

We do believe that the antiviral data are valuable and should be published within this work. We also agree with the comment that it should be clearly stated that the antiviral effect is not likely because of nsp10-16 inhibition and we will optimize that accordingly.

The structure of the paper and the language needs quite a lot of work to bring it to the expected quality.

We will go through the manuscript again and further improve the structure and language as much as possible

Technical point:

Refinement of crystallographic occupancies to single digit percentage is not normally supported by electron density.

We agree with that point and correct it in the revised version.

Once one is prescribed the drugs it is very difficult for them to withdraw as they are now accustomed to the feeling it gives them

DOI: 10.1016/j.celrep.2024.114514

Resource: AB_2836579

Curator: @scibot

SciCrunch record: RRID:AB_2836579

What is this?

DOI: 10.1016/j.ebiom.2024.105243

Resource: CVCL_5J15

Curator: @scibot

SciCrunch record: RRID:CVCL_5J15

What is this?

o que é?

Quanto ao

requisitados no mundo inteiro

um campo de batalha dos mais violentos

Por um lado, o restante do programa era, de antemão, impossível de ser aplicado, por outro lado,a

faz sua an[alise para o Coletivo Brasil

DOI: 10.1016/j.ebiom.2024.105212

Resource: (ECACC Cat# 05092802, RRID:CVCL_1045)

Curator: @scibot

SciCrunch record: RRID:CVCL_1045

What is this?

DOI: 10.1016/j.celrep.2024.114502

Resource: (SouthernBiotech Cat# 2072-02, RRID:AB_2795767)

Curator: @scibot

SciCrunch record: RRID:AB_2795767

What is this?

DOI: 10.1039/D4PM00054D

Resource: University of North Carolina at Chapel Hill Flow Cytometry Core Facility (RRID:SCR_019170)

Curator: @scibot

SciCrunch record: RRID:SCR_019170

What is this?

DOI: 10.1101/2024.07.08.602586

Resource: DESeq2 (RRID:SCR_015687)

Curator: @scibot

SciCrunch record: RRID:SCR_015687

What is this?

DOI: 10.1016/j.celrep.2024.114461

Resource: RRID:Addgene_70111

Curator: @scibot

SciCrunch record: RRID:Addgene_70111

What is this?

Como se hablo en clase, creo que la creatividad es el inicio de una escritura que emociona al lector, sin creatividad no podriamos cautivar la atencion ni el interes de unlector, si creamos un texto a base de imaginacion, podemos hacer verdaderas obras de arte, cuentos magicos, historias envolventes, podemos plasmar nuestras emociones y cada persina que lee, se personifica en cada palabra escrita y en la forma tan creativa que se daria

Es tan cierto como escritores como Vargas Llosa (2001 nos hacen caer en cuenta en como nos hemos acostumbrado solo a escribir mensajes de texto en vez de volver ha escribir cartas en las cuales podemos expresarnos emociones y pensamiento los cuales son muy dificles de expresar o talvez en adentrarnos en leer llibros que nos puede hacer imaginar otras mundos y realidades, mandamos de un 1000000 de mensajes al dia pero no somos capaces de leer un libro escribir algo a mano es por eso que hoy en dia prefieren darle a un niño un celular en vez de un cuaderno o un libro esto causa muchos problemos debido a que genera falencias en el aparendizaje y concentración por que los niños se enfocan en lo artifical a pensar en otras cosas.

Se refiere a que debemos tener la disposición y la motivación para iniciar nuevas ideas, proyectos o acciones sin esperar a que otros te digan qué hacer. Implica ser proactivo y estar dispuesto a tomar la iniciativa para explorar nuevas posibilidades, experimentar con diferentes enfoques y buscar soluciones innovadoras.

Este documento es muy interesante por que nos hace reflexionar y a su vez entender sobre la escritura en el aula de igual manera nos hacer ver como el ser humano es capaz de trasmitir emociones y sentimientos atravez de una escritura de hecho como es capaz de compartir sus pensamientos en una simple o compleja escritura es más si hoy en día lo replicaramos como en la antiguedad es más esta lectura nos caer en cuenta de tan importante que es fomentar la escritura creativa en los alumnos sobre todo nos ayuda a nosotros que vamos ha ser futuros maestros y as u vez nos reclaca lo negativo y positvo de las tecnologias o TICS en la educación pero a su expresando y proponiendo métodos efectivos para fomentar la escritura creatividad, partiendo de lo simple a lo complejo y permitiendo a los estudiantes escribir desde sus intereses pero sobre todo destacar que no debería ser por obligación sino por placer, por gusto por que les nace y les apasiona o como forma de expresar ,desahorgarse ante alguna situación .esto ayuda a cambiar el enfoque educativo hacia la creatividad e imaginación. El objetivo de nosotros como futuros docentes es transformar la educación y motivar a los estudiantes o alumn@s a disfrutar del proceso de escritura como una manera de desahogarse o expresar su vida no como una obligación por una nota si un simple placer por escribir y brindar información que para otros podria ser una salvación.

Acerca de este texto trata de decir que la escritura es considerada como una herramienta donde podemos expresar ideas, pensamientos, sentimientos o la información y debido esto es que nos podemos comunicar con otros. Debido a esto la escritura no debe ser vista únicamente como una actividad que se realiza por si misma, en tanto se puede decir que la escritura su principal objetivo es transmitir algo significativo y conectar con los demás.

La escritura creativa busca generar nuevas ideas, textos originales que pueden verse relacionadas con la poesía, la novela, el ensayo y otros textos que requieren de un pensamiento creativo.

En las ideas que plasma Álvarez, me doy cuenta de que la escritura creativa es un proceso artístico complejo que va más allá de la simple redacción. Reconozco que el desafío que implica transformar pensamientos y emociones abstractas en palabras concretas, especialmente ante la intimidante página en blanco. Me llamo mucho la atención que este acto de narración se considere un arte, ya que se eleva la escritura creativa a un nivel que trasciende la comunicación y abarca la expresión multidimensional en las ideas, sentimientos y experiencias personales.

Corrales (2001) explica que leer y escribir son habilidades importantes porque nos ayudan a entender el mundo y hablar sobre él, desde mi punto de vista esto es muy útil para mejorar nuestra escritura creativa, leer nos muestra diferentes formas de pensar y escribir, mientras que escribir nos permite expresar nuestras propias ideas sentimientos abriendo esa puerta para poder dar nuestra opinión o lo que nosotros creemos de algo, juntas, la lectura y la escritura nos ayudan a ser más creativos y pensar de manera crítica logrando que un día seamos los que con nuestra forma de pensar y expresarnos cambiemos el mundo en el que vivimos .

Este párrafo me intereso mucho ya que la importancia de su enfoque práctico en las Nuevas Tecnologías, considero que es acertado por su relevancia en el entorno actual del alumnado y su creciente presencia en el sistema educativo. Valoro positivamente esta consideración del contexto tecnológico de los estudiantes, ya que puede aumentar su interés y motivación. Sin embargo, tengo varias cuestiones sobre los desafíos que esta integración tecnológica puede suponer, como la formación del profesorado o la posible brecha digital entre estudiantes. A pesar de estos retos, veo en este enfoque una oportunidad para desarrollar formas de enseñanza y aprendizaje más dinámicas e interactivas, lo cual me parece una evolución necesaria en el ámbito educativo actual.

La escritura en el ser humano es, sin duda, importante. Cuando comencé a escribir, pude expresar mis sentimientos y pensamientos, siempre motivada para hacerlo. Hoy en día, muchos niños se limitan en la escritura debido a varios factores; uno de ellos puede ser la presión ejercida por los docentes. Para que un niño pueda producir un texto escrito, debe hacerlo sin ningún tipo de presión; de lo contrario, lo hará mal o simplemente no lo hará. Por lo tanto, como futuros docentes, debemos motivar a nuestros estudiantes a adentrarse en el maravilloso mundo de la escritura.

La escritura creativa se encuentra en una estrecha relación con los géneros literarios tradicionales. Si bien no se considera un género en sí misma, aporta un conjunto de herramientas y técnicas valiosas que enriquecen y potencian la expresión en diversos formatos literarios.

En el ámbito académico la escritura permite que los estudiantes se puedan comunicar, organicen sus pensamientos e ideas de manera más efectiva para mejor comprensión de sus compañeros y maestros. En el contexto familiar, la escritura puede facilitar la expresión de emociones, pensamientos y experiencias personales entre los miembros de la familia, escribiendo cartas o diarios se mejora la comunicación dentro del hogar y así se crea un espacio seguro para expresarse verbalmente.

Escribir no se trata de llenar páginas con palabras o frases dichas por alguien más ya que el escritor tiene la necesidad de expresar algo que realmente sea de su interés, que lo impulse a compartir sus ideas, sentimientos o experiencias con el mundo es importante la investigación que realizó,para empezar a escribir es necesaria la motivación y las ganas de empezar a través de la escritura y la lectura.

Me gusto mucho este documentoporque nos habla sobre cómo la escritura creativa puede resultar aburrido porque desde pequeños nos enseñan a escribir de manera muy monotona y tambien nos hacien aprender muchas reglas ortográficas, estas reglas a veces hace que no veamos la escritura como algo que nos permite expresar nuestras emociones o potenciar nuestra creatividad al máximo, en en este documento nos señala de manera muy importante que el problema es de ¿cómo enseñamos desde el principio? nostros como futuros docentes debemos utilizar métodos más divertidos en las clases, como inventar cuentos, realizar un club de lectura donde cada niño vaya llevando un libro de su mayor gusto, es una idea genial para hacer que la escritura sea más divertida y menos complicada.

Escribir me ha permitido expresar lo que siento y dejar volar mi creatividad, sin embargo, muchos estudiantes no quieren escribir en clase, aunque enseñar a comunicarse de manera escrita y oral es una prioridad en la escuela, como futuros docentes debemos sentirnos influenciados por cumplir con los objetivos para ayudar a los estudiantes a tomar gusto por escribir

La escritura surge de la necesidad de comunicar algo, de tener un mensaje, una historia o una idea que compartir. Es importante tener algo significativo que transmitir para poder plasmarlo en palabras.

En este documento de trabajo final he podido apreciar como el autor mediante la escritura nos atrapa de una y mil maneras poniéndonos a reflexionar de una u otra forma. En esta parte, me hace recordar la manera en la que aprendí a leer y a escribir, y como bien lo dice; tradicionalmente, la escuela cometía el error de concebir la escritura como un conjunto de reglas ortográficas que los alumnos debían memorizar, en mi ciclo escolar tuve la oportunidad de haberme encontrado con docentes que les apasionaba la parte literaria, en tercero o cuarto año de básica me encantaba observar cómo mis docentes nos promovían la parte lectora porque cuando ellas nos leían un cuento lo realizaban de una manera en la que uno como estudiante se impresiona y mágicamente vuela a esa lectura, dejándose llevar por la imaginación, ya más adelante la parte de escritura mi docente nos atrapaba fácilmente en el mundo de la poesía, lo cual era cautivador escuchar sus proclamaciones. Ya más tarde y como toda la parte mágica se volvió una parte obscura me tope más adelante con docentes que lamentablemente solo se preocupaban de dar su clase y ya, que a uno como estudiante lo que más importaba era obtener esa buena calificación como a dé lugar, y tengo que admitir que llegue a un punto en el que sentía miedo de expresarme de manera escrita y mucha más de manera verbal, porque para bien o para mal sentía que no era lo suficientemente buena en este ámbito. Dejé a un lado la parte de escribir, esto tiene que ver con el modo en que concibe la escritura en el colegio, ya que solo encontré presión. Pero aquí viene la parte interesante que comprendí con este documento, “la escritura” es la idea de verse como un medio de expresión y comunicación y no como un fin en sí mismo. Entender que es la parte creativa que tiene el ser humano y que como futuro docente se debe saber ser creativos, también para que los estudiantes puedan generar de buena manera el ámbito de transmitir sus conocimientos, ideas y pensamientos ya sea de manera oral o escrita con el fin de que esto no se pierda y pueda trasmitirse sin ningún miedo o presión.

Pienso que la educación es clave para desarrollar nuestras habilidades. Es súper importante hacer actividades que trabajen ambos hemisferios del cerebro,. Esto nos ayudara a ser más creativos y también a pensar de manera lógica y critica y pienso que una buena manera de estimular los hemisferios de nuestro cerebro es con la lectura

me pareció super interesante , creo que el documento toca un punto muy importante: la escritura creativa en los niños. Es súper esencial que como futuros docentes motivemos a los estudiantes a que se expresen y comuniquen de manera critica o por ellos mismos. Planificar bien y crear un ambiente seguro, sin presiones, es clave para que los niños vean la escritura como algo valioso y no una tarea pesada. Este enfoque puede realmente ayudar a mejorar sus habilidades de escritura y su actitud hacia ella. m gusto mucho ya que me ayudo ha descubrir nuevas técnicas que espero ponerlas en desarrollo en momento de trabajar con los niños

Existen tres enfoques diferentes para aprender a escribir: el enfoque léxico, que se centra en aprender palabras con apoyo visual; el enfoque fraseológico, que se basa en escribir frases dictadas o leídas por el maestro; y el enfoque contextual, que implica escribir una historia que ya ha sido contada.

Es muy interesante lo que el texto resalta la importancia crucial de dominar las habilidades fundamentales de interpretación y redacción inicial para cualquier persona, Estos tres enfoques ofrecen herramientas variadas para desarrollar ideas de manera espontánea y creativa, esto ayudar desglosar el contenido para identificar patrones y relaciones, y combinar ambos enfoques para equilibrar la innovación y la estructura. Esta clasificación no solo guía el proceso de escritura, sino que también enfatiza la importancia de una formación estricta y versátil para lograr una comunicación efectiva y significativa en lo académico o profesional.

Me parece muy interesante como habla sobre una definición de la escritura creativa y de como es de importante en el desarrollo emocional y cognitivo de los niños y en una parte práctica habla de propuestas didácticas sobre la escritura creativa las cuales son sacadas de un blog llamado EscCreativa dirigido a docentes, estas buscan fomentar la creatividad de los niños y mejorar sus competencias lingüísticas ayudados de técnicas innovadoras y nuevas tecnologías para las aulas. Con esto los profesores quieren despertar en los estudiantes su imaginación para escribir libremente para que vean la escritura como algo artístico y de libre expresión mas no como algo que hacer bajo presión o como castigo.

En el documento se nos presenta la escritura creativa y me parece muy novedoso, ya que, conforme se va leyendo el texto nos menciona como nosotros a futuros docentes deberían de practicar esta escritura creativa, debido a que también permitiría potenciar a los estudiantes. por que al tener esta escritura creativa nos pèrmitira ser mas abiertos y poder escribir ideas que sean claras y entendibles para nosotros mismos y así como docentes ser creativos también para buscar la manera en que la escritura llegue ha ser 'parte de los estudiantes por que es cierto que es muy difícil escribir pero si lo volvemos un hábito será mas fácil escribir, escritos académicos o simplemente para nuestra liberación de mente o pàra nosotros mismos. También se nos habla lo difícil que es para los docentes en la actualidad implementar la escritura creativa ya que aun se tiene una relación que con que el estudiante pueda leer será suficiente y dejan de un lado la importancia que es también saber escribir.

Es cierto que muchas veces los docentes solo le limitan a que los alumnos aprendan a leer pero no escribir y como bien se nos ha venido mencionado en todo el articulo el poder escribir es una forma de liberarse o una manera de expresión así de esta forma como futuros docentes debemos buscar técnicas, metodologías y sobre todo ser creativos para poder impulsar la manera la lectura y la escritura en nuestras aulas y ser unos profesores de cambio y llegar a profundizar en los estudiantes con cada cosa que ellos aprendan.

La escritura en sí es importante por diferentes motivos, la comunicación, transmisión de ideas, perpetuar estas misma, pero sobre todo nos hace reflexionar y crear desde nuestro ser. En la escuela escribir es esencial, por ello nos lo enseñan, no solamente para copiar lo que vemos sino para que nuestra mente pueda producir textos propios de nuestro pensar si bien escribir no es tan fácil como hablar o pintar, es un arte que también transmite y tiene que ser hecho con un pensamiento crítico.

Muchas veces tomar consciencia de mis errores y aciertos es difícil, ya que, solo nos gusta tomar en cuenta lo que hacemos bien, pero sin embargo como bien menciona el texto debemos o tenemos que fijarnos en nuestros errores para ser creativos por que de los aciertos no aprendemos mucho, mientras que con los errores miramos que hacemos mal y nos permite tener una visión más grande de todo lo que estamos haciendo así muchas veces de los errores se llega aprender mas que de los aciertos.

El escribir debe ir más allá de solo poner palabras por eso es que escribir debe ir con sentimientos o pensamientos abstractos para llegar a tener una idea clara y que no sea desmeritada del propio así es como el mensaje que deseemos impartir también debe de tener un sentido y creatividad para llegar a todas las personas

El documento me pareció bastante interesante, ya que destaca cómo enseñar la escritura creativa nos permite brindar a los niños de mejor manera. Asimismo, en cómo brindarles la oportunidad de poder expresar ideas de manera auténtica a través de los escritos que realicen. Es fundamental despertar el interés en la escritura en los estudiantes, ofreciéndoles oportunidades donde puedan expresar su imaginación, sus ideas, el lenguaje, entre otros. Para lograr una buena organización en la enseñanza de la escritura, es necesario incluir una planificación en la cual se generen ideas de manera más organizada. Por tanto, la escritura creativa va más allá de utilizarla como técnica; nos permitirá superar problemas en el momento de la enseñanza, evitando que sea algo riguroso o utilizado como una presión por cumplir impuesta por la institución educativa.

La escritura es la que permite hacer distinguir esas cualidades y rasgos, al escribir mostramos nuestros sentimientos y las palabras que utilizamos son el principal sello para saber quien escribió tal texto. Cuando escribimos tenemos una idea de la cual nos des playamos para dar a conocer, no todos vana escribir lo mismo sobre esa idea, lo que nos da a entender que cada persona piensa distinto y por tanto lo que escriba va ser el reflejo de su mente e ideologías que conlleva.

Duclaux, nos enseña en lo bonito que es la escritura y no solo hay un tipo de escritura en la que podemos agregar ideas de nosotros mismo, ideas de las que observamos etc

Esta técnica permite dirigir el pensamiento crítico y la imaginación porque cada persona se siente libre de expresar lo que piensa sobre una ilustración obteniendo diferentes puntos de vista y generando aprendizaje significativo al leer los demás comentarios.

Mediante la escritura se puede redactar opiniones, pensamientos, expresar emociones, deseos, anhelos donde cada ser humano escribe desde su entorno y promueve la diversidad de escritura y libre pensamiento.

No se trata de llenar páginas con palabras vacías o frases dichas por alguien más. El escritor tiene la necesidad de expresar algo que realmente sea de su interés, que lo impulse a compartir sus ideas, sentimientos o experiencias con el mundo. El algo que decir, puede ser una historia, una reflexión profunda, o el punto de vista de una investigación que realizó. Para empezar a escribir es necesaria la motivación y las ganas de empezar con la fascinante aventura de transportarse a través de la escritura y la lectura.

La idea de usar un blog en la enseñanza es muy importante. Un blog proporciona: un lugar donde la educación puede continuar de manera informal, donde los estudiantes pueden practicar la escritura de una forma auténtica y significativa. Además, nos ayuda a la comunidad colaborativa, donde cada comentario, es una crítica constructiva o bueno eso sería lo más ideal. Mejorando las habilidades de escritura, el pensamiento crítico y la capacidad de comunicarse. Los blogs son una herramienta que puede transformar la forma en que vemos la educación.

Para impulsar la creatividad nos podríamos permitirnos personalización de la enseñanza de la escritura para permitir a los alumnos expresar sus ideas y sentimientos muy importante en su proceso educativo. Al adaptar los métodos de enseñanza a las necesidades e intereses de cada uno los estudiantes, se les brinda la oportunidad de desarrollar su creatividad, expresarse auténticamente y generar un interés por la escritura y lectura. Así fomentaremos el desarrollo de habilidades lingüísticas, el autoconocimiento, creando un buen ambiente educativo.

El tema que se aborda me pareció muy importante y me lleva a pensar que como futuros profesores deberíamos interesarnos más en saber sobre esto ya que es un tema de gran utilidad y relevancia en la escuela, en el texto nos habla de que la escritura es algo muy importante para nosotros, ya que conforme hemos ido evolucionando esta se ha convertido en un medio de comunicación y no solo eso a través de esta también podemos expresarnos. Tiene mucha razón el texto cuando dice que muchas veces la enseñanza de la escritura simplemente se basa en saber las reglas que deben cumplir, o ver quien de todos escribe correctamente, sin duda es útil, pero lo que ganamos con esto es dejar completamente a un lado que los niños puedan escribir creativamente, que puedan desarrollar su habilitad creativa, la escritura nos ayuda en muchas cosas, nos enriquece, permite que exploremos y hagamos brillar esa imaginación que tenemos, contribuye a una capacidad de reflexión y un bienestar emocional. En el texto incluye las tics lo que me parece interesante, ya que de cierta manera las tics pueden afectar en una escritura libre y creativa, pero estamos ante una época más moderna en donde hay infinidad de recursos didácticos tecnológicos que podemos integrar en nuestras metodologías para enseñar, esto permitirá dejar a un lado el método tradicional y suplantarlo por un proceso de escritura más dinámico. Como futuros docentes me parece que debemos motivar y promover a nuestros estudiantes a que escriban y debemos ser los principales responsables de hacer que no solo aprendan a escribir correctamente y seguir reglar, más bien impulsar y promover su imaginación para que descubran el poder de expresión escrita.

Esta frase se refiere a que si en la escuela únicamente se enseña a los niños a leer y escribir sin ningún otro fin, ellos luego no tendrán la capacidad para poder comprender información, expresar sus pensamientos o emociones. Es por eso que para que esto no quede en algo pasajero debe ir mas allá, deben incluir la aplicación práctica en contextos reales, así los niños tendrán habilidades como pensamiento crítico y ayudara en su interacción de su vida diaria.

En este apartado nos deja en claro que la escritura además de ser algo creativo también debe centrarse en reglas gramaticales, por ejemplo la ortografía, la sintaxis, entre otras. Así podremos asegurar que la escritura sea mas adecuada según el contexto. Aplicando esto en cierta parte estaremos dejando aun lado la creatividad, la forma de poder expresarnos en forma personal, o tal vez ya no podamos escribir idea que sean mas originales, pero nosotros debemos optar por tener una escritura que sea creativa pero no solo eso sino que también cumpla normas y reglas.

Desde mi punto de vista deberíamos fomentar la creatividad en la escuela por que si bien es cierto esto impacta en el desarrollo de los estudiantes, además esto ayudara al pensamiento crítico, lo que ayuda a los niños poder crear o imaginar ideas que sean innovadoras. Al fomentar esta creatividad estamos enriqueciendo la escritura por que a traves de su creatividad los niños podrán escribir textos interesantes, para mi esta creatividad permitirá que el proceso de escritura sea mas gratificante.

La escritura como herramienta para el desarrollo de la expresión tiene un impacto significativo en el desarrollo personal de los estudiantes, permitiéndoles analizar información y expresar sus propias ideas, estructurando sus pensamientos de manera lógica. Escribir sobre varios temas les permite ampliar su conocimiento y plasmar historias o cuentos propios del escritor. Por lo tanto, es importante que los estudiantes vean la escritura como una herramienta de liberación o expresión y no como una obligación.

Creo que combinar los recursos de los métodos sintéticos y analíticos es una estrategia muy efectiva. En vez de centrarse solo en el proceso de lectura, estos métodos buscan darle un verdadero significado, lo que puede potenciar la escritura creativa. Al comprender mejor lo que leemos, no solo estamos memorizando palabras o frases, sino que realmente estamos captando su sentido. Esto hace que la lectura sea más interesante y enriquecedora, y nos permite aplicar lo aprendido de manera más creativa en nuestra escritura. Considero que esta combinación de métodos puede transformar el aprendizaje en una experiencia más dinámica y efectiva.

Es fundamental implementar la planificación dentro de las técnicas de escritura, ya que planificar nos brinda la oportunidad de tener una base bien estructurada al momento de organizar nuestras ideas o pensamientos, determinar propósitos, establecer las ideas en orden lógico, etc. Además, sin una adecuada técnica de organización, la escritura puede tornarse un tanto frustrante para el escritor, ya que el resultado que se espera conseguir al final no va a ser el adecuado.

Es importante que el escritor, en este caso el estudiante, produzca un texto con inspiración y de acuerdo a su interés. Es ahí donde se desarrollará la verdadera imaginación y se sentirá a gusto escribiendo.

Una parte fundamental es que en este trabajo la ejemplificación clara de como la lectura y escritura en muchos casos llega a ser muy cansada y estresante tanto para los docentes como para los estudiantes cuando no se aplica metodologías eficaces par poder transmitir y promulgar de forma eficiente esta importante e indispensable herramienta del ser humano capaz de llenar y trasmitir muchos aprendizajes significativos en la mente humana, sin embargo a partir de esta reflexión muestra diferentes propuestas a lo cual yo mencionare como herramientas tan eficientes al momento de aplicarlas que pueden llevar a un aprendizaje significativo en los estudiantes, como por ejemplo se encontraba muy bien ejemplificada la motivación al estudiante, el impulso y desarrollo de creatividad infaltable en el ser humano, la creatividad las herramientas las metodologías están allí la diferencia esta en nosotros si les damos un buen uso o simplemente nos encerramos a cumplir lo mínimo posible por humanos que necesitan de un guía.

Es interesante como actualmente el tradicionalismo aun posee fuerza en muchas instituciones educativas, siendo que los principales promotores sean los docentes y queda mas claro aun porque muchos de ellos no son docentes que se actualicen de manera regular porque no conocen muchas técnicas actuales de enseñanza que son mucho mejores que las que se utilizaban anteriormente.

La creatividad es un elemento podría mencionar privilegio del ser humano, porque es tan único y propio de el lo cual a permitido que este llegue a progresar en un sin fin de escenarios tanto espirituales como estructurales o sentimentales entre muchos mas, siendo mas precisos en este tema la creatividad debe de ser motivada constantemente en los estudiantes con el fin de fomentar un aprendizaje progresivo para el estudiante, donde el principal, objetivo es llegar a un aprendizaje significativo.

El ser humano es un ente social, por ende requiere dar uso a como su termino bien mencionado una de sus herramientas, motivado por la necesidad de transmitir sus conocimientos, transmitir sus ideas y pensamientos ante otros de manera oral o escrita como métodos para plasmar dicha información con el fin de que esta no se pierda y pueda trasmitirse.

Aprender a leer y escribir no se reduce únicamente a dominar la técnica; es un proceso que nos abre las puertas al conocimiento, nos conecta con otras mentes y culturas, y nos capacita para expresar nuestras ideas y emociones de manera profunda y significativa.

La creatividad no solo eleva la autoestima personal y promueve la felicidad y la salud psicológica, sino que también genera beneficios profesionales y económicos significativos. Socialmente, aporta ideas innovadoras que mejoran el bienestar colectivo, desde expresiones artísticas que enriquecen nuestra vida diaria hasta soluciones prácticas que salvan vidas. En todos los aspectos, la creatividad es una fuerza transformadora que impulsa el progreso individual y global.

El descuido hacia la fase de la planificación es muy evidente. Las etapas de escritura y revisión suelen recibir la mayor atención en las aulas, la planificación previa a la redacción es a menudo relegada a un segundo plano.

En el panorama educativo actual, marcado por la creciente digitalización, el blog emerge como una herramienta invaluable para acercar a los docentes estrategias y técnicas de escritura creativa. Esta plataforma accesible y versátil brinda a los maestros un espacio idóneo para acceder a una amplia gama de recursos, fomentando así el desarrollo de habilidades creativas en sus estudiantes.

La escritura creativa es muy importante y los maestros deben apoyar y desarrollar esa escritura en los estudiantes desde que son muy pequeños para que tengan la facilidad para escribir y expresar las ideas, pensamientos, emociones a través de textos narrati8vos, ensayos o un en un diario para que así en un futuro no tengan dificultad para expresarse ante los demás. Los docentes deben buscar diferentes formas, metodologías, didácticas como nos mostraba en los ejemplos en el documento de los acrósticos, tormenta de ideas, juegos de palabras que pueden ser muy útiles, y aun mas ejercicios para desarrollar los dos hemisferios del cerebro para empezar a realzar la habilidad de la escritura creativa. Para la escritura creativa no es necesario saber todas las reglas para escribir profesionalmente sino que se debe planificar las ideas y plasmarlas en textos.

El texto resalta cómo la escritura creativa se ha consolidado como una tradición académica en muchos países, integrándose en centros de Educación Secundaria y universidades desde los años sesenta. Sin embargo, en países como Francia, España y Argentina, la escritura creativa ha estado al margen de la enseñanza reglada. Esto refleja una diferencia en las prioridades educativas y tradiciones culturales, donde se ha privilegiado el análisis crítico de la literatura sobre la creación literaria, limitando así el desarrollo de habilidades creativas en los estudiantes. Incorporar la escritura creativa en estos países podría enriquecer su educación literaria, fomentando la creatividad y una comprensión más profunda de la literatura.

En la Educación actual sabemos que el cambio dramático de la educación ,llevo a desembocar algunos problemas como los mencionados y un claro ejemplo son los docentes los cuales al verse envueltos en esta situación optaron por abandonar sus puestos debido a que en ningún momento pensamos que hubiese este cambio debido a la pandemia . Lo que propone este documento propone algunas metodologías y propuestas para que los docentes puedan adaptarse en diferentes situaciones como las nuevas tecnologías diferentes ambientes del aula y demás. Por ende es muy importante que los profesionales de la educación salgan mejor preparados para todos los obstáculos que se le presenten en su vida profesional Y sobre todo incentiven a sus estudiantes a técnicas de lectura y así obtener un aprendizaje significativo.

Este artículo científico nos presenta una problemática que es causante de que la escritura creativa sea algo tedioso y que muchas veces se nos dificulta hacerlo, ya que todo viene desde nuestra formación académica en donde la escritura es algo sistemático y cotidiano con reglas ortográficas que hay que cumplir, causando que no vean a la escritura como una herramienta terapéutica que permite el desahogo de nuestras emociones o como también la potenciación de la creatividad, es por eso que todo parte de la enseñanza de la lectoescritura en donde se debe hacer uso de varias estrategias didácticas y pedagógicas, como también el uso de la lúdica, canciones, narración de cuentos que les llame la atención o crear cuentos con ellos, además de dejar a un lado métodos tradicionalistas que promueven la memorización que no fomentan el aprendizaje significativo que permite la aplicación de lo desarrollado en el salón de clases en su vida cotidiana esto irá relacionada con la producción de textos de manera recreativa y creativa a través de la planificación, organizando las ideas o temas que se quiere llegar a escribir siendo este uno de los pasos más complejos de lograr, ya que el currículo en muchas ocasiones no se centra en la potenciación de la creatividad e imaginación, puesto que se enfoca en dar cumplimiento a la creación de textos académicos con reglas sintácticas, sintaxis y tiempo establecido. Es por eso que también se debe potenciar la imaginación en los estudiantes, a través de la construcción de oraciones, o completar las frases dando palabras cables, la narración de cuentos pictográficos, poemas, llevar a cabo debates, conversatorios en el aula de clases, para ello el autor de este artículo nos presenta una novedosa página web, la cual se encuentra ordena por secciones o necesidades que el docente debe cumplir en sus estudiantes para promover en ellos la escritura creativa.

Me parece muy bueno el tema de la autora nos dice conceptos y también nos brinda ideas y maneras de como implementar la escritura creativa en una aula, nos habla de la importancia de saber los conceptos tanto de la escritura como de la creatividad, en resumen puedo decir que es trascendental incrementar este tipo de creatividad dejando a los niños fluir con sus palabras , sin limitaciones o privándolos de escribir algo que a ellos de verdad les gusta. En esto también se ve incluida la escritura creativa de forma digital ya que a si nos estaríamos adaptándonos a una nueva realidad porque no es la misma educación ahora que hacer 10 o 100 años y eso se debe reconocer muy bien en la educación.

Este documento me pareció súper interesante ya que resalta la importancia de fomentar la escritura creativa en los chicos. Hay que recalcar lo negativo que se han vuelto las TICS dentro de la escritura y lectura en el sistema educativo. Esto les limita la imaginación y creatividad al momento de escribir, si esto disminuyera los resultados al momento de realizar o generar escritura fueran diferentes. Por otro lado los métodos expuestos para realizar una escritura creativa son muy efectivos, empezando desde lo más simple a lo complejo. Los textos a generarse por los estudiantes deben ser partiendo desde su interés, experiencia y gusto por algún tema en específico y no limitar. Quiero mencionar algo muy importante que dice el texto y es sobre el no solo crear estos espacios de escritura creativa dentro del sistema educativo sino que se creen ambientes fuera de esto como algo extracurricular podría decirse y que también la creación de estos textos no sea por una nota o un mérito sino lo hagan como un gusto, un desahogo y como no también como una distracción. El escribir nos cambiará la vida y esto quiero implementar como futuro docente con mis estudiantes para cambiar ese chip de la educación y fomentar creatividad e imaginación, que no es que no exista, solo que hay que saber fomentarla y sacarle provecho.

La escritura es muy compleja ya que depende de la imaginación pero también tiene que tener coherencia y sintaxis para lograr la comprensión de otros lectores a futuro claro si es un texto público o la persona que lo vaya a leer.

Es uy importante entender que no debemos enseñar de la misma manera en la que nos enseñaron a nosotros porque son aproximadamente ya una década o más y ese es un error que muchos profesores cometen, es por eso que debemos implementar nuevas estrategias, nuevas maneras para así lograr ser maestros innovadores y para lograr esto si o si debemos incrementa la creatividad en las aulas y para lograr esto debemos darle espacio a los niños e imaginar, de creer y de escribir; los niños son niños, llenos di imaginación queriendo ser expresada y no debemos encerrarlos en una caja de cartón y decirles escriban de este tema o de aquello ya que si a este niño no le gusta dicho tema simplemente investiga y copia y no es lo que los profesores quieren con sus estudiantes; así que dejemos volar la imaginación de los niños y permitamos que el sea el protagonista de lo que escribe.

En este documento nos da a conocer que la escritura es importante en todos los aspectos ya sea para comunicarnos o expresar lo que sentimos y que presenta unos desafíos en la edad moderna al enseñarse con métodos tradicionales, donde los estudiantes no logran desarrollar esas habilidades creativas y expresivas, pero en este documento también se encuentra la solución el cual es hacer el uso de las TICS para que estos se motiven y les llame la atención aprender sobre la escritura.

Es la realidad, esto pasa en el sistema educativo ya aún existen algunos docentes que solo se guían en lo ya establecido, es decir, el currículo. Sería crucial que se creen espacios y se fomente la escritura no solo en el aula sino también en el aula de clases.

En esta frase hace referencia al proceso que tiene un escritor, las ideas que salen de su inspiración en momentos donde dominan las emociones o una mente clara y así, terminar redactando en un papel.

Siento que esta es una de las principales problemáticas causantes de que se vea a la escritura como algo sistemático y cotidiano, incluso es por eso que se a perdido la costumbre de leer novelas o diferentes textos creativos, la escritura trasciende mucho mas que una simple acción mecánica con reglas sintácticas que hay que cumplir, si no que esta nos ayuda en varios aspectos emocionales, psicológicos y creativos.

El texto nos menciona algo importante acerca de la escritura, ya que nos dice que es una herramienta esencial para el ser humano, ya que nos permite una comunicación perdurable y versátil, siendo una necesidad que se manifiesta en diversas formas para expresar, desahogarse y conectar con los demás a través de cartas, poesías, canciones y entre otros.

Es interesante como se destaca la escritura, como una herramienta fundamental en la expresión de nuestras emociones y creatividad. Sin embargo, me parece preocupante que muchos estudiantes se muestren reticentes a escribir en el ámbito escolar. Esto podría deberse a una falta de motivación o a la presión que sienten por cumplir con estándares académicos. Es crucial que los docentes encuentren maneras de fomentar el interés por la escritura, creando un ambiente más libre y creativo donde los alumnos se sientan cómodos expresándose.

El docente sabra en que momento utilizar diferentes teçnicas o formas de enselanza que le ayude a transmitir sus conocimientos para que asi pueda crear un aprendizaje significativo ademas exiten diferentes formas o tecnicas de enseñanza que le permitan genenar ese aprendizaje significativo en el texto nos narra diferentes maneras metodologias acorde a la edad para que asi los estudiantes acorde a su edad.

Realmente como futura maestra me hace un llamado a prepárame en todo aspecto literario, tomando en cuenta tener un pensamiento realmente con gran creatividad, buscando diferentes ideas para crear estrategias para adquirir en mis proximos estudiantes un hábito de escritura y lectura agradable e interesante

Me identifico profundamente con esto. La idea de hablar cara a cara con un terapeuta puede ser un poco incomodo, expresar mis sentimientos verbalmente se vuelve casi imposible

Escribir una carta en un "día bueno" para ti mismo puede ser una herramienta poderosa. En momentos de debilidad leer palabras de apoyo y comprensión que vengan de ti mismo te ayudará a encontrar fuerza y ánimo cuando más lo necesites.

En la actualidad la mayoría de personas no encuentra formas de expresión y este método ayuda a muchos adolescentes y personas que no tengan forma de asistir a un terapeuta el texto tiene toda la razón porque va a ayudar a aquellas personas que no son capaces de expresarse a escribir sus sentimientos sus emociones mediante la escritura mediante un diario personal que les va a ayudar a sentirse mejor y sí es una muy buena terapia Y como dice ahí puede ser la más poderosa pienso aplicar este método para mí misma.

Pues el texto tiene toda la razón ya que como lo menciona es súper importante que se exprese emocionalmente la persona que escribe y así logre una paz interior gracias a esta escritura terapéutica y me parece súper interesante que no tenga forma que no tenga parámetros para escribir que no sea basado en ningún reglamento sino en Cómo pensamos y en cómo nos expresamos cada una de las personas que elegimos escribir así para sanarnos emocionalmente.

Me parece muy interesante este método de utilizar la escritura terapéutica pienso que algunas personas no somos capaces de expresar nuestras emociones delante de alguien que nos quiera ayudar como un terapeuta pero si tal vez lo integramos como dice en el texto a utilizar esta escritura para ayudarnos a nosotros mismos y poder superar nuestras dificultades emocionales y también entender de un modo diferente las cosas y a las personas pienso que este método puede ayudar mucho a las futuras generaciones porque vivimos en un mundo rodeado de tecnología y es verdad que a veces no tenemos tiempo de agendar una cita con un terapeuta o a veces no nos animamos por miedo a decir que tal vez Estamos locos pero es una buena forma de ayudarnos a nosotros mismos y ayudar a las personas que nos rodean ya que si estamos nosotros mal emocionalmente también afectamos a las personas que también nos quieren implementar esta estrategia sería muy beneficioso en mi vida.

Como menciona el texto, se ha demostrado que la escritura terapéutica es eficaz en la recuperación de personas que enfrentan problemas de salud mental como la depresión o el trastorno de estrés postraumático, también es importante destacar que la escritura terapéutica puede ser especialmente beneficiosa para aquellas personas que encuentran dificultades para expresar sus sentimientos verbalmente, ya sea por ansiedad, angustia u otros motivos.

Este artículo trata sobre la escritura terapéutica y sus beneficios para la salud mental. Discute el uso de la escritura como herramienta para expresar emociones no expresadas o inexploradas. El autor describe el método del diario intensivo y otras formas de escritura expresiva. La escritura terapéutica puede ser útil para personas que sufren de depresión o trastorno de estrés postraumático. El artículo también menciona los beneficios de escribir cartas a uno mismo en los "días buenos" para leer en los "días malos".

La escritura, como lo menciona el texto en general, no siempre va a ser un método para transmitir hermosas poesías o los libros más largos llenos de conocimiento del mundo entero. Sino que a través de la escritura podemos expresarnos, dar a entender como nos sentimos, claro que no es lo mismo que hablar y que la otra persona vea los gestos que haces, pero las letras de alguna manera también pueden llegar a transmitir las emociones. No se necesita ser un experto literario para esto, como dicen por ahí se trata más de querer que poder. La escritura es una forma silenciosa de la comunicación, se dan ideas claras sin necesidad de hacerlo en voz alta, por ello utilizarla para quienes tienen terror de encontrarse con un terapeuta se convierte en una salida, no tan factible como el hablar en persona, pero es un comienzo para la mejora.

El escribir la carta de despedida me ayudó en muchos aspectos, pude decir adiós a una persona que nunca me pude despedir, el expresarme abiertamente me ayudó a desahogarme. A decirle cuánto lo extrañaba y la falta que me hace, que siempre fue un buen amigo, un gran compañero. Que me enseño lo valioso que es la vida, lo esencial de vivirla sin apuros, sino, más bien disfrutando de cada pequeña cosa que la hacen valiosa. Siento que escribir esta carta me ha servido de terapia para poder sanar el alma y expresar todo los sentimientos reprimidos que dentro de mi estaban.

En la escritura terapéutica, nos habla sobre que el objetivo de esta escritura no es crear una gran obra literario, mas bien se trata de que a través de las palabras escritas podemos desahogarnos, expresar lo que sentimos sin necesidad de hablar, lo cual ayuda mucho para las personas que no se sienten seguras hablando de como se siente.

Me llamo demasiado la atención este texto sobre la escritura terapéutica ya que como objetivo la escritura ha sido un medio para expresar nuestros sentimientos y emociones nuestras y de las personas que no han podido experimentar sus sentimientos o emociones y me incluyo yo por que hay momentos en los que no he podido expresar lo que siento ya sea por miedo o por el que dirá la gente y la escritura nos da un objetivo para expresarlo y tenerlo ocultos expresarlos en una silenciosa pero muy expresiva y ya que nos ayuda en el uso personal y bienestar emocional y sentimental.

Me llamo demasiado la atención este texto sobre la escritura terapéutica ya que como objetivo la escritura ha sido un media para expresar nuestros sentimiento y emociones nuestras y de las personas que no han podido experimentar sus sentimiento o emociones y me incluyo yo por que hay momentos en los que no he podido expresar lo que siento ya sea por miedo o por el que dirá la gente y la escritura nos da una objetivo para expresarlo y tener tenerlo ocultos expresarlos en una silenciosa pero muy expresiva y ya que nos ayuda en el uso personal y bienestar emocional y sentimental.

El poder de la escritura es un don o talento que mantiene vivo el expresarse abiertamente mediante las palabras, va mas halla de lo habitual ya que existe una accesibilidad de la escritura terapéutica, que brinda muchos beneficios para una mejor salud mental. Este articulo aborda la importancia de la escritura como una herramienta de autoexploración y expresión emocional ya que existen muchas formas de expresarse pero la escritura terapéutica forma nuevas fases de expresión en la vida mencionando mucho mas con su evolución y de su uso en la terapia moderna. La inclusión de simples ejemplos, como escribir cartas que ayudan en momentos difíciles, no solo aplicable para los lectores si no para todas las personas que quieren aprender a expresarse mucho mejor.

"Escritura Terapéutica: El Poder Sanador de la Expresión" probablemente aborda cómo la escritura puede ser utilizada como una herramienta para el bienestar emocional y mental. Los beneficios de la escritura terapéutica expresar pensamientos y sentimientos ayuda a procesar emociones, reducir el estrés y mejorar la salud mental. También nos habla sobre diarios, cartas no enviadas, o escribir sobre experiencias. Con esto ayuda a expresar el significado de la escritura terapéutica.

En esta lectura nos habla sobre la importancia de la lectura que esta es como una terapia y esta puede cambiar mucho en nuestro ánimo o estado emocional

En la lectura nos habla que la escritura como herramienta de expresión emocional es un recurso invaluable para el desarrollo personal, la autocomprensión y la sanación a través de la pluma o el teclado, nos embarcamos en un viaje introspectivo que nos permite conectar con nuestro yo más profundo, liberar nuestras emociones y construir un futuro más pleno y significativo.

Me parece muy interesante esta parte ya que, en el mundo, hay muchas personas que no expresamos nuestros sentimientos. Personalmente, creo que escribir es una manera muy buena de hacerlo, ya que nos ayuda a expresar lo que llevamos dentro. Además, nos permite articular pensamientos y emociones que, de otra manera, habrían permanecido ocultos. Es decir, la escritura nos ofrece un espacio seguro para explorar y liberar nuestros sentimientos.

Este texto muy importante y a la vez llamativo ya que nos habla sobre la escritura terapéutica la misma que nos ofrece un medio seguro para que las personas expresen y procesen sus emociones y experiencias. Este proceso de exteriorización puede aliviar el estrés y la ansiedad, proporcionando una salida constructiva para pensamientos y sentimientos difíciles, algunos estudios han mostrado que aquellos que practican la escritura terapéutica pueden experimentar una disminución en los síntomas de depresión y estrés postraumático, lo que sugiere que este método puede ser una valiosa herramienta complementaria en el tratamiento de estos trastornos.

La escritura ha sido una forma esencial de expresar emociones a lo largo del tiempo. Nos permite poner en palabras esos sentimientos que a veces no sabemos cómo compartir. Es una manera de explorar nuestro mundo interior, de entendernos mejor y de liberar lo que llevamos dentro. Cuando escribimos, encontramos un espacio seguro para ser sinceros con nosotros mismos. Incluso hoy, en un mundo lleno de tecnología, la escritura sigue siendo una herramienta poderosa para conectar con nuestras emociones y con los demás a un nivel más profundo. Nos ayuda a procesar lo que vivimos y a encontrar claridad en medio del caos emocional.

La escritura terapéutica es una forma de explorar y procesar emociones, pensamientos y experiencias a través de la escritura. Escribir puede ayudar a liberar tensiones, mejorar la autoconciencia y promover el bienestar emocional. Algunos beneficios incluyen liberar emociones reprimidas, mejorar el autoconocimiento, procesar experiencias difíciles, resolver problemas y mejorar la comunicación. Las técnicas incluyen diarios, cartas, poesía, cuentos y escritura creativa.

Escribirnos una carta en los momentos de calma y fortaleza emocional puede ser un regalo invaluable para nuestro yo del futuro en momentos de dificultad. Al leer esas palabras de aliento, recordatorios de nuestra fortaleza y ​​capacidades, podemos encontrar consuelo, motivación y una nueva perspectiva para afrontar los desafíos que se nos presenten. Esta carta se convierte en un ancla de esperanza y recordatorio de que somos capaces de superar incluso los momentos más difíciles.

La escritura terapéutica nos ayuda a poder transmitir nuestros pensamientos, emociones y sentimientos que solemos guardarnos, pero muchas de las veces lo transmitimos por medio de la escritura y esto suele sanarnos y tener una paz mental reduciéndonos así la ansiedad o estrés.

Este apartado me pareció interesante por que a la vez que nos brinda la oportunidad de sacar las preocupaciones y pensamientos que rondan en nuestra mente, permitiéndonos liberar esa carga emocional y mental.

DOI: 10.1093/genetics/iyab005

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @olekpark

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41556-020-00626-1

Resource: (BDSC Cat# 9688,RRID:BDSC_9688)

Curator: @olekpark

SciCrunch record: RRID:BDSC_9688

What is this?

DOI: 10.1038/s41556-020-00626-1

Resource: (BDSC Cat# 33966,RRID:BDSC_33966)

Curator: @olekpark

SciCrunch record: RRID:BDSC_33966

What is this?

DOI: 10.1083/jcb.202112094

Resource: (BDSC Cat# 24650,RRID:BDSC_24650)

Curator: @olekpark

SciCrunch record: RRID:BDSC_24650

What is this?

DOI: 10.1038/s41556-023-01110-2

Resource: RRID:BDSC_58449

Curator: @scibot

SciCrunch record: RRID:BDSC_58449

What is this?

DOI: 10.1038/s41467-019-12104-w

Resource: (BDSC Cat# 24651,RRID:BDSC_24651)

Curator: @scibot

SciCrunch record: RRID:BDSC_24651

What is this?

DOI: 10.1038/s41467-023-41540-y

Resource: (BDSC Cat# 62639,RRID:BDSC_62639)

Curator: @olekpark

SciCrunch record: RRID:BDSC_62639

What is this?

DOI: 10.1101/2023.10.04.560952

Resource: RRID:BDSC_50755

Curator: @scibot

SciCrunch record: RRID:BDSC_50755

What is this?

DOI: 10.3389/fphys.2023.1111244

Resource: (BDSC Cat# 55136,RRID:BDSC_55136)

Curator: @scibot

SciCrunch record: RRID:BDSC_55136

What is this?

DOI: 10.3389/fphys.2023.1111244

Resource: RRID:BDSC_38837

Curator: @scibot

SciCrunch record: RRID:BDSC_38837

What is this?

Can you spell out these acronyms here so the reader can refer to this section when interpreting the y-axes on Fig. 5 and 6? I see that they're listed in the abbreviations, but it would handy to have them written out in this section of the results.

DOI: 10.1016/j.celrep.2024.114453

Resource: (BioLegend Cat# 124802, RRID:AB_1279332)