Tomando en cuenta todo lo que nos menciona el texto e destacado este párrafo, ya que, bien nos dice sacar las cosas fuera de nuestra cabeza y eso es lo que más difícil resulta ser por que estamos llenos de ideas unas buenas otras malas pero el escribir nos permite liberarnos gracias a la escritura encontramos un lugar donde podemos desfogar todo lo que tenemos en nuestra mente así la escritura pasa a ser algo realmente beneficioso para todo lo que conlleva ser un ser humano y la escritura pasa a ser algo indispensable para nuestras vidas y eso es lo que deberíamos buscar que la escritura sea un lugar donde nos podemos desahogar y nos ayuda a nuestro propio desarrollo.

La habilidad de escribir, este echo es un arte para cada persona, en especial para las que pueden hacerlo de una manera mucho mas fluida y dando a comprender sus ideas, cada persona tiene en su mente el que quiere decir o expresar y asi mismo saben por que lo hacen, para ello, el echo de escribir les puede ayudar de manera terapeutica como nos menciona el texto y personalmente creo que esto les ayuda mucho mas de lo que se cree a las personas que tienen algun problema o algo asi.

También me encanto esta otra parte ya que no es nesesario tener habilidades literarias, sino más bien se puede escribir en silencio nuestro sentir

El arte de escribir como método terapéutico se percibe de diferentes formas, dependiendo de la persona que lo observe. Para algunos, puede parecer absurdo; para otros, es una herramienta fundamental para liberar ciertas cargas no físicas que, aunque no tienen una forma tangible, causan una angustia profunda. Esta lectura muestra un punto interesante en cuanto a su capacidad de ayudar e incluso salvar en algunos casos. Cuando escribí mi carta, no experimenté un cambio glorioso o un acontecimiento extraordinario, sin embargo, fue muy gratificante utilizar palabras capaces de evocar en los lectores sentimientos y sensaciones diferentes a las habituales, concebidas a través de acciones.

La escritura tiene que ser vista como un medio para la reflexión y en muchos casos para el desahogo. Tal vez el pensamiento de la sociedad es que si escribes algo es para ganar un premio o un reconocimiento, pero la realidad es que escribas para sentirse liberado. Desde mi experiencia al escribir cartas de despedida fue algo que me marcó ya que me di cuenta que es una forma de expresión y que en dicho papel estaba plasmado cosas que nunca pude decir personalmente, pero que por medio de la palabra me fue posible. En muchos casos la lectura y la escritura ha salvado vidas y como claramente dice que la lectura es un terapeuta.

Me gusto mucho esta parte de la lectura, si bien es cierto cuando uno escribe algo que no puede decirlo a alguien frente a frente a otra persona, por lo menos se desahoga y expresa sus sentimientos retenidos si verguenza alguna

E-atividade 2 – O funcionamento do cérebro Natureza da atividade: Individual

Fase 1 – Leia o artigo da autoria de Esperidião-Antonio, V. et al (2008) com ênfase nas áreas cerebrais envolvidas no processamento emocional (30 minutos). Faça uso das notas privadas para fazer comentários que o ajudem a concretizar segunda etapa desta e-atividade (produção de um infográfico ou mapa mental).

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

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Reply to the reviewers

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

The paper by Salinas-Rebolledo et al. describes a novel PROTAC approach in which the UBX domain of FAF1 is fused to a nanobody that recognizes the target protein. The idea is that the UBX domain will bind the fusion protein to the p97 ATPase, a major ATPase involved in the unfolding of many proteins. The target protein recognized by the UBX-nanobody fusion (UBX-Nb) is then supposed to be unfolded in a ubiquitin-independent manner and subsequently degraded by the proteasome.

The authors provide evidence that Ubx-Nb, containing a nanobody recognizing GFP, can colocalize with GFP fusion proteins in the nucleus and to liquid-liquid phase separation structures. Importantly, the fusion can reduce the cellular levels of the target proteins. The authors confirm that degradation triggered by Ubx-Nb is proteasome dependent. Ubx-Nb can also promote the degradation of model proteins that form aggregates relevant to neurodegenerative diseases.

__* The major issue with the paper is that it does not provide mechanistic insight into the degradation mechanism. First, the data implicating p97 in degradation are conflicting. On the one hand, siRNA of p97 compromises degradation (although degradation is not completely inhibited; see Figure 4G), but on the other hand, an inhibitor of p97 does not have an effect. The authors have not shown that target proteins are actually unfolded by their artificial adaptor (in vitro experiments would be required). __

__In addition, it would be important to show co-localization in vivo with p97. __

Thus, the role of p97 is not convincingly established. Another major question is how the unfolded, non-ubiquitinated proteins would be degraded by the 26S proteasome. Is there a ubiquitin ligase required after substrate unfolding?

Overall, the paper reports some intriguing effects of their designed PROTAC adaptor, but the mechanism by which it functions remains unclear. The findings of the manuscript appears too preliminary in its current version for it to be of value to the community.

Responses #1

We appreciate the detailed feedback provided and acknowledge the reviewers' concerns regarding the mechanistic insight into the degradation process. Below, we address each point raised:

We acknowledge that our current data do not fully elucidate the degradation mechanism involving p97. Our preliminary findings suggest that while siRNA of p97 compromises degradation, this effect is not absolute. Additionally, we recognize the inconsistency observed with the p97 inhibitor, which did not affect degradation. It is important to note that the selective inhibitor used in our study binds to the D2 domains of p97, as reported by Zhou et al. (J Med Chem. 2015). There are molecules that bind to the D1 domain and enhance p97-mediated degradation (Figuerola-Conchas et al., ACS Chem Biol. 2020). The primary ATPase function of p97 is associated with the D2 domain, but ATP binding to the D1 domain is crucial for hexamer formation and N-terminal domain conformation, which regulates cofactor binding and various functions of p97. Therefore, it is possible that our p97-PROTAC activates the D1 domain or interacts with cofactors enhancing protein degradation mediated by p97.

We plan to conduct in vitro experiments to demonstrate that target proteins are unfolded by our artificial adaptor. We have already cloned the degradation system into a bacterial expression vector to express and purify the system for these in vitro studies, which will be carried out in California. Furthermore, we aim to show in vivo co-localization with p97 in future studies.

*We understand the importance of establishing the role of p97 convincingly. Our preliminary data indicate the presence of residual p97 in siRNA experiments. Regarding the degradation of unfolded, non-ubiquitinated proteins by the 26S proteasome, there are studies indicating that various proteins are degraded by the proteasome independently of ubiquitin. Moreover, evidence suggests that different pools of the same protein can be directed to the proteasome via both ubiquitin-dependent and ubiquitin-independent mechanisms under the same cellular conditions. Also, prior research by Butler et al. (2016) demonstrated that fusing NbSyn87 with the mouse Ornithine Decarboxylase (ODC) PEST degron effectively reduced protein levels and this reduction was achieved by harnessing the innate cellular machinery responsible for ubiquitin-independent proteolysis. *

1. • Makaros Y, Raiff A, Timms RT, Wagh AR, Gueta MI, Bekturova A, Guez-Haddad J, Brodsky S, Opatowsky Y, Glickman MH, Elledge SJ, Koren I. Ubiquitin-independent proteasomal degradation driven by C-degron pathways. Mol Cell. 2023 Jun 1;83(11):1921-1935.e7. doi: 10.1016/j.molcel.2023.04.023. Epub 2023 May 17. PMID: 37201526; PMCID: PMC10237035.*
2. • Butler DC, Joshi SN, Genst E, Baghel AS, Dobson CM, Messer A. Bifunctional Anti-Non-Amyloid Component α-Synuclein Nanobodies Are Protective In Situ. PLoS One. 2016 Nov 8;11(11):e0165964. doi: 10.1371/journal.pone.0165964. PMID: 27824888; PMCID: PMC5100967.*
3. • Erales J, Coffino P. Ubiquitin-independent proteasomal degradation. Biochim Biophys Acta. 2014 Jan;1843(1):216-21. doi: 10.1016/j.bbamcr.2013.05.008. Epub 2013 May 14. PMID: 23684952; PMCID: PMC3770795.*
4. • Donghong Ju, Youming Xie, Proteasomal Degradation of RPN4 via Two Distinct Mechanisms, Ubiquitin-dependent and -independent*, Journal of Biological Chemistry, Volume 279, Issue 23, 2004, Pages 23851-23854, ISSN 0021-9258.*
5. *

We also speculate that the degradation may also occur via the autophagy-lysosome pathway. These speculations will be added to the discussion section, and we plan to investigate this pathway in detail in a subsequent scientific article focused on the mechanism of action of p97-PROTAC.

We recognize the need for further mechanistic studies and plan to publish these preliminary findings while continuing our research to elucidate the degradation mechanism. Our future work includes determining the crystal structure and conducting mass spectrometry to identify other proteins interacting with this complex, potentially aiding in degradation. We also plan to test the system in vivo using murine models with alpha-synuclein overexpression, in collaboration with researchers in Spain. This long-term project will form the basis of a subsequent publication.

We believe that our findings present an innovative and unique tool, and this preliminary data warrant publication. We appreciate the reviewers' comments and hope that our detailed response and future research plans address their concerns.

Minor points:

Fig. 1B: Although emerin is reported to be a nuclear envelope protein, it is not localized to the NE, but throughout the ER, likely because the protein was too highly expressed.

We agree with the reviewer, the overexpression of the NE could leak into the ER. We do not have specific Nanobodies to directly degrade Emerin. However, we would like to make the point that in both cases the protein will conserve a single transmembrane domain and even then, the GFP Nanobody fused to the UBX domain is able to trigger degradation

Fig. 1B: ETV co-localization is not obvious from the figure.

We are going to repeating the experiment and quantify the colocalization as suggested

Fig. 4: The depletion of p97 leads to cell death, so it is unclear whether the siRNA effect is specific.

We appreciate your comment and would like to clarify that there are published studies where the p97 gene has been silenced in HeLa cells without causing complete cell death. While it is true that a significant number of cells die post-transfection, we have observed that by changing the cell culture medium daily, the remaining cells start to grow again.

Studies supporting our findings include:

1. • Wójcik C, Yano M, DeMartino GN. RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis. J Cell Sci. 2004 Jan 15;117(Pt 2):281-92. doi: 10.1242/jcs.00841. Epub 2003 Dec 2. PMID: 14657277. *
2. • Beskow A, Grimberg KB, Bott LC, Salomons FA, Dantuma NP, Young P. A conserved unfoldase activity for the p97 AAA-ATPase in proteasomal degradation. J Mol Biol. 2009 Dec 11;394(4):732-46. doi: 10.1016/j.jmb.2009.09.050. Epub 2009 Sep 24. PMID: 19782090.*
3. • Yahiro K, Tsutsuki H, Ogura K, Nagasawa S, Moss J, Noda M. Regulation of subtilase cytotoxin-induced cell death by an RNA-dependent protein kinase-like endoplasmic reticulum kinase-dependent proteasome pathway in HeLa cells. Infect Immun. 2012 May;80(5):1803-14. doi: 10.1128/IAI.06164-11. Epub 2012 Feb 21. PMID: 22354021; PMCID: PMC3347452. Additionally, although our figure does not clearly show the band corresponding to p97, upon overexposing the film, we can detect a very faint band of the protein. This indicates that there is still residual expression of p97, albeit at a lower concentration, which is consistent with a significant reduction but not a complete elimination of the protein.*

Fig. 5C: The colocalization of GFP-HTT Q23 with UBX-Nb(GFP) is not entirely convincing.

We are going to repeating the experiment and quantify the colocalization as suggested

Reviewer #1 (Significance (Required)):

Overall, the paper reports some intriguing effects of their designed PROTAC adaptor, but the mechanism by which it functions remains unclear. The findings of the manuscript appears too preliminary in its current version for it to be of value to the community.

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

Summary:

The authors have developed a p97-directed proteolysis-targeting chimera (PROTAC) that operates independently of ubiquitin. This system employs a camelid nanobody to selectively recognize target proteins, tethered to p97 through the UBX domain of the p97 adapter FAF1. The anti-GFP nanobody effectively targets various GFP-fusion proteins for degradation via the proteasome, relying on p97 for its mechanism of action. The authors validate the presence of p97 in brain tissues of Non-Human Primates (Nhp) Macaca fascicularis, rat (Sprague Dawley), and mouse (C57BL6/C), supported by proteasome inhibition and p97 RNA silencing data. Importantly, the p97-PROTAC mechanism operates independently of ubiquitination, demonstrated through degradation of clinically relevant proteins such as alpha-synuclein using a camelid nanobody (NbSyn87).

Major comments:

* Anti-GFP Nanobody clarification: Details about the original anti-GFP nanobody are unclear, which makes reproducing the current work a challenge for outside labs.

o 20. Fulcher LJ, Macartney T, Bozatzi P, Hornberger A, Rojas-Fernandez A, Sapkota GP. An affinity-directed protein missile system for targeted proteolysis. Open Biol. Oct 2016;6(10)doi:10.1098/rsob.160255

o I assume that the anti-GFP nanobody is aGFP from supplementary figure #2 in the Fulcher manuscript but is unclear as they also have used anti-GFP nanobody aGFP16.

* Amino acids from FAF1-UBX domain: Further clarity is needed regarding the amino acid details from the FAF1-UBX domain, which may have been disclosed in a patent application but should be explicitly outlined in the methods section.

We appreciate your comment regarding the need for further clarity on the amino acid details from the FAF1-UBX domain. To address this, we have added a supplementary figure which includes a table outlining the amino acid sequences of our construct and the FAF1-UBX domain. This supplementary figure provides a detailed representation of the amino acid sequences. We hope this addition meets your requirements and provides the necessary information for a comprehensive understanding of our work.

__* Degradation of Proteins of Clinical Interest: The data presented is not convincing enough to support the stated claims that the PROTAC is clearing aggregated mutant HTT. In Figure 5, there is an abundance of GFP-HTT Q74 puncta. While the western blot data suggests a reduction of soluble GFP-HTT-Q74 protein levels, it does not account for aggregated HTT. Aggregated HTT does not efficiently enter the separating gel during electrophoresis. To make these claims the authors need to 1. Show the level of mHTT Q74 aggregation in the empty control groups so that a comparison can be visually made between empty control groups and UBX-Nb(GFP) treated groups. A similar comparison would be useful with the GFP-HTT Q23 treated cells as well. __

* Visualization of Aggregated Proteins: The continued visibility of puncta raises doubts about the system's efficacy in degrading aggregated proteins. Including comparisons between untreated and treated cells for all test systems would strengthen the argument. It would be useful to show a comparison between the untreated controls and UBX-Nb (GFP) treated cells for all the test systems shown.

*We acknowledge that the data presented in Figure 5 may not be sufficient to support the claim that the PROTAC is clearing aggregated mutant HTT. The western blot data suggests a reduction in soluble GFP-HTT-Q74 protein levels, but it does not account for aggregated HTT, which does not efficiently enter the separating gel during electrophoresis. We agree that more experiments will need to be performed to evaluate the impact of the p97 PROTAC on the direct turnover of the aggregates once those are form. *

Minor comments:

* The In-text citations should be placed outside of the sentence. Example: The ubiquitin-proteasome system (UPS) regulates protein abundance by specific E3 ubiquitin ligases, which catalyze ubiquitin chain formation on the substrates, inducing their proteasome mediated degradation. 1-4

* Sentence two in the introduction is missing a period. It is unclear whether sentence three is a heading or part of paragraph one.

* There are additional formatting issues. It would be easier to read the paper if there was a space between paragraphs. Page numbers would be helpful.

* Page 2. Missing word. Inclusion body myopathy associated with Paget's disease.

* Figure 1. D, F, and E. Missing annotation to denote significance.

* Figure 2G Missing annotation to denote significance.

* Figure 4. Missing annotation to denote significance for figures 4D, 4F, 4H.

* Is Figure 4I significantly different or not? In Figure 6, you use ns to denote not significant. This feels like it is an important point that you would want to make that the effect is dependent on p97. When you knock out p97 the degradation capacity of UBX-Nb is lost.

Response

These changes are going to be apply

Reviewer #2 (Significance (Required)):

* The p97-PROTAC system is an ubiquitin-independent approach to degrade intracellular proteins. This system was able to target proteins for degradation at diverse subcellular locations, integral membrane protein residing at the inner nuclear membrane, chromatin located, and liquid-liquid phase separated compartments. The ability to clear alpha-synuclein builds on previous research suggesting that ubiquitin-independent degradation of alpha-synuclein could be a therapeutic approach to treat synucleinopathies such as Parkinson's Disease. However, the ability of this approach to clear aggregated proteins is not convincing, given the presence of visible aggregates in the treated cells.

* The investigations with NbSyn87 build upon prior research by Butler et al. (2016), who fused NbSyn87 with the mouse Ornithine decarboxylase (ODC) PEST degron. This fusion strategy not only facilitates the targeting of alpha-synuclein but also harnesses the innate cellular machinery responsible for ubiquitin-independent proteolysis. The current approach demonstrates and alternative mechanism to direct alpha-synuclein (and other proteins) into the proteasome for ubiquitin-independent clearance.

* At the current state of development, this research is of interest to specialized audience with antibody engineering backgrounds; however, it holds translational potential for clearance of toxic proteins.

* My research interests is in the development of therapeutics for the treatment of neurodegenerative diseases including Huntington's Disease, Parkinson's Disease, and Alzheimer's Disease.

DOI: 10.3389/fcell.2019.00129

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bpowell22

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1016/j.chembiol.2024.06.004

Resource: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)

Curator: @scibot

SciCrunch record: RRID:AB_262044

What is this?

Author response:

Reviewer #2 (Public Review):

I have two significant concerns that I believe can be resolved on the timescale of review.

1) The work identifies substantial thinning in one leaflet. Lipids expand as they thin. Given this, are there too few lipids in this leaflet (which would also indicate thinning)? I would expect their deformations depend strongly on the number-balance of lipids in each leaflet. The authors should check if thinning, and the boundary, is sensitive to inter-leaflet-lipid imbalance.

We thank Reviewer #2 for this insight, as it led us to evaluate the leaflet tensions in our restrained 2L0J simulation. We found there was an imbalance in the leaflet packing, which we addressed with an extensive set of new simulations and new analysis aimed at generating balanced leaflets.

See Page 6-8, Appendix Section 1, Appendix – figures 1, 2. We discuss these findings in the new Results section “Protein footprint asymmetry can lead to differential leaflet stresses” and accompanying appendix. Many of the bilayer features in the repacked simulations are consistent with our original submission, but not all. For instance, while we continue to see large tilt immediately around the amphipathic helices in the lower leaflet and little in the upper leaflet, tilts in both leaflets decay to similar values at the box edge (Appendix - figure 2). The degree of membrane pinch along the membrane-protein contact boundaries are less sensitive to the leaflet packing, as demonstrated by the surface heights (Appendix - figure 1).

Determining the proper change in leaflet count is quite difficult. We are actively extending our continuum model to address questions of differential leaflet strain and coupled lipid tilt, which may allow us to estimate changes in leaflet-count, but this is a significant undertaking beyond the scope of this resubmission.

2) By constraining the pore to have 2-fold symmetry, the authors remove a large entropic penalty disfavoring such a conformation, and thus presumably disfavoring the negative- gaussian-curvature it induces. For example, if the free energy surface for the fluctuations were rather flat, and only 1% of the conformations were consistent with 2-fold symmetry, the coupling to NGC may be reduced by -kT log( 1 % ), neglecting enhancement by coupling to NGC. Therefore, I predict that the coupling to NGC would be reduced further were the constraint removed.

We agree with the reviewer that if the 2-fold states are highly disfavored for entropic or enthalpic reasons, it would directly reduce the coupling to NGC. However, we don’t know the free energy difference between these states, and it is hard to calculate them from all-atom and beyond our current scope. While our unrestrained simulations are not converged, they demonstrate that there is a wide range of orientations for the amphipathic helices that are energetically accessible (see Figure 2, Appendix Section 1, and Appendix - figure 4). Still, the DEER data from the Howard lab (Kim et al., 2015) would be better described by further symmetry-broken states with greater inter-AH distances, suggesting that such conformations are not well represented in our equilibrium ensemble.

Reviewer #3 (Public Review):

Helsell et al. uses atomistic molecular dynamics simulations to characterize the structural dynamics of the M2 protein together with continuum elastic models to evaluate the energetic cost of the protein-induced bilayer deformations. Using unbiased simulations (without constraints on the protein) they show that the M2 structure is dynamic and that the AH helices are mobile (though they tend to retain their secondary structure), in agreement with experimental observations. Then, using simulations in which the peptide backbone was restrained to the starting structure, they were able to quantitatively characterize the protein- induced bilayer deformations as well as the acyl chain dynamics.

Both the atomistic simulations and the continuum-based determinations of the bilayer deformation energies are of high quality. The authors are careful to note that their unbiased simulations do not reach equilibrium, and the authors' conclusions are well supported by their results, though some issues need to be clarified.

1) P. 7: Choice of lipid composition: POPC:POPG:Cholesterol 0.56:0.14:0.3. This lipid composition (or POPC:POPG 0.8:0.2) has been used in a number of experimental studies that the authors use as reference. It differs, however, substantially from the lipid composition of the influenza membrane (Gerl et al., J Cell Biol, 2012; Ivanova et al., ACS Infect Dis, 2015), which is enriched in cholesterol, has a 2:1 ratio of phosphatidylethanolamine to phosphatidylcholine, and almost no PG. The choice of lipid composition is unlikely to impact the authors' major conclusions, but it should be discussed briefly. As noted by Ivanova et al., the lipids of the influenza membrane are enriched in fusogenic lipids. How will that impact the authors results.

As noted by the Reviewer, the lipid composition we explored was based on DEER studies from Kathleen Howard. While there is a lot of cholesterol in our simulations, it is lower than the lipidomics papers suggest for the viral membrane (Gerl et al., 2012; Ivanova et al., 2015). We hypothesize that further increasing cholesterol would stiffen the membrane even more and cause the energy differences we report here to become even larger – accentuating our finding. We employ 14% POPG and the Simons lab finds about 14% PS. Chemically these headgroups are similar, but the size and spontaneous curvature difference could be a concern. This is the the different intrinsic curvatures of PE versus PC. However, we have not considered spontaneous curvature in our continuum calculations, so we cannot predict how this will influence our results.

See Appendix - figure 6. We added a new panel to this figure with continuum parameters intended to mimic a high 50 % cholesterol membrane reported for viral coats, and we show that the curvature sensing of symmetry-broken states increases as the cholesterol content increases.

See Page 25. We added text in the Discussion concerning the difference in lipids found in the virus versus those compositions employed in experiment and here.

2) The definition of the lipid tilt needs to be revisited. On P. 13 (in the Pdf received for review, the authors do not provide page numbers), the tilt is defined/approximated as "the angle between the presumed membrane normal (aligned with the Z axis of the box) and the vector pointing from each phospholipid's phosphate to the midpoint between the last carbon atoms of the lipid tails." This (equating the normal to the interface with the Z axis of the simulation box) may be an acceptable approximation for the lower leaflet, which is approximately flat, but probably not for the upper leaflet where the interface is curved in the vicinity of the protein. The authors should, at least, discuss the implications of their approximation in terms of their conclusion that there is little lipid tilt in the upper leaflet.

We agree that our lipid tilt calculations are approximate since we assume the membrane normal points along the z direction. We have now restated this assumption in the Results when we start to discuss tilt. Different models define lipid tilt in different ways, but the work of Deserno defines it with respect to the bilayer mid-plane which is a shared surface for the upper and lower leaflets. Thus, tilt would be moderately impacted in both leaflets. Examining the snapshots at the top of Figure 7, we surmise that the calculated tilts in both leaflets adjacent to the protein would be slightly reduced, leaving the values at the boundary unaffected. Thus, the upper leaflet likely experiences even less tilt than calculated.

See Page 16. We have added the discussion above to the section on lipid tilt. Also, we have added page numbers to the resubmission.

3) P. 14, last paragraph, Figure 5 and 6: The snapshots in Figure 5 are too small to see what the authors refer to when they write "tilt their lipid tails to wrap around the helices." The authors should consider citing the work of H W. Huang, e.g., Huang et al. (PRL, 2004), who introduced the notion of curvature stress induced by antimicrobial peptides, a concept similar to what the present authors propose.

See Page 17. We have now drawn the connection between what our simulations are showing and the earlier work by Huey Huang on antimicrobial peptides.

See Figure 7. To make the lipid deformations easier to see, we are attaching the full-size versions of each snapshot to the figure as supplemental data.

4) P. 17-18, Figure 7: The authors introduce the bilayer midplane, which becomes important for the determination of the deformation energy in the (unnumbered) equation on P. 17, but do not specify how it is determined. This is a non-trivial undertaking, but critical for the evaluation of the deformation energy; please add the necessary details.

See Pages 15 and 20. In the continuum model, we define CM (the compression surface) following the work of May and colleagues (and other groups) as the areal compression weighted mean of the upper and lower surface. In the MD simulation results in Figure 6, we define leaflet thickness as the absolute difference between the interpolated leaflet hydrophobic surface (calculated using the first carbon atoms of each POPC and POPG lipid tail) and the interpolated bilayer midplane surface (calculated as the average of the upper and lower leaflet tail surfaces, each interpolated based on the last carbon atoms of each POPC and POPG lipid tail for each leaflet, respectively). These two leaflet-based definitions are different, and a more sophisticated continuum model of the upper and lower leaflet coupling would require the incorporation of lipid tilt, which we do not currently have.

5) P. 18-19, Figure 8: The comparison of the MD and continuum membrane deformations is very informative, but the authors should discuss the implications of the increased symmetry further in terms of the estimated deformation energies. (I do not believe the authors really mean that they predicted the energies, they estimated/approximated them.)

The Reviewer is correct, we are not predicting the energies of the actual MD generated bilayers, but rather we are estimating the energies of these shapes using a continuum-based approximation. The good agreement between the MD generated surfaces and the continuum predicted surfaces suggested that the model is capturing the underlying physics. We argued that the increased symmetry of the continuum surfaces compared to the MD surfaces was due to incomplete sampling in the MD. We were right about that. Please see revised Figure 10 with new data and some longer simulations, where the symmetry in the MD is now apparent and the match between continuum and MD is even better. Frankly, we are very pleased with these new results.

See Page 18 and Figure 10. We have changed language throughout moving away from “predicting” to “estimating”. The new MD generated data shows much greater symmetry reflected in the starting structures, and better agreement with model predictions.

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Kim, S. S., Upshur, M. A., Saotome, K., Sahu, I. D., McCarrick, R. M., Feix, J. B., Lorigan, G. A., & Howard, K. P. (2015). Cholesterol-Dependent Conformational Exchange of the C- Terminal Domain of the Influenza A M2 Protein. Biochemistry, 54(49), 7157-7167. https://doi.org/10.1021/acs.biochem.5b01065

Kučerka, N., Tristram-Nagle, S., & Nagle, J. F. (2006). Structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains. J Membr Biol, 208(3), 193-202.

Latorraca, N. R., Callenberg, K. M., Boyle, J. P., & Grabe, M. (2014). Continuum approaches to understanding ion and peptide interactions with the membrane. J Membr Biol, 247(5), 395-408. https://doi.org/10.1007/s00232-014-9646-z

Liu, J., Kaksonen, M., Drubin, D. G., & Oster, G. (2006). Endocytic vesicle scission by lipid phase boundary forces. Proc Natl Acad Sci U S A, 103(27), 10277-10282. https://doi.org/10.1073/pnas.0601045103

Pan, J., Tristram-Nagle, S., & Nagle, J. F. (2009). Effect of cholesterol on structural and mechanical properties of membranes depends on lipid chain saturation. Phys Rev E Stat Nonlin Soft Matter Phys, 80(2 Pt 1), 021931. https://doi.org/10.1103/PhysRevE.80.021931

Rawicz, W., Olbrich, K. C., McIntosh, T., Needham, D., & Evans, E. (2000). Effect of chain length and unsaturation on elasticity of lipid bilayers. Biophys J, 79(1), 328-339. https://doi.org/10.1016/S0006-3495(00)76295-3

Sun, D., Peyear, T. A., Bennett, W. F. D., Andersen, O. S., Lightstone, F. C., & Ingolfsson, H. I. (2019). Molecular Mechanism for Gramicidin Dimerization and Dissociation in Bilayers of Different Thickness. Biophys J, 117(10), 1831-1844. https://doi.org/10.1016/j.bpj.2019.09.044

Tzlil, S., Deserno, M., Gelbart, W. M., & Ben-Shaul, A. (2004). A statistical-thermodynamic model of viral budding. Biophys J, 86(4), 2037-2048. https://doi.org/10.1016/S0006- 3495(04)74265-4

Ursell, T. S., Klug, W. S., & Phillips, R. (2009). Morphology and interaction between lipid domains. Proc Natl Acad Sci U S A, 106(32), 13301-13306. https://doi.org/10.1073/pnas.0903825106

Veatch, S. L., & Keller, S. L. (2003). Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys J, 85(5), 3074-3083. https://doi.org/10.1016/S0006-3495(03)74726-2

Venable, R. M., Brown, F. L. H., & Pastor, R. W. (2015). Mechanical properties of lipid bilayers from molecular dynamics simulation. Chem Phys Lipids, 192, 60-74. https://doi.org/10.1016/j.chemphyslip.2015.07.014

Reviewer #1 (Public Review):

Summary:

Bowler et al. present a thoroughly tested system for modularized behavioral control of navigation-based experiments, particularly suited for pairing with 2-photon imaging but applicable to a variety of techniques. This system, which they name behaviorMate, represents a valuable contribution to the field. As the authors note, behavioral control paradigms vary widely across laboratories in terms of hardware and software utilized and often require specialized technical knowledge to make changes to these systems. Having a standardized, easy-to-implement, and flexible system that can be used by many groups is therefore highly desirable. This work will be of interest to systems neuroscientists looking to integrate flexible head-fixed behavioral control with neural data acquisition.

Strengths:

The present manuscript provides compelling evidence of the functionality and applicability of behaviorMate. The authors report benchmark tests for real-time update speed between the animal's movement and the behavioral control, on both the treadmill-based and virtual reality (VR) setups. Further, they nicely demonstrate and quantify reliable hippocampal place cell coding in both setups, using synchronized 2-photon imaging. This place cell characterization also provides a concrete comparison between the place cell properties observed in treadmill-based navigation vs. visual VR in a single study, which itself is a helpful contribution to the field.

Documentation for installing and operating behaviorMate is available via the authors' lab website and linked in the manuscript.

Weaknesses:

The following comments are mostly minor suggestions intended to add clarity to the paper and provide context for its significance.

(1) As VRMate (a component of behaviorMate) is written using Unity, what is the main advantage of using behaviorMate/VRMate compared to using Unity alone paired with Arduinos (e.g. Campbell et al. 2018), or compared to using an existing toolbox to interface with Unity (e.g. Alsbury-Nealy et al. 2022, DOI: 10.3758/s13428-021-01664-9)? For instance, one disadvantage of using Unity alone is that it requires programming in C# to code the task logic. It was not entirely clear whether VRMate circumvents this disadvantage somehow -- does it allow customization of task logic and scenery in the GUI? Does VRMate add other features and/or usability compared to Unity alone? It would be helpful if the authors could expand on this topic briefly.

(2) The section on "context lists", lines 163-186, seemed to describe an important component of the system, but this section was challenging to follow and readers may find the terminology confusing. Perhaps this section could benefit from an accompanying figure or flow chart, if these terms are important to understand.

(2a) Relatedly, "context" is used to refer to both when the animal enters a particular state in the task like a reward zone ("reward context", line 447) and also to describe a set of characteristics of an environment (Figure 3G), akin to how "context" is often used in the navigation literature. To avoid confusion, one possibility would be to use "environment" instead of "context" in Figure 3G, and/or consider using a word like "state" instead of "context" when referring to the activation of different stimuli.

(3) Given the authors' goal of providing a system that is easily synchronizable with neural data acquisition, especially with 2-photon imaging, I wonder if they could expand on the following features:

(3a) The authors mention that behaviorMate can send a TTL to trigger scanning on the 2P scope (line 202), which is a very useful feature. Can it also easily generate a TTL for each frame of the VR display and/or each sample of the animal's movement? Such TTLs can be critical for synchronizing the imaging with behavior and accounting for variability in the VR frame rate or sampling rate.

(3b) Is there a limit to the number of I/O ports on the system? This might be worth explicitly mentioning.

(3c) In the VR version, if each display is run by a separate Android computer, is there any risk of clock drift between displays? Or is this circumvented by centralized control of the rendering onset via the "real-time computer"?

Parece ser um erro l: independencia não é nessa data.... Essa data é a festa da viotoria militar do Imperio em 1824 contra a Republica nordestina !!! Acho que muita gente fica ignorante do que essa data não é feliz : é a vitoria do esclavagismo contra a revolta revolucionaria do povo noderstino. E uma data fundamental para entender e desprezo do Nordeste no Brasil desde então. Tem quie mudar isso e até questionar o esquecimento da historia a tal ponto que o prprio Lula veio a Slvador ha uma semana para comemorar essa vitoria milita do Imperio contra a Republica... Se for, Luiz, tem que escrever uma nota sobre o ssunto...

que tantas repetições !!! tem que denflatar muito o texto tudo !! E fora isso, ne, se sabe até aqui no texto o que é o FIOCRUZ...

Condensar : o autor fala 3 vesez a mesma coisa sem organisação nenhuma. O até suprimis se você verificar que na verdade esse conteudo tem outras formulaçes no texto. O pragrafo seguinte seria um inicio melhor

Es como si la Fundación Wikimedia se limitara a ofrece servicios de gestión a la comunidad Wikimedia, representada por el Consejo Global.

No entiendo si se refiere a la distribución que establezca el Consejo Global, o los recursos que establezca el Consejo Global.

"la implementación y aplicación" o "implementando y haciendo cumplir"

Если имеется ввиду нулевая матрица конкретного размера, то это обычно обозначается \(O_{m,n}\) или \(O_{mn}\). И еще иногда букву \(Z\) используют, можно тоже про это написать, чтобы было понятнее, если где-то встретится

Em regra, não se admite contrato verbal com a Administração. Porém, será válido para contratações abaixo de 10.000 reais.

Isto é, em regra, no Direito Administrativo, vigora o Princípio da Solenidade das Formas, ao contrário do Direito Privado, em que se admite o Princípio da Instrumentalidade das Formas.

Entretanto, em casos definidos em lei, poderá haver produção de efeitos jurídicos de ato administrativo não escrito, como na hipótese destacada, admitindo contrato verbal, ou mesmo nas situações de sinalização por placas, apitos e gestos.

1. Finding: co-emotional vulnerability might lead to moral authority or the rejection of it. Remaining vulnerable is emotional work.

Este texto fez-me pensar no papel que o ensino híbrido pode ter na transformação em direção a uma aprendizagem mais inclusiva. Por variadas razões, que vão desde problemas de saúde (imunodeficiências, tratamentos agressivos contra certas doenças, etc.), a deficiências, condições climáticas que impossibilitem o acesso à escola física, distância geográfica (ex.: nas regiões mais remotas do Canadá, muitas são as crianças que não têm outra opção), entre outras, há situações onde/quando/em que o estudante não pode ter acesso ao ensino face-a-face. Por outro lado, a utilização de ferramentas digitais que caracteriza o ensino híbrido pode permitir a estudantes portadores de deficiências deslocarem-se em cenários, manipularem objetos e terem acesso a recursos diversificados que lhes permitem escolher aquele(s) que melhor se adaptam às suas circunstâncias pessoais. Contudo, isto só acontecerá se o desenho dos cursos, das unidades curriculares e das atividades tiver em conta, verdadeiramente, este aspeto (Blakeborough & Rosaloo, 2023; DOI: 10.1007/978-981-99-5540-4_6), o que nos leva à necessidade de empregar o conceito de Universal Design for Learning.

"e que as modalidades ao longo do caminho de aprendizado de cada estudante em um curso ou matéria estejam conectados, oferecendo uma experiência integrada". Ou seja, no ensino híbrido, o todo deve ser maior do que as partes. Não é simplesmente uma "salada" (uma mistura) de formatos, metodologias e atividades, mas a utilização de cada um destes tem de ser criteriosa e integrada numa visão geral do que se pretende da aprendizagem.

Boa tarde, Esta fusão a que o texto se refere suscita-me refletir sobre o 3.º mundo que é criado através do ensino híbrido e para o qual nos alerta o texto de José António Moreira e Maria João Horta (2020). De acordo com estes autores (Moreira & Horta, 2020), o ensino blended learning oferece o melhor dos dois mundos, físico e online, fazendo gerar um 3.º mundo, que favorecer uma experiência de ensino integrado, e, por consequência, no meu entender, mais rica, porque potencia a construção do conhecimento na sua globalidade. Ou seja, favorece o desenvolvimento de um conjunto articulado de lentes epistemológicas, em que online e ambiente físico formam uma conjuntura de aprendizagem coerente, em que o aluno, mediado pelo professor, participa, reflete e (re)constrói conhecimento. Esta perspetiva é de facto muito favorável ao desenvolvimento do cidadão autónomo, crítico e criativo que defende as principais linhas orientadoras do processo educativo em Portugal, mas implica uma mudança de atitude do professor. Implica que este desenvolva não apenas uma literacia digital, mas também uma literacia educativa e pedagógica. Do nosso ponto de vista, e fazemos esta afirmação com base na nossa experiência de trabalho com os professores no âmbito do projeto curricular integrado em ambientes físico, é importante que o professor conceba o processo educativo como um processo de construção, em que a criação das experiências de aprendizagens blended learning se afirma como um todo inteligível, com processos de planificação de competências muito concretos, que visam, efetivamente, o desenvolvimento da autonomia e da capacidade de reflexão dos alunos. Na verdade, pensamos que a riqueza deste processo ensino aprendizagem blended learning impõe um conjunto de pressupostos por parte do professor: a) Que o professor reconheça que o aluno aprende mesmo que o professor não exponha a matéria; b) Processo educativo deve favorecer o desenvolvimento de competências como autonomia, espírito crítico e reflexivo, problematização da realidade. Para tal é importante que o docente pense e promova um processo educativo mediado por metodologias ativas que favoreçam a construção autónoma do conhecimento por parte dos alunos, o desenvolvimento competências como a resolução de problemas e o desenvolvimento do pensamento crítico e a proatividade cidadã; c) O professor reconheça o seu papel como de investigador reflexivo, que promove uma ação praxeológica, na qual ele desempenha o de mediador do processo de ensino aprendizagem dos alunos. Referência Moreira, J.A. & Horta, M.J. (2020). Educação em ambientes híbridos de aprendizagem. Um processo de inovação sustentador. Revista UEG, V. 2: e66027.

Nas leituras que realizei sobre ensino híbrido pude concluir o ensino híbrido, apesar de ter recebido um grande impulso com a pandemia, se tornou uma necessidade do processo educativo do século XXI por vivermos num período em que as crianças e jovens são efetivamente nativos digitais (Link, Washington & Lopes,2022) Em virtude desta característica dos nosso alunos, mas também à resposta aos desafios da sociedade contemporânea e à urgência de desenvolvermos o aluno enquanto cidadão global ativo, o ensino híbrido tornou-se um forma de promover um processo de ensino-aprendizagem em que o aluno é o protagonista. Parte-se do interesse dos alunos enquanto nativos digitais para promover um processo educativo integrado e significativo.

DOI: 10.1093/molbev/msz274

Resource: BDSC_9299

Curator: @bpowell22

SciCrunch record: RRID:BDSC_9299

What is this?

DOI: 10.1038/s41598-024-56343-4

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @anisehay

SciCrunch record: RRID:SCR_006457

What is this?

Para fins de Despesa com Pessoal: * 1) indenização por demissão, * 2) incentivos à demissão voluntária, * 3) convocação extraordinária do Congresso Nacional, (Não Recepcionado EC 50/2006) * 4) decorrente de decisão judicial, * 5) inativos e pensionistas * = não comporão o limite de gastos com pessoal do Ente da Federação, que 50% para União e 60% para os demais.

Na conclusão, o autor também lembra do propósito desse trabalho. No entanto, em alguns trechos há menções ao valor histórico de maneira mais acentuada.

skaityt toliau dar, daugiau paaiškinama. IR BŪTINAI: pradėt iteracijas su generacija, o ne seed'u!

different uses of Wikidata properties

Do they mean the same property used in different contexts, such as in statement o reference auxiliary nodes?

Não conhecia o recurso Hypothes.is e parece-me bastante útil para favorecer a leitura e interpretação de conteúdos, favorecendo a reflexão colaborativa entre pares. Relativamente ao excerto que escolhi, considerando que a minha formação base é Marketing, inevitavelmente, quando analisei o texto, fiz um paralelismo com esta área. Assim, um gestor de marketing, antes de lançar um serviço, tem que previamente, estudar as necessidades, desejos e expectativas dos clientes, criando serviços com atributos valorizados pelos clientes. Os consumidores têm diferentes objetivos e necessidades quando adquirirem um produto. Têm características sociodemográficas, culturais e económicas distintas, portanto, é necessário personalizar e adaptar o serviço às necessidades e características dos clientes. Somente, com esta abordagem, é possível gerar o interesse na experimentação do serviço e fomentar o desejo à compra/ação. Se o serviço corresponder às suas expectativas de qualidade e resolver um problema/necessidade, poderá motivar o consumidor para voltar a comprar (fidelização). Neste sentido, o mesmo é aplicável ao Ensino a Distância e Digital. Se produzirmos E-Atividades cujos conteúdos são ajustados às necessidades e perfil dos alunos, nomeadamente, quanto ao seu estilo de aprendizagem, poderemos estimular a sua interação e motivação para aprender. Deste modo, o processo ensino aprendizagem poderá ser mais eficaz.

Atualmente, a escassez de recursos torna fundamental o uso eficiente dos mesmos. Um exemplo dessa eficiência foi a transformação do Curso de Comando e Direção Policial (CCDP) ministrado no ISCPSI, que, antes de 2018, era exclusivamente presencial. Com a implementação da Portaria n.º 101/2018, o curso adotou o modelo em blended-learning, combinando aulas online síncronas com períodos presenciais reduzidos, através de um Sistema de Gestão de Aprendizagem. Apenas esta mudança permitiu a admissão de 153 Comissários em seis turmas, com aulas presenciais concentradas em apenas três semanas por turma, resultando em 18 semanas presenciais no total, em comparação com as 42 semanas que seriam necessárias anteriormente. Ora esta redução no tempo de permanência dos alunos, de todo o país, nas instalações do ISCPSI, tanto para aulas quanto para alojamento, refletiu-se numa considerável economia de recursos, além de mitigar o impacto nos transportes e serviços de apoio. Outro resultado precioso foi a diminuição do tempo de ausência dos alunos de seus locais de trabalho, o que trouxe benefícios para o serviço policial.

O Feedback imediato (ou quase) é, na minha perspetiva, um elemento chave nos modelos assíncronos, porque é claramente uma interação desejada (da minha experiência) por pessoas em formação. Esse feedback pode funcionar entre pares também e isso pode ser estimulado na discussão em fórum em torno de um tema em discussão, por exemplo.

Esta ferramenta é interessante, embora se me afigure mais pobre (tendo em conta, obviamente, os meus contextos como docente e formadora) do que o VideoAnt, porque os comentários surgem mais atomizados o que não favorece tanto as interações. Os retornos são mais escassos e, sobretudo, mais delimitados.

Não deixo de registar como uma ferramenta a utilizar eventualmente com textos mais curtos (pensando sempre nos meus contextos (áreas científicas, UC ou cursos de formação, etc.).

Teresa Pinto

Totalmente de acordo. Tenho por hábito referir aos meus formandos que "para quem não sabe para onde vai qualquer caminho serve" (relembrando as sábias palavras do Coelho em Alice do País das Maravilhas) e daí a importância de uma planificação adequada que obrigatoriamente envolve o desenvolvimento de e-atividades com intencionalidade pedagógica.

A formulação de questões, a investigação e o desenvolvimento do pensamento crítico tem sido apontada como um ponto fundamental nos processos de ensino-aprendizagem, na medida em que promove um tipo de raciocínio de complexidade e qualidade superiores potenciado a tomada de decisões a vários níveis e normalmente associada a resultados positivos.

Acabei de ler o capítulo sobre e-atividades em ambientes digitais do documento fornecido. Achei muito interessante como as e-atividades são destacadas como essenciais para promover uma aprendizagem online ativa e participativa.

O texto enfatiza a importância de diversificar os formatos para atender a diferentes estilos de aprendizagem e a necessidade de oferecer feedback imediato aos alunos.

Além disso, a flexibilidade e acessibilidade dessas atividades são pontos fortes, especialmente para estudantes com agendas mais apertadas.

Gostaria de perguntar como podemos implementar estas práticas de forma eficaz nos nossos próprios cursos. Alguém já tem experiência com isso e poderia partilhar insights?

Cumprimentos, André Calvinho

Acabei de ler o capítulo sobre e-atividades em ambientes digitais do documento fornecido. Achei muito interessante como as e-atividades são destacadas como essenciais para promover uma aprendizagem online ativa e participativa.

O texto enfatiza a importância de diversificar os formatos para atender a diferentes estilos de aprendizagem e a necessidade de oferecer feedback imediato aos alunos.

Além disso, a flexibilidade e acessibilidade dessas atividades são pontos fortes, especialmente para estudantes com agendas mais apertadas.

Gostaria de perguntar como podemos implementar estas práticas de forma eficaz nos nossos próprios cursos. Alguém já tem experiência com isso e poderia partilhar insights?

Cumprimentos, André Calvinho

Contudo não podemos esquecer que os objetivos das e-atividades variam de acordo com cada disciplina/módulo. Tendo sempre em conta que devem promover a aprendizagem ativa, desenvolver habilidades especificas, avaliar o conhecimento, estimular a interação de todos num ambiente colaborativo, flexibilizando sempre a aprendizagem, adaptando-a a cada aluno/estilo de vida.

Este parágrafo resume aquilo que tem vindo a ser apresentado sobre a docência digital ao longo deste curso e mudou muito a minha percepção sobre a docência digital. Vai também muito ao encontro daquilo que já há muito tempo defendo para o modo convencional de aprendizagem presencial onde há claramente uma orientação para o modelo expositivo em que o trabalho fica quase exclusivamente do lado do aluno.

Esta ferramenta parece-me muito interessante, e pode ajudar a construir uma e-actividade que responda a estes requisitos aqui sintetizados neste parágrafo.

A true

DOI: 10.26508/lsa.202302458

Resource: BDSC_55271

Curator: @DavidDeutsch

SciCrunch record: RRID:BDSC_55271

What is this?

DOI: 10.3892/or.2024.8760

Resource: (Cell Signaling Technology Cat# 9102, RRID:AB_330744)

Curator: @scibot

SciCrunch record: RRID:AB_330744

What is this?

Preferir 60/90min de 0 counts per minute para determinar Non Wear Time. Falta determinar si es con o sin allowance de pequeños movimientos por max 2 min

tipo de azúcar

I don't think that is a valid comparison but o well

i agree

• Reintegração: Esbulho
• Manutenção: Turbação
• Interdito Proibitório: justo receio de molestar posse.

Vide art. 1.210/CC

SÚMULA 637/STJ - O ente público detém legitimidade e interesse para intervir, incidentalmente, na ação possessória entre particulares, podendo deduzir qualquer matéria defensiva, inclusive, se for o caso, o domínio.

It can be useful to take some mineral spirits, naphtha, or paint thinner and a tooth brush (or, even better, a brass bristle brush) to your typeface every now and then to clean the ink, dirt, paper, bits of ribbon, dust, etc. out of it. Doing it after changing ribbon is always a good idea. If you're really hard pressed, nail polish remover (acetone) or rubbing alcohol and a cotton ball along with a small pin tip for the loops of letters like 'a', 'o', 'p', 'g', etc. can be used.

How well (or not) your typewriter works from a print perspective can also change with the type of paper you're using, what your ribbon is made out of (usually nylon, silk, or cotton), how much ink it's got in it and how old/worn it may be. A good backing sheet behind your typing paper can also be helpful.

reply to u/kirrachristine at https://www.reddit.com/r/typewriters/comments/1dtuksy/one_letter_weirdly_thicksmudged/ RE: one letter weirdly thick/smudged

SEÇÃO III

Da locação não residencial

Art. 51. Nas locações de imóveis destinados ao comércio, o locatário terá direito a renovação do contrato, por igual prazo, desde que, cumulativamente:

• I - o contrato a renovar tenha sido celebrado por escrito e com prazo determinado;

• II - o prazo mínimo do contrato a renovar ou a soma dos prazos ininterruptos dos contratos escritos seja de cinco anos;

• III - o locatário esteja explorando seu comércio, no mesmo ramo, pelo prazo mínimo e ininterrupto de três anos.

图的说明： 在这个模型中，main reactor（主反应堆）主要负责接受新的客户端连接请求。这包括处理accept事件，接受新连接，并将新连接分派给sub reactor（子反应堆）。

sub reactor则负责处理具体的I/O事件，如读事件（read）、写事件（write）、以及错误事件（error）。这些I/O事件的处理由sub reactor中的各个EventLoop实例来完成，每个实例都会有自己的Poller和Channel来处理具体的事件。

总结来说： - main reactor负责处理新的连接请求（连接事件）。 - sub reactor负责处理已建立连接的I/O事件（可读、可写、错误事件）。

populated sea shore is worth documenting reinforces that idea the battery park ocean is important

persistent workers reveals importance of water and cargos on ships

commune based workers

populated ocean by traders and merchants

like minded social members

environment takes social awareness

ocean environment

Author response:

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

Reviewer #1:

(1) Point for more elaborate discussion: Apparently the timescale of negative feedback signals is conserved between endothelial cell migration in vitro (with human cells) and endothelial migration during the formation of ISVs in zebrafish. What do you think might be an explanation for such conserved timescales? Are there certain processes within cytoskeletal tension build up that require this quantity of time to establish? Or does it relate to the time that is needed to begin to express the YAP/TAZ target genes that mediate feedback?

The underlying mechanisms responsible for the conserved timescale is a major direction that we continue to explore. Localization of YAP/TAZ to the nucleus is likely not rate-limiting. We showed previously that acute RhoA activation produced significant YAP/TAZ nuclear localization within minutes, while subsequent co-transcriptional activity aligned with the gene expression dynamics observed here (Berlew et al., 2021). We hypothesize that the dynamics of YAP/TAZdependent transcription and the translation of those target genes are rate-limiting for initial feedback loop completion (tic = 4 hours). This is supported by work from us and others in a variety of cell lines showing YAP/TAZ transcriptional responses take place during the first few hours after activation. (Franklin et al., 2020; Mason et al., 2019; Plouffe et al., 2018) While our data identify mediators of initial feedback loop completion, the molecular effectors that determine the timescale of new cytoskeletal equilibrium establishment (teq = 8 hours) remain unclear.

(2) Do you expect different timescales for slower endothelial migratory processes (e.g. for instance during fin vascular regeneration which takes days)?

We selected the ISV development model because it exhibits similar migratory kinetics to our previously-explored human ECFC migration in vitro. The comparable kinetics allowed us to study dynamics of the feedback loop in vivo on similar time scales, but we have not explored models featuring either slower or faster dynamics. 

It would be interesting to test how feedback dynamics are impacted in distinct endothelial migratory processes. Our data suggest that the feedback loop is necessary for persistent migration; however, YAP and TAZ respond to a diversity of upstream regulators in addition to mechanical signals, which might depend on the process of vascular morphogenesis. For example, after fin amputation, inflammation and tissue regeneration may impact the biochemical and mechanical environment experienced by the endothelium. Additionally, cells display different migratory behaviors in ISV morphogenesis compared to fin regeneration. During ISV formation, sprouting tip cells migrate dorsally through avascular tissue, followed by stalk cells. (Ellertsdóttir et al., 2010) In contrast, the fin vasculature regenerates by forming an intermediate vascular plexus, where some venous-derived endothelial cells migrate towards the sprouting front, while others migrate against it. (Xu et al., 2014) We are excited to study the role of this feedback loop in these different modes of neovessel formation in future studies.

(3) Is the ~4hrs and 8hrs feedback time window a general property or does it differ between specific endothelial cell types? In the veins the endothelial cells generate less stress fibers and adhesions compared to in the arteries. Does this mean that there might be a difference in the feedback time window, or does that mean that certain endothelial cell types may not have such YAP/TAZcontrolled feedback system?

Recent studies suggest that venous endothelial cells are the primary endothelial subtype responsible for blood vessel morphogenesis. (Lee et al., 2022, 2021; Xu et al., 2014) They are highly motile and mechanosensitive, migrating against blood flow. (Lee et al., 2022) The Huveneers group has shown that the actin cytoskeleton is differently organized in adult arteries and veins in response to biomechanical properties of its extracellular matrix, rather than intrinsic differences between arterial and venous cells. (van Geemen et al., 2014) This suggests that arterial and venous cells have distinct cytoskeletal setpoints due to mechanical cues in their environment (Price et al., 2021). We expect this to impact the degree of cytoskeletal remodeling and cell migration at equilibrium, rather than the kinetics of the feedback loop per se, though we have not yet tested this hypothesis. Testing these predictions on cytoskeletal setpoint stability and adaptation is a major direction that we continue to explore. 

(4) The experiments are based on perturbations to prove that transcriptional feedback is needed for endothelial migration. What would happen if the feedback systems is always switched on? An experiment to add might be to analyse the responsiveness of endothelial cells expressing constitutively active YAP/TAZ.

This is a problem that we are actively pursuing. Though the feedback system forms a coherent loop, we anticipate that the identity of the node of the loop selected for constitutive activation will influence the outcome, depending on whether that node is rate-limiting for feedback kinetics and the extent of intersection of that node with other signaling events in the cell. For example, we have observed that constitutive YAP activation drives profound changes to the transcriptional landscape including, but not limited to, RhoA signaling (Jones et al., 2023). We further anticipate that constitutive activation of feedback loop nodes may alter feedback dynamics, while dynamic or acute perturbation will be required to dissect these contributions in real time. For these reasons, ongoing work in the lab is pursuing these questions using optogenetic tools that enable precise spatial and temporal control (Berlew et al., 2021).   

(5) To investigate the role of YAP-mediated transcription in an accurate time-dependent manner the authors may consider using the recently developed optogenetic YAP translocation tool: https://doi.org/10.15252/embr.202154401

We are enthusiastic about the power of optogenetics to interrogate the nodes and timescales of this feedback system, and we are now funded to pursue this line of research. 

Reviewer #2:

The idea is intriguing, but it is not clear how the feedback actually works, so it is difficult to determine if the events needed could occur within 4 hrs. Specifically, it is not clear what gene changes initiated by YAP/TAZ translocation eventually lead to changes in Rho signaling and contractility. Much of the evidence to support the model is preliminary. Some of the data is consistent with the model, but alternative explanations of the data are not excluded. The fish washout data is quite interesting and does support the model. It is unclear how some of the in vitro data supports the model and excludes alternatives.

Major strengths:

The combination of in vitro and in vivo assessment provides evidence for timing in physiologically relevant contexts, and a rigorous quantification of outputs is provided. The idea of defining temporal aspects of the system is quite interesting.

Major weaknesses:

The evidence for a "loop" is not strong; rather, most of the data can also be interpreted as a linear increase in effect with time once a threshold is reached. Washout experiments are key to setting up a time window, yet these experiments are presented only for the fish model. A major technical challenge is that siRNA experiments take time to achieve depletion status, making precise timing of events on short time scales problematic. Also, Actinomycin D blocks most transcription so exposure for hours likely leads to secondary and tertiary effects and perhaps effects on viability. No RNA profiling is presented to validate proposed transcriptional changes.

We thank the reviewer for these helpful suggestions. We have expanded our explanation of the history and known mediators of the feedback loop in the introduction. We and, independently, the Huveneers group recently reported that human endothelial cells maintain cytoskeletal equilibrium for persistent motility through a YAP/TAZ-mediated feedback loop that modulates cytoskeletal tension. (Mason et al., 2019; van der Stoel et al., 2020) Because YAP and TAZ are activated by tension of the cytoskeleton (Dupont et al., 2011), suppression of cytoskeletal tension by YAP/TAZ transcriptional target genes constitutes a negative feedback loop (Fig. 1A). We described key components of this cell-intrinsic feedback loop, which acts as a control system to maintain cytoskeletal homeostasis for persistent motility via modulation of Rho-ROCK-myosin II activity. (Mason et al., 2019) Both we and the Huveneers group found that perturbation of genes and pathways regulated by YAP/TAZ mechanoactivation can functionally rescue motility in YAP/TAZ-depleted cells (e.g., RhoA/ROCK/myosin II, NUAK2, DLC1). (Mason et al., 2019; van der Stoel et al., 2020) We further showed previously that both YAP/TAZ depletion and acute YAP/TAZ-TEAD inhibition consistently increased stress fiber and FA maturation and arrested cell motility, accounting for these limitations of siRNA. (Mason et al., 2019)

Enduring limitations to the temporal, spatial, and cell-specific control of the genetic and pharmacologic methods have inspired us to initiate alternative approaches, which are the subject of ongoing efforts. Further research will be necessary in the zebrafish to determine the extent to which the observed migratory dynamics are driven by cytoskeletal arrest. 

To identify early YAP/TAZ-regulated transcriptional changes, we have added RNA profiling of control and YAP/TAZ depleted cells cultured on stiff matrices for four hours. Genes upregulated by YAP/TAZ depletion were enriched for Gene Ontology (GO) terms associated with Rho protein signal transduction, vascular development, cellular response to vascular endothelial growth factor (VEGF) stimulus, and endothelial cell migration (Fig. 9B). These data support a role for YAP and TAZ as negative feedback mediators that maintain cytoskeletal homeostasis for endothelial cell migration and vascular morphogenesis.  

Reviewer #3:

The authors used ECFC - endothelial colony forming cells (circulating endothelial cells that activate in response to vascular injury).

Q: Did the authors characterize these cells and made sure that they are truly endothelial cells - for example examine specific endothelial markers, arterial-venous identity markers & Notch signalling status, overall morphology etc prior to the start of the experiment. How were ECFC isolated from human individuals, are these "healthy" volunteers - any underlying CVD risk factors, cells from one patient or from pooled samples, what injury where these humans exposed to trigger the release of the ECPFs into the circulation, etc. The materials & methods on ECFC should be expanded.

Human umbilical cord blood-derived ECFCs were isolated at Indiana University School of Medicine and kindly provided by Dr Mervin Yoder. Cells were cultured as described by the Yoder group (Rapp et al., 2011) and our prior paper (Mason et al., 2019). We have expanded the materials and methods section to describe the source and characterization of these cells.

The authors suggest that loss of YAP/TAZ phenocopies actinomycin-D inhibition - "both transcription inhibition and YAP/TAZ depletion impaired polarization, and induced robust ventral stress fiber formation and peripheral focal adhesion maturation". However, the cell size of actinomycin-D treated cells (Fig. 1B, top right panel), differs from the endothelial cell size upon siYAP/TAZ (Fig. 1E, top right panel) - and vinculin staining seems more pronounced in actinomycin-D treated cells (B, bottom right) when compared to siYAP/TAZ group. Cell shape is defined by acto-myosin tension.

Q: Besides Fraction of focal adhesion >1um; focal adhesion number did the authors measure additional parameters related to cytoskeleton remodelling / focal adhesions that can substantiate their statement on similarity between loss of YAP/TAZ and actinomycin-D treatment. Would it be possible to make a more specific genetic intervention (besides YAP/TAZ) interfering with the focal adhesion pathway as opposed to the broad spectrum inhibitor actinomyocin-D.

Our previous paper (Mason et al., 2019) delineated the mechanistic relationships between YAP/TAZ signaling, focal adhesion turnover, actomyosin polymerization, and the intervening mechanisms of myosin regulation. Specifically, we demonstrated that YAP/TAZ regulate the myosin phosphatase kinase, NUAK2, and ARHGAP genes to mediate this feedback. Expanding on this work, the current study aimed to define the temporal kinetics of the cytoskeletal mechanotransductive feedback in vitro and in vivo. We used actinomycin-D and YAP/TAZ depletion to interrogate the role of transcriptional regulation and YAP/TAZ signaling, respectively. In this revision, we have added RNA profiling that identifies early YAP/TAZ-regulated transcriptional changes and further points to other molecular mediators of focal adhesions (e.g. TRIO, RHOB, THBS1) that will be the subjects of future studies.    

Q: Does the actinomycin-D treatment affect responsiveness to Vegf? induce apoptosis or reduce survival of the ECFC?

We have not looked specifically at the effect of actinomycin-D treatment on responsiveness to VEGF. However, actinomycin-D has been reported to reduce transcription of VEGF receptors (E et al., 2012). In contrast, we found that YAP/TAZ depletion upregulated GO terms associated with endothelial cell migration and response to VEGF stimulus (Fig. 9B), as well as receptors to angiogenic growth factors, including KDR and FLT4 (Fig. 9E). These results suggest YAP/TAZ depleted cells may be more sensitive to VEGF stimulation but remain nonmotile due to cytoskeletal arrest.

We showed previously that long-term treatment with actinomycin-D reduces ECFC survival (Mason et al., 2019).

Q: Which mechanism links ECM stiffness with endothelial surface area in the authors scenario. In zebrafish, activity of endothelial guanine exchange factor Trio specifically at endothelial cell junctions (Klems, Nat Comms, 2020) and endoglin in response to hemodynamic factors (Siekmann, Nat Cell Biol 2017) have been show to control EC shape/surface area - do these factors play a role in the scenario proposed by the authors.

Our new transcriptional profiling indicates both Trio and endoglin are regulated through YAP and TAZ in human ECFCs. We plan to follow up on these findings.

Q: The authors report that EC migrate faster on stiff substrate, and concomitantly these cells have a larger surface area. What is the physiological rationale behind these observations. Did the authors observe such behaviors in their zebrafish ISV model? How do these observations integrate with the tip - stalk cell shuffling model (Jakobsson & Gerhardt, Nat Cell Biol, 2011) and Notch activity in developing ISVs.

This question raises important distinctions between the mode of migration in ISV morphogenesis and endothelial cells adherent to substrates. Cells behave and respond to mechanical cues differently in 2D vs. 3D matrices. (LaValley and Reinhart-King, 2014) Additionally, the microenvironment in vivo is much more complex, combining numerous biochemical signals and changing mechanical properties. (Whisler et al., 2023) We are actively investigating the downstream targets of YAP/TAZ mechanotransduction and how that integrates with other pathways known to regulate vascular morphogenesis, such as Notch signaling. 

The authors examined the formation of arterial intersegmental vessels in the trunk of developing zebrafish embryos in vivo. They used a variety of pharmacological inhibitors of transcription and acto-myosin remodelling and linked the observed morphological changes in ISV morphogenesis with changes in endothelial cell motility.

Q: Reduced formation and dorsal extension of ISVs may have several reasons, including reduced EC migration and proliferation. The Tg(fl i1a:EGFP) reporter however is not the most suitable line to monitor migration of individual endothelial cells. Can the authors repeat the experiments in Tg(fl i1a:nEGFP); Tg(kdrl:HRAS-mCherry) double transgenics to visualize movement-migration of the individual endothelial cells and EC proliferation events, in the different treatment regimes.

So far, we have not tracked individual endothelial cells during ISV morphogenesis. We agree this is the best approach and are pursuing a similar technique for these experiments.

ISV formation is furthermore affected by Notch signalling status and a series of (repulsive) guidance cues.

Q: Does de novo blockade of gene expression with Actinomycin D affect Notch signalling status, expression of PlexinD - sFlt1, netrin1 or arterial-venous identify genes.

While we have not performed gene expression analysis under the Actinomycin D condition, Actinomycin D functions as a broad transcription inhibitor. We are currently pursuing the downstream targets of YAP/TAZ mechanotransduction in both ECFCs and zebrafish.

Remark: The authors may want to consider using the Tg(fl i1:LIFEACT-GFP) reporter for in vivo imaging of actin remodelling events.

We thank the reviewer for their helpful suggestion.

Remark: the authors report "As with broad transcription inhibition, in situ depletion of YAP and TAZ by RNAi arrested cell motility, illustrated here by live-migration sparklines over 10 hours: siControl: , siYAP/TAZ: (25 μm scale-bar: -)". Can the authors make a separate figure panel for this, how many cells were measured?

Please refer to our previous publication for the complete details on this data (Mason et al., 2019). We have added the citation in the text.

Remark: in the wash-out experiments, exposure to the inhibitors is not the same in the different scenarios - could it be that the longer exposure time induces "toxic" side effect that cannot be "washed out" when compared to the short treatment regimes?

This is a possible limitation of the pharmacological approach and have included it in the discussion section. We are currently exploring alternative approaches to interrogate the timescale of the feedback loop more precisely.  

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Chris foi repreendido por ela pela primeira vez por distribuir cópias do seu boletim informativo "Sonichu's News Dash", mas o golpe mais devastador ocorreu quando Chris estava perambulando com seu habitual Cartaz de Atração; de acordo com Chris, Walsh rasgou seu cartaz e disse-lhe que ele nunca conseguiria uma namorada desse jeito, ou de fato de qualquer maneira. Por mais verdadeira que essa alegada declaração pudesse ser, há praticamente nenhuma chance de Mary ter dito isso, pois seria altamente inapropriado e colocaria seu emprego em risco.

Por causa de todo o estresse causado por Mary Lee Walsh, Chris remoía e obsessivamente pensava sobre sua derrota "ESMAGADORA DO CORAÇÃO" e começou a reinventar todo o cenário para que ele saísse como o vencedor. Assim, Walsh começou a aparecer em muitas das obras de Chris como uma antagonista principal, especificamente se opondo à sua Busca pelo Amor.

Posteriormente seu nome seria trocado por Slaweel Ryam na Saga Myamoto

Para além das vantagens referidas, penso que também deverá ser identificada a mais valia de não existir necessidade de uma deslocação física. A aprendizagem através de um ambiente digital permite que alargar esse conhecimento, bastando que seja necessário a existência de internet.

Achei bastante interessante esta aplicação Hypothes.is. Trata-se de uma ferramenta bastante útil que, em minha opinião, facilita bastante a leitura e a discussão de textos na web. Sobre o texto destacado, esse pensamento reflete uma visão moderna e dinâmica sobre o ensino e a aprendizagem, especialmente no contexto digital. Concordo com a ideia de que precisamos ir além da análise clássica de ambientes de aprendizagem e focar nas novas perspetivas oferecidas pelas tecnologias digitais e pelas e-atividades.

Anderson S. Araújo

A utilização de quizzes como forma de sistematização da aprendizagem é uma forma muito eficaz de, por um lado permitirmos aos alunos/formandos fazerem uma auto-avaliação da sua aprendizagem e, por outro lado, o professor/formador disponibilizar uma espécie de súmula daquilo que são os principais takeaways sobre um determinado tópico.

Saudações académicas, José Martins

A parte da avaliação no ensino a distância foi algo que sempre me suscitou algumas dúvidas. É verdade que através das e-atividades podes fazer avaliação, mas o que é que nos garante que estamos a avaliar quem queremos avaliar, já que não sabemos quem está efetivamente a fazer a e-atividade? Por exemplo, um teste feito no moodle, como posso saber se quem o fez foi mesmo o formando/aluno que quero avaliar? Parece-me que a única forma seria num ambiente síncrono, com câmara ligada, fazer questões diretamente ao aluno. Ou existe outra forma?

O link, no meu caso , não abriu. Só abre em http://www.gillysalmon.com/ (http sem segurança).

Um dos recursos para a criação de fóruns de discussão síncronos que muito me surpreendeu , pela positiva, foi o kumospace . Criei um forum de discussão no âmbito de uma e-atividade de uma formação anterior e do qual partilho o link. https://www.kumospace.com/invite/AfjVspXSZg06kxCNNjmM

Olá, Esta ferramenta parece bastante útil :) .

Ao ler o capítulo fico com a questão se derivar E-atividades em E-exercícios ou E-tarefas será útil ou acabam por ser conceitos redundantes e que apenas adicionam complexidade à taxonomia usada no contexto dos ambientes digitais.

Obrigado! Luis Dias

Considero que o cerne da questão colocada acima pela colega Idalina Santos(relativamente à reflexão proposta sobre se os estilos de aprendizagem são verdadeiros ou mitos) está nesta afirmação. Mais do que rotular alunos e processos de ensino aprendizagem é fundamental diversificar e ampliar o potencial da aprendizagem fazendo uso de uma diversidade de recursos, sem nunca esquecer a intecionalidade pedagógica que temos vindo a discutir ao longo das últimas semanas!

Concordo, que neste tipo de ensino, mais do que no ensino presencial e com estas e-atividades, o aluno tem que ser mais autónomo na busca de informação, na aprendizagem de novas ferramentas tecnológicas.

NOOO

so he was somewhat honorble

ufisflc;o;sfnjeROBB

that guy...

that kids mental health is down the drain

bro

nah they kinda have chemistry...

nooo but i wanna meet edmure

ooo them i know theyd get the job done

yess

cut their heads off....

YESS

yay hes not scared of wolves

oh i didnt know he was a noble

YUP

he's trying his best

the wolves match them all so perfectly

ravens...

he loves his siblings so much omg

supersinly sweet in a way but atleast she can rest now

YES CAT

oh what??

help this kid is built different

oh! uhhh

pls dont

no aftercare??

NOOO

baratheons did kill ned

fr

now thats a REAL leader

crazy

DOI: 10.3390/bios14060284

Resource: Addgene (RRID:SCR_002037)

Curator: @olekpark

SciCrunch record: RRID:SCR_002037

What is this?

DOI: 10.1016/j.isci.2024.110132

Resource: (Cell Signaling Technology Cat# 2348, RRID:AB_331212)

Curator: @scibot

SciCrunch record: RRID:AB_331212

What is this?

Muito interessante ao pensarmos na vertente do ensino universitario, para que o ensino universitário permaneça relevante e competitivo, é essencial que as instituições adotem uma abordagem mais aberta às inovações disruptivas. Isso requer não apenas a implementação de novas tecnologias e métodos, mas também uma reavaliação das estratégias educacionais e da estrutura organizacional para fomentar a criatividade e a inovação. Assim, as universidades podem equilibrar a busca por resultados imediatos com a exploração de novas oportunidades, garantindo sua relevância e impacto no futuro da educação.

De acordo com o estudo "A Study Comparing Traditional and Hybrid Internet-Based Instruction in Introductory Statistics Classes" (https://doi.org/10.1080/10691898.2003.11910722) "Performance of students in the hybrid offering equaled that of the traditional students, but students in the hybrid were slightly less positive in their subjective evaluation of the course".

Talvez uma das razões para esta avaliação menos positiva seja a necessidade/dificuldade em haver esta conexão entre as modalidades referidas aqui e o próprio processo de adaptação dos docentes a esta modalidade de ensino.

Mudança, Transformação, Flexibilidade, Interação, Qualidade de Aprendizagem, Personalização, são algumas palavras-chave que fazem parte das estratégias para desenvolver modelos pedagógicos inovadores e plenamente integrados com a atual sociedade no século XXI. Devemos adotar uma estratégia de inovação sustentada ou disruptiva? A experiência, proveniente da indústria, das soluções híbridas e da sua adequação aos mercados onde não há não-consumidores, leva-nos a considerar como adequados modelos pedagógicos híbridos, que combinam metodologias online com metodologias presenciais, procurando combinar o melhor de cada uma, numa estratégia simultaneamente integradora e gradualmente disruptiva.

Por uma questão de clareza pareceu-me adequado incluir, como anotação, as várias definições resumidas dos modelos referidos na nota de rodapé.

Rotação por Estações: os alunos alternam entre atividades online e offline dentro da sala de aula. O espaço é dividido em estações, e pelo menos uma delas deverá ser obrigatoriamente online. Cada estação aborda o mesmo tema central da aula, mas de maneiras diferentes para atender aos diversos estilos de aprendizagem. Os alunos percorrem todas as estações durante a aula, como se fosse um circuito.

Rotação Individual: os alunos têm um cronograma personalizado de rotação entre diferentes atividades, adaptado às suas necessidades de aprendizado.

Laboratório Rotacional: a rotação dos alunos ocorre entre a sala de aula e um laboratório de aprendizagem, como uma sala de informática. Esse modelo combina características da sala de aula tradicional e do ensino online.

Sala de Aula Invertida: os alunos estudam o conteúdo em casa (geralmente por meio de vídeos ou materiais online) e, em seguida, aplicam o conhecimento na sala de aula, onde o professor pode esclarecer dúvidas e promover discussões.

Flex: permite que os alunos escolham entre aulas presenciais e online, adaptando o ensino às suas preferências e necessidades individuais.

À La Carte: um programa no qual os alunos participam e um ou mais cursos inteiramente online, com um professor online, e ao mesmo tempo continuam a ter experiências educacionais em escolas tradicionais. Os alunos podem participar das aulas online tanto no campus físico como em outros lugares.

Virtual Enriquecido: combina aulas online com experiências práticas, como projetos, laboratórios ou atividades em grupo.

A ênfase que tem sido colocada na sustentabilidade, tanto no que respeita ao desenvolvimento da sociedade e da economia, como da educação e do uso das tecnologias digitais, nomeadamente por parte de organismos internacionais de referência, é digna de destaque. Podemos verificar isto mesmo na Agenda 2030 da Organização das Nações Unidas (ONU, 2016), pela definição de 17 objetivos que definem as prioridades e aspirações para o desenvolvimento sustentável a nível global, bem como no Relatório da UNESCO (2022) “Reimaginar nossos futuros juntos. Um novo contrato social para a educação”, onde se apela a um novo contrato social para a educação, fundamentado nos direitos humanos e na ação coletiva para a paz, a justiça e a sustentabilidade. Ainda a OECD (2023), recomenda aos decisores políticos e às instituições educativas o desenvolvimento de uma visão estratégica e coordenação de políticas, visando a integração sustentada das tecnologias digitais na educação.

A disrupção, ao tornar os produtos mais baratos, veio trazer novas oportunidades, ao nível da educação, que há alguns anos seriam impossíveis de implementar. Um professor terá de estar sempre atento às desigualdades sociais no acesso à educação: famílias com menor poder aquisitivo geralmente possuem menos acesso a materiais didáticos adequados e oportunidades extracurriculares, perpetuando ciclos de pobreza e desigualdade. Uma atividade só poderá ser planeada quando se assegura que todos os estudantes terão a possibilidade de acesso aos recursos que permitem a sua execução. A disrupção é um processo contínuo que está constantemente moldando o panorama de produtos, serviços e modelos de negócios. É importante estar atento às novas tendências e tecnologias para se aproveitar as oportunidades que a disrupção pode trazer.

O modelo híbrido, também conhecido como "blended learning", combina atividades presenciais e online, aproveitando os benefícios de ambos os formatos para criar uma experiência de aprendizagem mais rica e flexível. Este modelo tem-se tornado mais popular devido à sua capacidade em adaptar-se às necessidades individuais dos estudantes e às exigências de um mundo cada vez mais digital.

Quais as características do Modelo Híbrido? 1) A flexibilidade, uma vez que permite que os estudantes escolham quando e onde aprender, combinando as sessões presenciais com as atividades online. Isso é particularmente útil para os estudantes que precisam de horários flexíveis ou que têm outras responsabilidades, como o trabalho ou a atenção e o tempo dedicado à família.

2) A personalização, já que a aprendizagem híbrida possibilita a personalização do conteúdo de acordo com o ritmo e as preferências de aprendizagem de cada estudante. As ferramentas de e-learning podem adaptar o material às necessidades individuais, oferecendo uma abordagem mais centrada no aluno.

3) A interação e a colaboração, dado que mesmo no formato online, o modelo híbrido promove a interação e a colaboração entre os estudantes. As ferramentas como os fóruns de discussão, os chats e as videoconferências permitem que os alunos trabalhem juntos em projetos e que possam trocar ideias.

4) O acesso a recursos diversificados, pois os estudantes têm acesso a uma variada gama de recursos educativos online, desde vídeos e podcasts até a artigos científicos e e-books. Isso enriquece a experiência de aprendizagem e proporciona múltiplas fontes de informação.

Como implementar o Modelo Híbrido?

Para implementar, de forma eficaz, o modelo híbrido, é necessário um planeamento cuidadoso e a escolha das ferramentas adequadas, como por exemplo o planeamento de quais as partes ou secções do conteúdo da unidade curricular deverão trabalhadas online e quais as que deverão ser abordadas presencialmente; a seleção das plataformas e das ferramentas que irão ser úteis na aprendizagem online; a capacitação dos docentes para a utilização as tecnologias e metodologias de ensino híbrido de forma eficaz; e a implementação de métodos de avaliação que integrem tanto atividades online quanto atividades presenciais. Note-se que o feedback contínuo é fundamental para ajustar o processo de ensino-aprendizagem e para garantir que os objetivos educacionais sejam alcançados.

Referências Utilizadas https://blogs.worldbank.org/es/education/que-es-el-aprendizaje-hibrido-como-pueden-los-paises-implementarlo-de-manera-efectiva https://ciberespiral.org/es/modelos-hibridos-para-promover-el-aprendizaje/ https://ec.europa.eu/education/education-in-the-eu/digital-education-action-plan_en https://eadbox.com/o-que-e-ensino-hibrido/ https://tutormundi.com/blog/ensino-hibrido/ https://observatoriodeeducacao.institutounibanco.org.br/em-debate/ensino-hibrido https://www.arvore.com.br/blog/aprendizagem-hibrida https://revistaft.com.br/o-ensino-hibrido-sob-a-otica-do-professor-desafios-estrategias-e-reflexoes/ https://blog.eadplataforma.com/educacao/modelos-de-ensino-hibrido/ https://www.christenseninstitute.org/publication/ensino-hibrido/ https://iave.pt/wp-content/uploads/2022/08/1_ensaio.pdf https://desafiosdaeducacao.com.br/ensino-hibrido-guia/

Arriscar-me-ia a dizer que toda esta questão que orbita a inovação disruptiva se pode sintetizar em evolução. Quando o projetor de slides surgiu, estivemos na presença de uma disrupção (evoluímos, introduzimos, resistimos, utilizamos, adaptamos e extraímos o seu potencial em matéria de prática pedagógica). O mesmo aconteceu com o projeto de vídeo e o diapositivo, assim como aconteceu, mais recentemente, com o Zoom, o Teams, e tudo aquilo que está para vir. A disrupção julgo resultar apenas do grau de desconforto que o reconhecimento da necessidade de mudar, e os próprios processos de mudança, geram em nós. Por isso destaco o meu entendimento em como este processo de mudança não é mais do que um processo evolutivo, pelo qual, analogamente, outros já passaram no passado, pelo qual estamos a passar (com a exposição a esta nova realidade e à responsabilidade de a integrar na nossa prática pedagógica), e pelo qual vamos continuar a passar até ao fim das nossas carreiras. É a “prática antiga” com um gostinho de presente, olhando para o futuro. Relativamente ao hypothes.is, não conhecia o recurso, mas estou muito interessado em explorá-lo. Vejo grande potencial naquilo que é o acompanhamento do trabalho dos estudantes quando disponibilizamos textos de apoio de especial relevância, designadamente pela possibilidade de feedback “in situ”, utilizando uma expressão familiar ao domínio da saúde.

I would set the scene around who Dan is first... something like: Meet your fellow ADHDer Dan.

Infopiguła:

Liczba Polaków zarabiających ponad 10 tys. zł / mc wzrosła w 2022 r. o 51% rdr., do 1,5 mln osób. Na koniec 2022 r. było w Polsce 90 tys. milionerów dolarowych, 10% mniej niż rok wcześniej.

Pensje zarabiających ponad 10 tys. wzrosły o 10% do ok. 375 mld zł. Głównym czynnikiem była wysoka inflacja, ale też napływ Ukraińców z dobrymi zarobkami w korporacjach.

Liczba osób z zarobkami 20-50 tys. zł / mies. wzrosła o 38% do 440 tys., tych z zarobkami między 50 a 83 tys. zł wzrosła o 1% do 84 tys., a osób z zarobkami ponad 83 tys. zł (czyli 1 mln rocznie) spadła o 5% do 35 tys. O prawie 2% wzrosła liczba osób z majątkiem ponad 50 mln $.

MDMA-assisted therapy for severe PTSD: a randomized, double-blind, placebo-controlled phase 3 study

• Jennifer M. Mitchell, 
• Michael Bogenschutz, 
• Alia Lilienstein, 
• Charlotte Harrison, 
• Sarah Kleiman, 
• Kelly Parker-Guilbert, 
• Marcela Ot'alora G., 
• Wael Garas, 
• Casey Paleos, 
• Ingmar Gorman, 
• Christopher Nicholas, 
• Michael Mithoefer, 
• Shannon Carlin, 
• Bruce Poulter, 
• Ann Mithoefer, 
• Sylvestre Quevedo, 
• Gregory Wells, 
• Sukhpreet S. Klaire, 
• Bessel van der Kolk, 
• Keren Tzarfaty, 
• Revital Amiaz, 
• Ray Worthy, 
• Scott Shannon, 
• Joshua D. Woolley, 
• ...
• Rick Doblin 

Show authors

Nature Medicine volume 27, pages1025--1033 (2021)Cite this article

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Matters Arising to this article was published on 11 October 2021

Matters Arising to this article was published on 11 October 2021

Abstract

Post-traumatic stress disorder (PTSD) presents a major public health problem for which currently available treatments are modestly effective. We report the findings of a randomized, double-blind, placebo-controlled, multi-site phase 3 clinical trial (NCT03537014) to test the efficacy and safety of 3,4-methylenedioxymethamphetamine (MDMA)-assisted therapy for the treatment of patients with severe PTSD, including those with common comorbidities such as dissociation, depression, a history of alcohol and substance use disorders, and childhood trauma. After psychiatric medication washout, participants (n = 90) were randomized 1:1 to receive manualized therapy with MDMA or with placebo, combined with three preparatory and nine integrative therapy sessions. PTSD symptoms, measured with the Clinician-Administered PTSD Scale for DSM-5 (CAPS-5, the primary endpoint), and functional impairment, measured with the Sheehan Disability Scale (SDS, the secondary endpoint) were assessed at baseline and at 2 months after the last experimental session. Adverse events and suicidality were tracked throughout the study. MDMA was found to induce significant and robust attenuation in CAPS-5 score compared with placebo (P < 0.0001, d = 0.91) and to significantly decrease the SDS total score (P = 0.0116, d = 0.43). The mean change in CAPS-5 scores in participants completing treatment was -24.4 (s.d. 11.6) in the MDMA group and -13.9 (s.d. 11.5) in the placebo group. MDMA did not induce adverse events of abuse potential, suicidality or QT prolongation. These data indicate that, compared with manualized therapy with inactive placebo, MDMA-assisted therapy is highly efficacious in individuals with severe PTSD, and treatment is safe and well-tolerated, even in those with comorbidities. We conclude that MDMA-assisted therapy represents a potential breakthrough treatment that merits expedited clinical evaluation.

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Main

PTSD is a common and debilitating condition with immeasurable social and economic costs that affects the lives of hundreds of millions of people annually. There are a number of environmental and biological risk factors that contribute to the development and maintenance of PTSD1, and poor PTSD treatment outcomes are associated with several comorbid conditions that include childhood trauma2, alcohol and substance use disorders3, depression4, suicidal ideation5 and dissociation6. It is therefore imperative to identify a therapeutic that is beneficial in those individuals with the comorbidities that typically confer treatment resistance.

The selective serotonin reuptake inhibitors (SSRIs) sertraline and paroxetine are Food and Drug Administration (FDA)-approved first-line therapeutics for the treatment of PTSD. However, an estimated 40--60% of patients do not respond to these compounds7. Likewise, although evidenced-based trauma-focused psychotherapies such as prolonged exposure and cognitive behavioral therapy are considered to be the gold standard treatments for PTSD8, many participants fail to respond or continue to have significant symptoms, and dropout rates are high9,10. Novel cost-effective therapeutics are therefore desperately needed11.

The substituted amphetamine 3,4-methylenedioxymethamphetamine (MDMA) induces serotonin release by binding primarily to presynaptic serotonin transporters12. MDMA has been shown to enhance fear memory extinction, modulate fear memory reconsolidation (possibly through an oxytocin-dependent mechanism), and bolster social behavior in animal models13,14. Pooled analysis of six phase 2 trials of MDMA-assisted therapy for PTSD have now shown promising safety and efficacy findings15.

Here, we assess the efficacy and safety of MDMA-assisted therapy in individuals with severe PTSD. Participants were given three doses of MDMA or placebo in a controlled clinical environment and in the presence of a trained therapy team. Primary and secondary outcome measures (CAPS-5 and SDS, respectively) were assessed by a centralized pool of blinded, independent diagnostic assessors. MDMA-assisted therapy for PTSD was granted an FDA Breakthrough Therapy designation, and the protocol and statistical analysis plan (SAP) were developed in conjunction with the FDA16.

Results

Demographics

Participants were recruited from 7 November 2018 to 26 May 2020, with the last participant visit conducted on 21 August 2020. A total of 345 participants were assessed for eligibility, 131 were enrolled, 91 were confirmed for randomization (United States, n = 77; Canada, n = 9; Israel, n = 5), and 46 were randomized to MDMA and 44 to placebo (Fig. 1).

Fig. 1: Procedure timeline and study flow diagram.

a, Procedure timeline. Following the screening procedures and medication taper, participants attended a total of three preparatory sessions, three experimental sessions, nine integration sessions and four endpoint assessments (T1--4) over 18 weeks, concluding with a final study-termination visit. IR, independent rater; T, timepoint of endpoint assessment; T1, baseline; T2, after the first experimental session; T3, after the second experimental session; T4, 18 weeks after baseline. b, CONSORT diagram indicating participant numbers and disposition through the course of the trial.

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Study arms were not significantly different in terms of race, ethnicity, sex, age, dissociative subtype, disability or CAPS-5 score (Table 1). The mean duration of PTSD diagnosis was 14.8 (s.d. 11.6) years and 13.2 (s.d. 11.4) years in the MDMA and placebo groups, respectively. Of note, six participants in the MDMA group and 13 participants in the placebo group had the dissociative subtype according to CAPS-5 score.

Table 1 Demographics and baseline characteristics

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Efficacy

MDMA significantly attenuated PTSD symptomology, as shown by the change in CAPS-5 total severity score from baseline to 18 weeks after baseline. Mixed model repeated measure (MMRM) analysis of the de jure estimand (that is, the effects of the drug if taken as directed) showed a significant difference in treatment arms (n = 89 (MDMA n = 46), P < 0.0001, between-group difference = 11.9, 95% confidence interval (CI) = 6.3--17.4, d.f. = 71) (Fig. 2a). MMRM sensitivity analysis of the de facto estimand (that is, the effects of the drug if taken as assigned, regardless of adherence) showed a significant difference in treatment arms (n = 90, P < 0.0001, d.f. = 72).

Fig. 2: Measures of MDMA efficacy in the MDMA-assisted therapy group and the placebo group.

a, Change in CAPS-5 total severity score from T1 to T4 (P < 0.0001, d = 0.91, n = 89 (MDMA n = 46)), as a measure of the primary outcome. Primary analysis was completed using least square means from an MMRM model. b, Change in SDS total score from T1 to T4 (P = 0.0116, d = 0.43, n = 89 (MDMA n = 46)), as a measure of the secondary outcome. Primary analysis was completed using least square means from an MMRM model. c, Change in BDI-II score from T1 to study termination (t = -3.11, P = 0.0026, n = 81 (MDMA n = 42)), as a measure of the exploratory outcome. Data are presented as mean and s.e.m.

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The mean change in CAPS-5 scores from baseline to 18 weeks after baseline in the completers (per protocol set) was -24.4 (s.d. 11.6) (n = 42) in the MDMA-assisted therapy group compared with -13.9 (s.d. 11.5) (n = 37) in the placebo with therapy group.

The effect size of the MDMA-assisted therapy treatment compared with placebo with therapy was d = 0.91 (95% CI = 0.44--1.37, pooled s.d. = 11.55) in the de jure estimand and d = 0.97 (95% CI = 0.51--1.42) in the de facto estimand. When the within-group treatment effect (which included the effect of the supportive therapy that was administered in both arms) was compared between the MDMA and placebo groups, the effect size was 2.1 (95% CI = -5.6 to 1.4) in the MDMA group and 1.2 (95% CI = -4.9 to 2.5) in the placebo group.

Over the same period, MDMA significantly reduced clinician-rated functional impairment as assessed with the SDS. MMRM analysis of the de jure estimand showed a significant difference in treatment arms (n = 89 (MDMA n = 46), P = 0.0116, d.f. = 71, effect size = 0.43, 95% CI = -0.01 to 0.88, pooled s.d. = 2.53) (Fig. 2b). The mean change in SDS scores from baseline to 18 weeks after baseline in the completers was -3.1 (s.d. 2.6) (n = 42) in the MDMA-assisted therapy group and -2.0 (s.d. 2.4) (n = 37) in the placebo with therapy group.

MDMA was equally effective in participants with comorbidities that are often associated with treatment resistance. Participants with the dissociative subtype of PTSD who received MDMA-assisted therapy had significant symptom reduction on the CAPS-5 (mean MDMA Δ = -30.8 (s.d. 9.0), mean placebo Δ = -12.8 (s.d. 12.8)), and this was similar to that in their counterparts with non-dissociative PTSD (mean MDMA Δ = -23.6 (s.d. 11.7), mean placebo Δ = -14.3 (s.d. 11.2)). The benefit of MDMA therapy was not modulated by history of alcohol use disorder, history of substance use disorder, overnight stay or severe childhood trauma. Results were consistent across all 15 study sites with no effect by study site (P = 0.1003). In MMRM analysis there was no obvious impact of SSRI history on effectiveness of MDMA (Supplementary Table 2).

MDMA therapy was effective in an exploratory endpoint analysis of the reduction of depression symptoms (using the Beck Depression Inventory II (BDI-II)) from baseline to study termination of the de jure estimand (mean MDMA Δ = -19.7 (s.d. 14.0), n = 42; mean placebo Δ = -10.8 (s.d. 11.3), n = 39; t = -3.11, P = 0.0026, d.f. = 79, effect size = 0.67, 95% CI = 0.22--1.12) (Fig. 2c).

Clinically significant improvement (a decrease of ≥10 points on the CAPS-5), loss of diagnosis (specific diagnostic measure on the CAPS-5), and remission (loss of diagnosis and a total CAPS-5 score ≤ 11) were each tracked. At the primary study endpoint (18 weeks after baseline), 28 of 42 (67%) of the participants in the MDMA group no longer met the diagnostic criteria for PTSD, compared with 12 of 37 (32%) of those in the placebo group after three sessions. Additionally, 14 of 42 participants in the MDMA group (33%) and 2 of 37 participants in the placebo group (5%) met the criteria for remission after three sessions (Fig. 3).

Fig. 3: Treatment response and remission for MDMA and placebo groups as a percentage of total participants randomized to each arm (MDMA, n = 46; placebo, n = 44).

Responders (clinically significant improvement, defined as a ≥10-point decrease on CAPS-5), loss of diagnosis (specific diagnostic measure on CAPS-5), and remission (loss of diagnosis and a total CAPS-5 score of ≤11) were tracked in both groups. Non-response is defined as a <10-point decrease on CAPS-5. Withdrawal is defined as a post-randomization early termination.

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Safety

Treatment-emergent adverse events (TEAEs, adverse events that occurred during the treatment period from the first experimental session to the last integration session) that were more prevalent in the MDMA study arm were typically transient, mild to moderate in severity, and included muscle tightness, decreased appetite, nausea, hyperhidrosis and feeling cold (Supplementary Table 3). Importantly, no increase in adverse events related to suicidality was observed in the MDMA group. A transient increase in vital signs (systolic and diastolic blood pressure and heart rate) was observed in the MDMA group (Supplementary Table 4). Two participants in the MDMA group had a transient increase in body temperature to 38.1 °C: one had an increase after the second MDMA session, and one had an increase after the second and third MDMA sessions.

Two participants, both randomized to the placebo group, reported three serious adverse events (SAEs) during the trial. One participant in the placebo group reported two SAEs of suicidal behavior during the trial, and another participant in the placebo group reported one SAE of suicidal ideation that led to self-hospitalization. Five participants in the placebo group and three participants in the MDMA group reported adverse events of special interest (AESIs) of suicidal ideation, suicidal behavior or self-harm in the context of suicidal ideation. One participant in the placebo group reported two cardiovascular AESIs in which underlying cardiac etiology could not be ruled out (Table 2). One participant randomized to the MDMA group chose to discontinue participation due to being triggered by the CAPS-5 assessments and to an adverse event of depressed mood following an experimental session; this participant met the criterion as a non-responder, which was defined as having a less than 10-point decrease in CAPS-5 score. MDMA sessions were not otherwise followed by a lowering of mood.

Table 2 Participants with treatment-emergent SAEs and AESIs

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Suicidality was tracked throughout the study using the Columbia Suicide Severity Rating Scale (C-SSRS) at each study visit. More than 90% of participants reported suicidal ideation in their lifetime, and 17 of 46 participants (37%) in the MDMA group and 14 of 44 participants (32%) in the placebo group reported suicidal ideation at baseline. Although the number of participants who reported suicidal ideation varied throughout the visits, prevalence never exceeded baseline and was not exacerbated in the MDMA group. Serious suicidal ideation (a score of 4 or 5 on the C-SSRS) was minimal during the study and occurred almost entirely in the placebo arm (Fig. 4).

Fig. 4: Number of participants reporting the presence of suicidal ideation as measured with the C-SSRS at each visit and separated by treatment group.

C-SSRS ideation scores range from 0 (no ideation) to 5. A C-SSRS ideation score of 4 or 5 is termed 'serious ideation'. The number of participants endorsing any positive ideation (>0) is shown by the colored bars and noted in the table below the graph. The number of participants endorsing serious ideation is given in parentheses in the table.

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Discussion

Here, we demonstrate that three doses of MDMA given in conjunction with manualized therapy over the course of 18 weeks results in a significant and robust attenuation of PTSD symptoms and functional impairment as assessed using the CAPS-5 and SDS, respectively. MDMA also significantly mitigated depressive symptoms as assessed using the BDI-II. Of note, MDMA did not increase the occurrence of suicidality during the study.

These data illustrate the potential benefit of MDMA-assisted therapy for PTSD over the FDA-approved first-line pharmacotherapies sertraline and paroxetine, which have both exhibited smaller effect sizes in pivotal studies16. Previous comparison of change in CAPS score between sertraline and placebo showed effect sizes of 0.31 and 0.37 (ref. 16). Similarly, comparison of change in CAPS score between paroxetine and placebo showed effect sizes of 0.56, 0.45 and 0.09 (ref. 16). By contrast, the effect size of 0.91 demonstrated in this study between MDMA-assisted therapy and placebo with therapy was larger than that for any other previously identified PTSD pharmacotherapy16,17,18. To directly assess superiority, a head-to-head comparison of MDMA-assisted therapy with SSRIs for PTSD would be needed. Although the present study tested the effects of MDMA using a model in which both treatment groups received supportive therapy, participants who received MDMA and supportive therapy (d = 2.1) had greater improvement in PTSD change scores compared with those who received placebo with supportive therapy (d = 1.2), suggesting that MDMA enhanced the effects of supportive therapy. In clinical practice, both MDMA and supportive therapy will be components of this PTSD treatment.

Previous research on MDMA for PTSD has suggested that those with a recent history of SSRI treatment may not respond as robustly to MDMA18. Given that 65.5% of participants in the current trial have a lifetime history of SSRI use, it is difficult to separate the ramifications of long-term SSRI treatment from the effects of treatment resistance. However, there was no obvious effect of previous SSRI use on therapeutic efficacy in this trial. Similarly, although years of PTSD diagnosis or age of onset may affect treatment efficacy, no obvious relationship was seen here between duration or onset of PTSD diagnosis and treatment efficacy.

Serotonin and the serotonin transporter are of particular importance in the generation, consolidation, retrieval and reconsolidation of fear memories19,20. Reduced serotonin transporter levels (which result in greater amounts of extracellular serotonin) have been shown to predict propensity to develop PTSD21, increase fear and anxiety-related behaviors22, and induce greater amygdalar blood oxygenation level-dependent (BOLD) activity in response to fearful images23. There is extensive serotonergic innervation of the amygdala, and amygdalar serotonin levels have been shown to increase following exposure to stressful and fear-inducing stimuli24. MDMA enhances the extinction of fear memories in mice through increased expression of brain-derived neurotrophic factor in the amygdala, and human neuroimaging studies have demonstrated that MDMA is associated with attenuated amygdalar BOLD activity during presentation of negative emotional stimuli25. Together these data suggest that MDMA may exert its therapeutic effects through a well-conserved mechanism of amygdalar serotonergic function that regulates fear-based behaviors and contributes to the maintenance of PTSD. Perhaps by reopening an oxytocin-dependent critical period of neuroplasticity that typically closes after adolescence15, MDMA may facilitate the processing and release of particularly intractable, potentially developmental, fear-related memories.

It is intriguing to speculate that the pharmacological properties of MDMA, when combined with therapy, may produce a 'window of tolerance,' in which participants are able to revisit and process traumatic content without becoming overwhelmed or encumbered by hyperarousal and dissociative symptoms26. MDMA-assisted therapy may facilitate recall of negative or threatening memories with greater self-compassion27 and less PTSD-related shame and anger28. Additionally, the acute prosocial and interpersonal effects of MDMA25,29 may support the quality of the therapeutic alliance, a potentially important factor relating to PTSD treatment adherence30 and outcome31. Indeed, clinicians have suggested that "MDMA may catalyze therapeutic processing by allowing patients to stay emotionally engaged while revisiting traumatic experiences without becoming overwhelmed"32.

Given that PTSD is a strong predictor of disability in both veteran and community populations33, it is promising to note that the robust reduction in PTSD and depressive symptoms identified here is complemented by a significant improvement in SDS score (for example, work and/or school, social and family functioning). Approximately 4.7 million US veterans report a service-related disability[34](https://www.nature.com/articles/s41591-021-01336-3#ref-CR34 "Bureau of Labor and Statistics. Employment Situation of Veterans---2020. News release, 18 March 2021; https://www.bls.gov/news.release/pdf/vet.pdf

            "), costing the US government approximately $73 billion per year[35](https://www.nature.com/articles/s41591-021-01336-3#ref-CR35 "Congressional Budget Office. Possible Higher Spending Paths for Veterans' Benefits (2018);
              https://www.cbo.gov/publication/54881

            "). Identification of a PTSD treatment that could improve social and family functioning and ameliorate impairment across a broad range of environmental contexts could provide major medical cost savings, in addition to improving the quality of life for veterans and others affected by this disorder.

PTSD is a particularly persistent and incapacitating condition when expressed in conjunction with other disorders of mood and affect. In the present study, perhaps most compelling are the data indicating efficacy in participants with chronic and severe PTSD, and the associated comorbidities including childhood trauma, depression, suicidality, history of alcohol and substance use disorders, and dissociation, because these groups are all typically considered treatment resistant2,3,4,5,6. Given that more than 80% of those assigned a PTSD diagnosis have at least one comorbid disorder3, the identification of a therapy that is effective in those with complicated PTSD and dual diagnoses could greatly improve PTSD treatment. Additional studies should therefore be conducted to evaluate the safety and efficacy of MDMA-assisted therapy for PTSD in those with specific comorbidities.

Although recent research suggests that dissociative subtype PTSD is difficult to treat36, participants with the dissociative subtype who received MDMA-assisted therapy had significant symptom reduction that was at least similar to that of their counterparts with non-dissociative PTSD. Given that this covariate was significant, it warrants further study. Furthermore, given that other treatments for PTSD are not consistently effective for those with the dissociative subtype, these data, if replicated, would indicate an important novel therapeutic niche for MDMA-assisted therapy for typically hard-to-treat populations.

Importantly, there were no major safety issues reported in the MDMA arm of this study. Although abuse potential, cardiovascular risk and suicidality were recorded as AESIs, MDMA was not shown to induce or potentiate any of these conditions. In addition, although there was often a transient increase in blood pressure during MDMA sessions, this was expected based on phase 2 data and previous studies in healthy volunteers37. These data suggest that MDMA has an equivalent, if not better, safety profile compared with that of first-line SSRIs for the treatment of PTSD, which are known to carry a low risk of QT interval prolongation38.

There are several limitations to the current trial. First, due to the coronavirus disease 2019 (COVID-19) pandemic, the participant population is smaller than originally planned. However, given the power noted in this study, it is unlikely that population size was an impediment. Second, the population is relatively homogeneous and lacks racial and ethnic diversity, which should be addressed in future trials. Third, this report describes the findings of a short-term pre-specified primary outcome, 2 months after the last experimental session and 5 weeks since the final integrative therapy session; long-term follow-up data from this controlled trial will be collected to assess durability of treatment. Fourth, safety data were by necessity collected by site therapists, perhaps limiting the blinding of the data. To eliminate this effect on the primary and secondary outcome measures, all efficacy data were collected by blinded, independent raters. Last, given the subjective effects of MDMA, the blinding of participants was also challenging and possibly led to expectation effects14. However, although blinding was not formally assessed during the study, when participants were contacted to be informed of their treatment assignment at the time of study unblinding it became apparent that at least 10% had inaccurately guessed their treatment arm. Although anecdotal, at least 7 of 44 participants in the placebo group (15.9%) inaccurately believed that they had received MDMA, and at least 2 of 46 participants in the MDMA group (4.3%) inaccurately believed that they had received placebo.

We may soon be confronted with the potentially enormous economic and social repercussions of PTSD, exacerbated by the COVID-19 pandemic. Overwhelmingly high rates of psychological and mental health impairment could be with us for years to come and are likely to impart a considerable emotional and economic burden. Novel PTSD therapeutics are desperately needed, especially for those for whom comorbidities confer treatment resistance.

In summary, MDMA-assisted therapy induces rapid onset of treatment efficacy, even in those with severe PTSD, and in those with associated comorbidities including dissociative PTSD, depression, history of alcohol and substance use disorders, and childhood trauma. Not only is MDMA-assisted therapy efficacious in individuals with severe PTSD, but it may also provide improved patient safety. Compared with current first-line pharmacological and behavioral therapies, MDMA-assisted therapy has the potential to dramatically transform treatment for PTSD and should be expeditiously evaluated for clinical use.

LE - 218. Encarnado, conserva o Espírito algum vestígio das percepções que teve e dos conhecimentos que adquiriu nas existências anteriores? “Guarda vaga lembrança, que lhe dá o que se chama ideias inatas.” a) Não é, então, quimérica a teoria das ideias inatas? “Não; os conhecimentos adquiridos em cada existência não mais se perdem. Liberto da matéria, o Espírito sempre os tem presentes. Durante a encarnação, esquece-os em parte, momentânea.

Importante!

O sea llama más la atención del ojo pro no sé si va a ser más memorable...

La onicorrexis son fisuras o roturas longitudinales o transversales de las uñas, llamadas también distrofía media canaliforme de heller.

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Reply to the reviewers

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

#1) Summary: The transport of effector proteins across membranes from the producing bacterium into a target cell is at the core of bacterial secretion systems. How an additional layer in form of a capsule affects effector export and the susceptibility towards effector import is not fully understood. Here, Flaugnatti and colleagues combined bacterial genetics with phenotypic assays and electron microscopy to demonstrate a dual role of a bacterial capsule in preventing T6SS-mediated effector export and promoting protection from effector import by another bacterium's T6SS. The wide variety of methods used, complementation of the mutants, and validation of the findings across strains strengthen the author's conclusions. Although the main conclusions seem straight forward, the authors unravel the unexpected complexity underlying these phenotypes with strong mechanistic work. In brief, a capsule-deficient mutant (∆itra) is shown to assemble its T6SS similar to the WT, yet secretes more Hcp than the WT and is better in T6SS-mediated killing of other bacteria. A capsule-overproducing mutant (∆bfmS) shows both, a partial deficiency in T6SS assembly and an additional reduction in exported Hcp, and is worse in T6SS-mediated killing than the WT. A mutant with a capsule similar to WT and deficient in cell sensing (∆tslA) forms the least T6SS apparatuses and is yet better in T6SS-mediated killing than the overcapsulated mutant. Together, these data show an effect of the capsule on (i) T6SS apparatus assembly, (ii) effector export, (iii) effector import, and (iv) the need for clearance of accumulating non-secreted Hcp by ClpXP. The work on a clinical isolate of Acinetobacter tumefaciens and the data on an impaired T6SS activity on other cells by antibiotic-induced capsulation is a strong demonstration of the work's clinical relevance in addition to the findings' conceptual novelty.

• In my view, the manuscript is outstanding with very high quality of experimental data, very well written text and very clear presentation of the data in figures. A few minor comments and suggestions below that I think would strengthen the manuscript.*

__ Authors’ reply #1: __We thank the reviewer for their enthusiasm.

• *

Major comment:

#2) OPTIONAL: Fig. 4c/l. 320: Having an indirect effect of an antibiotic on T6SS activity by antibiotic-induced capsule formation is very intriguing and contributes to the clinical relevance of the overall findings. When I saw the data in Fig. 4c, the graph instantaneously reminded me of the panel in Fig. 2a, where a similar phenotype is observed by changing the predator:prey ratio in the absence of any antibiotic. The authors themselves comment on the possibility of antibiotic-induced, reduced predator growth (and thereby a change in predator:prey ratio) as a one factor impacting the phenotype here. I am wondering if this data could be strengthened or better disentangled to test more precisely if it is the antibiotic induced capsule formation per se that affects T6SS-mediated killing by A. baumanii in the presence of antibiotics. Would it help to take the bfmS mutant along as a control for direct comparison to see if antibiotic-induced capsule formation of the WT to similar levels of the mutant results in the same killing phenotype? Would it help to test for T6SS-mediated killing in the presence and absence of antibiotics at multiple predator:prey ratios? Could the effect of the antibiotic on A. baumanii growth be measured and considered when choosing the ratio at which the bacteria are mixed?

__ Authors’ reply #2: __The point raised by the reviewer is very important. As we have stated in the manuscript, the capsule-induced production using antibiotics impacts the growth of A. baumannii and could therefore change the predator-prey ratio, potentially affecting the observed phenotype. However, the antibiotic is expected to equally impact the non-encapsulated ΔitrA strain, yet this strain maintains very strong T6SS killing activity in the presence of chloramphenicol. Thus, we do not believe the predator-prey ratio is causing the observed effect. To address this point more directly, we nonetheless propose to: i) repeat the experiments with different predator-prey ratios (1:1, 2:1, and 5:1), and ii) include a bfmS mutant as a control.

Minor comments:

#3) Figure 1D, l. 155, I might have missed this, do the authors happen to have the numbers of E. cloacae as well? This would strengthen the claim on A. baumannii survival because of E. cloacae is being killed.

__ Authors’ reply #3: __The reviewer is correct; we did not include the survival of E. cloacae in the initial manuscript due to technical reasons (counter-selection of E. cloacae). However, we propose to repeat the experiment using an E. cloacae strain carrying a plasmid conferring kanamycin resistance. This will allow us to counter-select E. cloacae after contact with the A. baumannii predator to determine if E. cloacae is killed by A. baumannii in a T6SS-dependent manner.

#4) Figure 2, I suggest to write out the species name of the prey in the box with the ratio. With E. cloacae being referred to in the previous figure and starting with similar letters than E. coli, I wasn't sure at first sight what E. c. refers to.

__ Authors’ reply #4: __We appreciate the comment and will revise the figure as suggested.

#5) use of the term "T6SS activity" throughout the manuscript (e.g. l. 182, l. 187). I leave this up to the authors. To me, it seems like an umbrella term for the initial observation and I see that such a term can be very handy for the writing. I just would like to mention that the use of the term was not always intuitive to me and sometimes even a bit misleading. For example, l. 182 refers to "increased T6SS activity". As a reader, I only know about 'T6SS activity on other cells' or 'a T6SS-mediated effect on other cells' at this point. T6SS apparatus assembly/firing activity is tested for specifically later and it turns out to differ between mutants. By the time the term is used in the discussion, it captures multiple nuanced phenotypes described by then. The more precise definition of the term in l. 200 helped to capture what exactly is meant by the authors.

__ Authors’ reply #5: __We propose rephrasing the sentences to include the term "T6SS-secretion activity" when referring to Hcp secretion assays and "T6SS-mediated killing activity" when referring to killing experiments.

__#6) __l. 198-199 "Collectively, our findings indicate that CPS does not hinder the secretion process of the T6SS or the consequent elimination of competing cells". I might be missing something, I cannot entirely follow this sentence. Didn't the authors just show that the CPS does hinder T6SS-mediated elimination of competing cells in panel 2A and less secreted Hcp in the encapsulated WT compared to the non-encapsulated mutant in panel 2B?

__ Authors’ reply #6:__ We thank the reviewer for this comment. We realize that the sentence wasn’t well phrased, resulting in confusion. What we meant was that the T6SS is functional regarding its T6SS-mediated killing and secretion in the WT strain, while we also showed that the non-capsulated strain kills and secretes more T6SS material in the supernatant. Thus, there seems to be a balance between capsule production and T6SS activity in the WT. We will revise the sentence to better reflect this meaning.

#7) l. 224, typo, "in"

__ Authors’ reply #7:__ We will correct this typo. Thank you.

• *

#8) Two connected comments: l. 338, Just a thought, I am wondering about the title of the section. After reading it a second time, I think it is technically correct. When reading it first, I was a bit confused when getting to the data because apparatus assmebly is impaired in the capsule-overproducing strain and although "preserved", doesn't the data indicate that there is less T6SS assembly in the bfmS mutant and that this might be because of less cell sensing and isn't this a main point that there is a difference in apparatus assembly in the capsule overproducing strain compared to WT (other than no difference in apparatus assembly in the strain without capsule)? To me it seems not fully acknowledged as a finding in the interpretation of the data that less cells of the bfmS mutant have a T6SS apparatus. Isn't that interesting? A title along the lines of "Capsule-overproducing strain has preserved sensory function and assembles less T6SS apparatuses" would have been more intuitive for me. l. 352, In case I didn't miss a reference to this data earlier in the manuscript, I am wondering if it would be worth mentioning the finding on the reduced apparatus assembly of the bfmS mutant earlier, together with Figure 3 already. At least a sentence that mentions already that there is more coming later. When I got to this line in the manuscript and read the findings on the apparatus assembly, I first needed to go back to figure 3 and look at the data there again in light of this finding. It is mentioned here on the side but I think very important for the interpretation of the phenotypic data of the bfmS mutant shown earlier, isn't it? The tslA mutant is used beautifully here.

__ Authors’ reply #8:__ We thank the reviewer for the suggestion and the kind comment about the beautiful usage of the tslA mutant. We will modify the title of the corresponding paragraph as suggested to make it more intuitive.

        Regarding the comment about mentioning the T6SS apparatus assembly defect in the *bfmS* mutant earlier, we respectfully disagree. While we agree that this point is important and can partially explain the difference in killing activity, we believe that showing it together with the *tslA* mutant (Figure 5) makes more sense and is easier for the reader to understand.

#9) Discussion: optional comment. On the one hand, I like the concise discussion. On the other hand, I see more potential here for bringing it all together (potentially at the expense of shortening some of the introduction). I think the subtleties of the findings are complex. For example, I could envision a graphical summary with a working model on all the effects of a capsule on the T6SS and its potential clinical relevance making the study accessible to even more readers.

__ Authors’ reply #9: __In the revised manuscript, we will include a graphical summary/model.

Significance

#10) General assessment: I consider the story very strong in terms of novelty, experimental approaches used, quality of the data, quality of the writing and figures of the manuscript. In my view, the aspects that could be improved are optional/minor and concern only one figure and some phrasing.

• Advance: I see major advance in the findings (i, mechanistic) on the mechanism of how the capsule interferes with T6SS, (ii, fundamental) on the discovery of ClpXP degrading Hcp, and (iii, clinical) on the meaning of antibiotic treatment for the T6SS of this clinically relevant and often multi-drug resistant bacterial species, which strongly complements existing work on the T6SS and antibiotics in A. baumanii (e.g. of the Feldman group). As the authors write themselves, the starting points of the study of a capsule protecting from a T6SS and the effect of a T6SS on other cells being negatively impacted by a capsule were known, although not studied in one species and not understood mechanistically.*

• Audience: I see the result of interest to a broad audience in the fields of bacteria-bacteria interactions, Acinetobacter baumanii, type VI secretion, antimicrobial resistance, bacterial capsules.*

__ Authors’ reply #10: __We once again thank the reviewer and highly appreciate their positive and constructive feedback on our work. We hope the reviewer will be satisfied with the revised version of our manuscript.

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

#11) In the manuscript by Flaugnatti et al., the authors provide clear evidence of the interplay between capsule outer coat production and the Type VI secretion system (T6SS) in Acinetobacter baumannii. The authors demonstrate that the presence of the capsule or the activity of the T6SS enhances survival against attacking bacteria. However, they also show that in their model bacterium, the (over)production of the capsule likely hinders T6SS dynamics, thereby reducing overall killing efficiency. Additionally, they reveal that the amount of the T6SS component Hcp is regulated in cells that can no longer assemble and/or secrete via the T6SS, presumably by the ClpXP protease. Overall, the experiments are well designed, and most conclusions are supported by the data and appropriate controls. I have however some suggestions that could further strengthen the manuscript prior to publication.

__ Authors’ reply #11: __We are grateful for the reviewer’s enthusiasm and will implement their comments and suggestions in the revised version of the manuscript.

Major comments:

#12) Line 164. The authors use E. coli as prey to test the T6SS activity of A. baumannii. Why not directly use the E. cloacae strain (with or without T6SS) for this purpose? This would provide direct evidence that A. baumannii uses its T6SS to kill E. cloacae, thus confirming the authors conclusions in this section.

__ Authors’ reply #12: __We thank the reviewer for this comment. We used E. coli to assess the functionality of the T6SS in different strains of A. baumannii, as it is commonly done in the T6SS field. However, as suggested by reviewer 1 (see comment #3) and in response to this query, we will also provide survival data of E. cloacae in the revised manuscript using a plasmid-carrying E. cloacae derivative that allows direct selection.

#13) In Figure 2, the authors show that a non-capsulated strain kills more effectively and secretes more than a WT, but has a similar number of T6SS. They suggest in their conclusion that "the observed increase in T6SS activity in the non-capsulated strain suggests a compensatory mechanism for the absence of the protective capsule layer." This conclusion implies the presence of an "active" regulatory mechanism that would increase the number of successful T6SS firing events, which has not been demonstrated. Could it not simply be that the capsule blocks some shots that cannot penetrate and are therefore ineffective? This hypothesis is mentioned in lines 204-208. The authors should clarify the conclusion of this section. Given the challenge this may pose in A. baumannii, I suggest that the authors quantify the assembly/firing dynamics of the T6SS under WT and ΔitrA conditions. This would help distinguish between the two hypotheses explaining better firing in non-capsulated cells: i.e., if the number of assembled T6SS is the same in both strains (Fig 2C & 2D), do non-capsulated cells assemble/fire faster, indicating an adaptation in regulation, or do we observe the same dynamics, suggesting a simple physical barrier blocking the passage of certain T6SS firing events?

__ Authors’ reply #13:__ We realize that the sentence, and more specifically the word "compensatory," might have been misleading and thank the reviewer for bringing this to our attention. What we meant to convey is that there is a balance between capsule production and T6SS activity; if disturbed, the balance shifts in one direction or the other. Specifically, there is more protection through the production of a thicker capsule (e.g., in the ∆bfmSmutant or under sub-MIC conditions of antibiotics, regulated by the Bfm system, as mentioned in the text) or more T6SS activity when less capsule is present (e.g., in the ΔitrA mutant, which we propose is caused by the lack of the steric hindrance). We will rephrase this sentence in the revised manuscript to better convey this message.

        Regarding the quantification of T6SS dynamic assembly/firing events between the capsulated (WT) and non-capsulated (ΔitrA) strains, we do not think this is required for this study, as the amount of secreted Hcp reflects the overall activity of the system. Importantly, we also do not have the technical means to quantify assembly/firing rates under Biosafety 2 conditions, as this requires specialized microscopes with very fast acquisition options (see, for instance, Basler, Pilhofer *et al.*, 2012, *Nature*). Indeed, very few labs in the T6SS field have been able to measure such rates.

#14) Line 428-429. It is mentioned that the deletion of lon does not have a notable effect. However, I observe that the absence of Lon alone causes a more rapid degradation of Hcp in the cells compared to the WT strain (Fig 7B). How do the authors explain that the absence of this protease (whether under conditions of Hcp accumulation or not) increases the degradation of this protein in the cell? This explanation should be included in the manuscript.

__ Authors’ reply #14: __That’s a fair point. We didn’t address this point further, as the deletion of lon didn’t resolve the issue of why Hcp is degraded. In fact, the opposite seems to be the case, as there is less Hcp in the ∆lon strain compared to the WT. While this observation is not directly relevant to the question of why Hcp is degraded late during growth in secretion-impaired strains, we will properly mention it in the revised manuscript.

        Please also note that a strong growth defect of a Δ*lon*Δ*clpXP* double mutant impaired further investigation in this direction.

• *

Minor comments:

#15) Throughout the manuscript, the authors use the term "predator" to refer to A. baumannii. Predation is a specific phenomenon that involves killing for nourishment. To my knowledge, the T6SS has never been shown to be a predation weapon but rather a weapon for interbacterial competition, which is a different concept. If this has not been demonstrated in A. baumannii, the authors should replace the term "predator" with "attacker" (or an equivalent term) to clarify the context.

__ Authors’ reply #15: __We thank the reviewer for this comment. The term “predator,” as highlighted by the reviewer, typically implies killing for nourishment/cellular products. In the context of T6SS, it facilitates the killing of competitors, releasing DNA into the environment that can subsequently be acquired through natural competence for transformation, as observed in species like Vibrio cholerae (our work by Borgeaud et al., 2015, Science) or other Acinetobacter species such as Acinetobacter baylyi (Ringel et al., 2017, Cell Rep.; Cooper et al., 2017, eLife). The acquisition of DNA reflects the killing for cellular products of the prey. As most A. baumannii strains are also naturally competent, this justifies the usage of the predator and prey nomenclature.

        Apart from this fact, it seems to be a matter of nomenclature, with many papers in the field using one term or the other. Yet, ultimately, this doesn’t change any of the scientific findings. Therefore, to satisfy the reviewer, we will change “predator” to “attacker” throughout the revised manuscript.

#16) Line 274. Since the authors stated that in the Wzc mutant, the capsule is "predominantly found in the supernatant and only loosely attached to the cell," this result is not unexpected. This finding is also consistent with the previous results from Fig. 3A & B, which show sensitivity to complement-mediated killing and the weak amount of (ab)normal CPS produced in that strain, further confirmed by Fig. 3E.

__ Authors’ reply #16__: We fully agree with the reviewer’s suggestion and will remove the statement.

#17) Line 299. The authors speculate that "... T6SS may deploy through gaps akin to arrow-slits in the capsule's mesh...". However, this is very unlikely since a WT strain kills (Fig. 3C) and secretes (Fig. 2B & 3D) less effectively than the itrA mutant, suggesting that the T6SS is not assembled in the "right places" devoid of CPS; otherwise, we would expect similar T6SS activity. Based on the results in Fig. 2 (and my earlier comment), this implies that A. baumannii assembles its T6SS randomly, and in the presence of the capsule, its shots would need to be in the right place to penetrate the envelope and reach the target. Could the authors comment on this point and provide a model figure to better visualize the interplay between the capsule and T6SS under the three major conditions: WT, non-capsulated, and capsule overproduction?

__ Authors’ reply #17: __We thank the reviewer and agree with their comment. We discussed the hypothesis of T6SS deployment through gaps, drawing a parallel to what was proposed for biofilm and T6SS in V. cholerae(Toska et al., 2018, PNAS). However, as mentioned earlier, we believe that the effect of the capsule on T6SS activity is primarily due to steric hindrance, which increases the distance between the T6SS apparatus and the prey cell. To clarify our findings further, we will include a model summarizing our results, as requested by reviewer 1 (see comment #9).

---

__ #18)__ In Fig. 5A, the microscopy panels should be adjusted to the same dynamic range as the WT (which represents a true signal), which does not appear to be the case for the tlsA mutant panel for instance. The image gives the impression of a large amount of free TssB-msfGFP in the cytoplasm. However, this effect is due to the dynamic range being adjusted to display a signal. This observation is consistent with the fact that the amount of TssB-msfGFP protein is identical across all strains (Fig. S2F).

__ Authors’ reply #18: __We will adjust the images to the range of the WT in the revised manuscript, as suggested. However, regardless of how these images are presented, the enumeration of T6SS structures will remain unchanged, which was the sole point of this experiment.

• *

#19) Unless I am mistaken, the authors do not comment on the fact that in a ΔbfmS strain, the number of T6SS is halved compared to a WT or ΔitrA strain. If capsule overproduction only partially limits the TslA-dependant T6SS assembly, how can this result be explained? Is it related to the degradation of Hcp in this background, which ultimately limits the formation of T6SS? If so, it would be interesting to mention this connection in the section "Prolonged secretion inhibition triggers Hcp degradation”

__ Authors’ reply #19: __We did mention that the T6SS assembly of the ΔbfmS mutant is reduced compared to the WT (or ΔitrA), likely due to the defect in sensing the prey (lines 369-374 and 468-472 of the initial manuscript). However, we will revise the sentence to improve clarity in the revised version of the manuscript.

Significance

#20) This work is highly intriguing as it not only delves into the specific mechanisms involved but also connects fundamental elements in bacterial competition, i.e., the necessity for self-protection and aggression for survival. The manuscript offers valuable insights into cellular dynamics at a microscale level and prompts new inquiries into the regulation of these systems on a population scale. The work is well-done and the writing is also clear. I am convinced that this work represents another significant step towards understanding bacterial mechanisms and will undoubtedly spark considerable interest in the field.

__ Authors’ reply #20: __We sincerely thank reviewer #2 for their constructive inputs, which will improve our manuscript.

• *

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

#21) The manuscript by Flaugnatti et al investigates the relationship between functions of the T6SS in A. baumannii and production of capsular polysaccharide. The manuscript argues that (1) capsule protects A. baumannii against T6SS-mediated attack by other bacteria, (2) capsule also interferes with the bacterium's own T6SS activity, and (3) the T6SS inner tube protein Hcp is regulated by degradation by ClpXP. The main critiques regard the first two conclusions, which seem to be based solely on use of a mutant that has a confounding effect as described below; and to strengthen the third claim by further exploring the results of overexpressing Hcp and by determining whether there is a fitness benefit for Hcp regulation.

__ Authors’ reply #21: __We thank reviewer #3 for their relevant input. We will conduct additional experiments based on their comments, and these will be incorporated into the revised manuscript.

• *

__Main items:____ __

#22) Throughout the paper, an itrA deletion mutant is used as the capsule-deficient strain and conclusions are drawn about role of capsule based on this mutant. However, itrA deletion also eliminates the protein O-glycosylation pathway (Lees-miller et al 2013), a potential confounder. Analysis of mutants specifically deficient in the high-molecular weight capsule but not protein glycosylation, and/or mutants in the protein o-glycosylation enzyme, should be incorporated into the study to enhance the ability to make conclusions about the role of the capsule.

__ Authors’ reply #22: __Fair point. We thank the reviewer for this important suggestion. To distinguish between the O-glycosylation pathway and capsule production, we will generate a ∆pglL strain (specific to O-glycosylation), as suggested, and will repeat the key experiments (similar to Fig. 2A and 2B). We are almost done with the engineering of this mutant strain and therefore don’t expect any major delays.

#23) Evidence could be provided to support the idea raised in lines 482-483 that T6SS component accumulation is toxic ("degradation [of T6SS components] could serve as a strategy to alleviate proteotoxic stress..."). For example, growth curves of ∆clpXP strains with and without hcp could be analyzed, to determine how degrading Hcp is helping the bacteria.

__ Authors’ reply #23: __We will perform growth curves of ΔclpXP strains with and without hcp, as suggested by the reviewer. However, we are uncertain whether we will be able to observe differences between these strains, as the conditions under which such degradation is significant may be challenging to replicate under standard laboratory conditions.

__#24) __The possible ClpXP recognition sequence identified at the C terminus of Hcp is interesting-does overexpression of an Hcp variant lacking/altered in this motif alter its protein levels compared to WT Hcp?

__ Authors’ reply #24: __We thank the reviewer for this suggestion. We are in the process of performing the suggested experiment and will include the data in the manuscript.

__Minor items:____ __

#25) *A better explanation could be provided for why overexpressing hcp in WT but not in ∆hcp leads to increased Hcp protein levels. There is a statement about Hcp being regulated post transcriptionally, possibly by degradation (lines 422-423), but would that not also result in regulation in the WT strain? *

__ Authors’ reply #25: __The reviewer is absolutely correct here. Despite careful genetic engineering, we believe that the hcp mutant used may have a polar effect, causing Hcp accumulation only in the ∆hcp + p-hcp strain but not in the WT + p-hcp strain, which remains capable of secretion. The ∆hcp strain therefore mimics the secretion-impaired tssB mutant. We will clarify this in the revised manuscript.

#26) *An untreated control is needed in Fig. 4B. *

__ Authors’ reply #26: __The untreated samples were shown in all previous figures. However, we understand the reviewer's point and will repeat the experiment with the untreated control included in the same experiment.

#27) *line 179: please clarify "reflecting better invading bacteria" *

__ Authors’ reply #27: __We appreciate the reviewer mentioning this oversight. We meant to compare this to a situation where a bacterium invades an already existing community, resulting in a predator-prey ratio below 1. We will clarify this further in the revised manuscript.

#28) *line 351: consider rewording the statement that ∆tslA results in decreased in T6SS assembly and activity using the tssB-msfGFP microscopy assay; it is not clear that activity is measured in this assay. *

__ Authors’ reply #28: __The reviewer is correct. We will revise the sentence accordingly to better reflect the T6SS assembly.

#29) *lines 260-265: This experiment could use clarifying, but it would seem that it requires analysis of the secreted capsule levels in the tssB mutant to show it does not produce extracellular capsule to the same extent that ∆bfmS does. *

__ Authors’ reply #29: __We thank the reviewer for the suggestion and will include these experimental data in the revised manuscript.

#30) *Fig. 6C and 7A labelling could be improved to avoid potential confusion that the bar graphs are quantifying the western blot. E.g., could add a corresponding vertical label to the Western data, or consider changing "relative expression of hcp" to something reflecting analysis of transcript levels. *

__ Authors’ reply #30: __We will improve this figure by splitting the qPCR and Western blot data into independent panels. This will eliminate any confusion.

#31) lines 416-417 and Fig. 7A: states that "hcp mRNA levels increased significantly", but more careful wording could be used because the WT's transcript change is not significant after overexpression (though it is significant in ∆hcp).

__ Authors’ reply #31: __Point well taken. We will improve the sentence (and Figure) to make its meaning unambiguous.

• *

#32) lines 479-480 states that in secretion-impaired strains accumulation of Hcp is mitigated by ClpXP; while this was shown for ∆tssB, was this also the case for ∆bfmS?

__ Authors’ reply #32: __This is indeed an interesting suggestion. We are in the process of generating the double mutant ∆bfmS∆clpXP and will include the experimental results in the revised manuscript.

Significance

#33) *The strengths of the study are the focus on a clinically significant pathogen, the potential novel roles for the important capsule virulence factor of A. baumannii, and the identification of novel points of control of the T6SS. The analyses of T6SS function are thorough and carefully performed. *

__ Authors’ reply #33: __We thank the reviewer for their comments, which we believe will significantly strengthen our work, particularly regarding the capsule aspect.

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Referee #3

Evidence, reproducibility and clarity

The manuscript by Flaugnatti et al investigates the relationship between functions of the T6SS in A. baumannii and production of capsular polysaccharide. The manuscript argues that (1) capsule protects A. baumannii against T6SS-mediated attack by other bacteria, (2) capsule also interferes with the bacterium's own T6SS activity, and (3) the T6SS inner tube protein Hcp is regulated by degradation by ClpXP. The main critiques regard the first two conclusions, which seem to be based solely on use of a mutant that has a confounding effect as described below; and to strengthen the third claim by further exploring the results of overexpressing Hcp and by determining whether there is a fitness benefit for Hcp regulation.

Main items:

• Throughout the paper, an itrA deletion mutant is used as the capsule-deficient strain and conclusions are drawn about role of capsule based on this mutant. However, itrA deletion also eliminates the protein O-glycosylation pathway (Lees-miller et al 2013), a potential confounder. Analysis of mutants specifically deficient in the high-molecular weight capsule but not protein glycosylation, and/or mutants in the protein o-glycosylation enzyme, should be incorporated into the study to enhance the ability to make conclusions about the role of the capsule.
• Evidence could be provided to support the idea raised in lines 482-483 that T6SS component accumulation is toxic ("degradation [of T6SS components] could serve as a strategy to alleviate proteotoxic stress..."). For example, growth curves of ∆clpXP strains with and without hcp could be analyzed, to determine how degrading Hcp is helping the bacteria.
• The possible ClpXP recognition sequence identified at the C terminus of Hcp is interesting--does overexpression of an Hcp variant lacking/altered in this motif alter its protein levels compared to WT Hcp?

Minor items:

• A better explanation could be provided for why overexpressing hcp in WT but not in ∆hcp leads to increased Hcp protein levels. There is a statement about Hcp being regulated post transcriptionally, possibly by degradation (lines 422-423), but would that not also result in regulation in the WT strain?
• An untreated control is needed in Fig. 4B.
• line 179: please clarify "reflecting better invading bacteria"
• line 351: consider rewording the statement that ∆tslA results in decreased in T6SS assembly and activity using the tssB-msfGFP microscopy assay; it is not clear that activity is measured in this assay.
• lines 260-265: This experiment could use clarifying, but it would seem that it requires analysis of the secreted capsule levels in the tssB mutant to show it does not produce extracellular capsule to the same extent that ∆bfmS does.
• Fig. 6C and 7A labelling could be improved to avoid potential confusion that the bar graphs are quantifying the western blot. E.g., could add a corresponding vertical label to the Western data, or consider changing "relative expression of hcp" to something reflecting analysis of transcript levels.
• lines 416-417 and Fig. 7A: states that "hcp mRNA levels increased significantly", but more careful wording could be used because the WT's transcript change is not significant after overexpression (though it is significant in ∆hcp)
• lines 479-480 states that in secretion-impaired strains accumulation of Hcp is mitigated by ClpXP; while this was shown for ∆tssB, was this also the case for ∆bfmS?

Significance

The strengths of the study are the focus on a clinically significant pathogen, the potential novel roles for the important capsule virulence factor of A. baumannii, and the identification of novel points of control of the T6SS. The analyses of T6SS function are thorough and carefully performed.

Dado un rasgo concreto, no se va a poder refutar la tesis, pues el rasgo concreto que ha de darse para refutarla reside en la propia definición que se da prestablecidamente en las premisas. Se equiparan la sintiencia animal A(x) a la humana H(x) como si estan no presentaran T(x) (rasgos) que establecieran diferencias entre ellas. He en estos rasgos donde se puedan incluir objeciones o distinciones tanto en cuanto a diferencias cuatificables, observables y empíricas significativas en términos de procesamiento del dolor, percepción del yo, intereses (voluntad), metacognición, inteligencia, etc.

BIG IDEA OF THIS SECTION

District Two in New York City is an example of a school system that has strong internal accountability and thus is seeing concurrent improvement in learning outcomes.

BIG IDEA OF THIS ARTICLE

School improvement efforts rarely improve actual learning and teaching because they do not fundamentally alter how educators think about and do their work.

I am not sure where you get a list of "the electors" who of course are appointed for life, and I am not really sure by whom.

They of course are something similar to the "Supreme Soviet" they are tasked with doing nothing but voting in accordance with the states laws regarddddddddddddddddddddddddding the popular vote, whether to split the elector count based on popular vote or go "all in" per state.

I am downloading a list of all "elected or hopefully nominatively born representatives of each country" to "accompany" THE ACADEMY which glows very bright as a kind of arm or hand or ... mouthpiece of even brain of God in the words of "how to build heaven" and "hwo to do it right" when you get into the message of reading history and reading the difference between "replicators" and "fed five thousand with something like a fish."

fish, shoes, the woman that lived in shoe ... the frog that turned from a tad pole into a t-rex ... there's all kinds of stories about the "letter alpha."

AND The OMEGA.

it doesn't look like my "wget -3" is going to grab a list of presidents, viice presidents or electors, though it will get to this page, and perhaps get all the current senators, and representatives.

Who knows if there's a big difference between this list and the 114th Congress that happened to synchronistically and coincidentally "reign" in the year 2014.

I jsut remember cuz it's the "ADth" you know, like the year Cristobol Colon sailed the Ocean Blue, ADIB, or 1492.

In the year of our lord, Anno Domini; which ChatGPT assures me is "really good latin" for those words, even though it doesn't relaly look like any kind of conjugation or strrrrrrrrrrrrrrrange tense for "annum dominus" which is my latin for saying "year lord" in the latin that ive discerned over the ... years since the news papers stopped putting "A.D." postfixing the year since 0 ... aroud when "after the death of .." or the "before Christ" had something to do with an alternative calendar that goes back another 3 thousand or so years.

It is 5700 something, in Jewish years. Who knows why.

El sdrife es una reacción medicamentosa cutánea intertriginosa y autolimitada, se caracteriza por una erupción eritematosa simétrica que involucra los pliegues intertriginosos, los glúteos y las áreas perigenitales. Representa un trastorno inofensivo y raro que se produce por exposición sistémica a fármacos, independientemente de si hay o no sensibilización previa.

El eritrasma es una infección intertriginosa causada por Corynebacterium minutissimum, muy frecuente en pacientes con diabetes y en individuos que viven en climas más cálidos. El diagnóstico es clínico. El tratamiento se realiza con antibióticos tópicos o con claritromicina por vía oral.

El eritema infeccioso, también conocido como megaloeritema o 5.º enfermedad, es una enfermedad infecciosa producida por el virus de DNA Parvovirus B19,2​ de la familia Parvoviridae.

por calor o radiación uv

La dermatitis artefacta (DA) es un diagnóstico excepcional, que genera perplejidad y ansiedad al dermatólogo al encontrase ante una patología autoprovocada y de la que el paciente sabe más que el médico en cuanto a su etiología. Al contrario que otras dermatosis en las que existen pruebas histológicas o bioquímicas, en la DA el diagnóstico es de exclusión, lo que exige un gran consumo de recursos y de tiemp

Royal Model "O" Typewriter Adjust Ribbon Lift for each Type Bar Lever, Works on Quite Deluxe too. by [[Phoenix Typewriter]]

Forming individual type bars, particularly for capital letters to get the tops to print evenly when they're more faint than other similar typefaces.

Qual imagem ilustrará o texto? Podemos talvez produzir uma...

Vejam meu comentário abaixo para a última imagem

Não podemos por Fonte: fornecido pelos autores. Isso não diz nada. Ou a imagem da plantação de dendê foi feita por eles ou eles fornecem o nome de que a fotografou ou da insituição responsável

Se for possível, dar maior espaço do final do texto para a foto. Me pergunto se, a nível de diagramação, não pomos a imagem do Edson alinhada à esquerda e a do Márcio à direita para distribuir o peso da página

aqui seria ou óleo de palma, não? Seria interessante dar o significado/etimologia da palavra 'dendê'

Isso é uma afirmação? uma realidade? ou ainda uma possibilidade? Se for o caso, por o verbo 'poderia'. Aí é preciso dar um fecho ao resumo introdutório, fazendo uma 'chamada' para o texto propriamente dito.

Por então entre vírgulas

Author response:

Reviewer #1 (Public Review):

Abbasi et al. assess in this MEG study the directed connectivity of both cortical and subcortical regions during continuous speech production and perception. The authors observed bidirectional connectivity patterns between speech-related cortical areas as well as subcortical areas in production and perception. Interestingly, they found in speaking low-frequency connectivity from subcortical (the right cerebellum) to cortical (left superior temporal) areas, while connectivity from the cortical to subcortical areas was in the high frequencies. In listening a similar cortico-subcortical connectivity pattern was observed for the low frequencies, but the reversed connectivity in the higher frequencies was absent.

The work by Abbasi and colleagues addresses a relevant, novel topic, namely understanding the brain dynamics between speaking and listening. This is important because traditionally production and perception of speech and language are investigated in a modality-specific manner. To have a more complete understanding of the neurobiology underlying these different speech behaviors, it is key to also understand their similarities and differences. Furthermore, to do so, the authors utilize state-of-the-art directed connectivity analyses on MEG measurements, providing a quite detailed profile of cortical and subcortical interactions for the production and perception of speech. Importantly, and perhaps most interesting in my opinion, is that the authors find evidence for frequency-specific directed connectivity, which is (partially) different between speaking and listening. This could suggest that both speech behaviors rely (to some extent) on similar cortico-cortical and cortico-subcortical networks, but different frequency-specific dynamics.

These elements mentioned above (investigation of both production and perception, both cortico-cortical and cortico-subcortical connectivity is considered, and observing frequency-specific connectivity profiles within and between speech behaviors), make for important novel contributions to the field. Notwithstanding these strengths, I find that they are especially centered on methodology and functional anatomical description, but that precise theoretical contributions for neurobiological and cognitive models of speech are less transparent. This is in part because the study compares speech production and perception in general, but no psychophysical or psycholinguistic manipulations are considered. I also have some critical questions about the design which may pose some confounds in interpreting the data, especially with regard to comparing production and perception.

(1) While the cortico-cortical and cortico-subcortical connectivity profiles highlighted in this study and the depth of the analyses are impressive, what these data mean for models of speech processing remains on the surface. This is in part due, I believe, to the fact that the authors have decided to explore speaking and listening in general, without targeting specific manipulations that help elucidate which aspects of speech processing are relevant for the particular connectivity profiles they have uncovered. For example, the frequency-specific directed connectivity is it driven by low-level psychophysical attributes of the speech or by more cognitive linguistic properties? Does it relate to the monitoring of speech, timing information, and updating of sensory predictions? Without manipulations trying to target one or several of these components, as some of the referenced work has done (e.g., Floegel et al., 2020; Stockert et al., 2021; Todorović et al., 2023), it is difficult to draw concrete conclusions as to which representations and/or processes of speech are reflected by the connectivity profiles. An additional disadvantage of not having manipulations within each speech behavior is that it makes the comparison between listening and speaking harder. That is, speaking and listening have marked input-output differences which likely will dominate any comparison between them. These physically driven differences (or similarities for that matter; see below) can be strongly reduced by instead exploring the same manipulations/variables between speaking and listening. If possible (if not to consider for future work), it may be interesting to score psychophysical (e.g., acoustic properties) or psycholinguistic (e.g., lexical frequency) information of the speech and see whether and how the frequency-specific connectivity profiles are affected by it.

We thank the reviewer for pointing this out. The current study is indeed part of a larger project investigating the role of the internal forward model in speech perception and production. In the original, more comprehensive study, we also included a masked condition where participants produced speech as usual, but their auditory perception was masked. This allowed us to examine how the internal forward model behaves when it doesn't receive the expected sensory consequences of generated speech. However, for the current study, we focused solely on data from the speaking and listening conditions due to its specific research question. We agree that further manipulations would be interesting. However, for this study our focus was on natural speech and we avoided other manipulations (beyond masked speech) so that we can have sufficiently long recording time for the main speaking and listening conditions.

(2) Recent studies comparing the production and perception of language may be relevant to the current study and add some theoretical weight since their data and interpretations for the comparisons between production and perception fit quite well with the observations in the current work. These studies highlight that language processes between production and perception, specifically lexical and phonetic processing (Fairs et al., 2021), and syntactic processing (Giglio et al., 2024), may rely on the same neural representations, but are differentiated in their (temporal) dynamics upon those shared representations. This is relevant because it dispenses with the classical notion in neurobiological models of language where production and perception rely on (partially) dissociable networks (e.g., Price, 2010). Rather those data suggest shared networks where different language behaviors are dissociated in their dynamics. The speech results in this study nicely fit and extend those studies and their theoretical implications.

We thank the reviewer for the suggestion and we will include these references and the points made by the reviewer in our revised manuscript.

(3) The authors align the frequency-selective connectivity between the right cerebellum and left temporal speech areas with recent studies demonstrating a role for the right cerebellum for the internal modelling in speech production and monitoring (e.g., Stockert et al., 2021; Todorović et al., 2023). This link is indeed interesting, but it does seem relevant to point out that at a more specific scale, it does not concern the exact same regions between those studies and the current study. That is, in the current study the frequency-specific connectivity with temporal regions concerns lobule VI in the right cerebellum, while in the referenced work it concerns Crus I/II. The distinction seems relevant since Crus I/II has been linked to the internal modelling of more cognitive behavior, while lobule VI seems more motor-related and/or contextual-related (e.g., D'Mello et al., 2020; Runnqvist et al., 2021; Runnqvist, 2023).

We thank the reviewer for their insightful comment. The reference was intended to provide evidence for the role of the cerebellum in internal modelling in speech. We do not claim that we have the spatial resolution with MEG to reliably spatially resolve specific parts of the cerebellum.

(4) On the methodological side, my main concern is that for the listening condition, the authors have chosen to play back the speech produced by the participants in the production condition. Both the fixed order as well as hearing one's own speech as listening condition may produce confounds in data interpretation, especially with regard to the comparison between speech production and perception. Could order effects impact the observed connectivity profiles, and how would this impact the comparison between speaking and listening? In particular, I am thinking of repetition effects present in the listening condition as well as prediction, which will be much more elevated for the listening condition than the speaking condition. The fact that it also concerns their own voice furthermore adds to the possible predictability confound (e.g., Heinks-Maldonado et al., 2005). In addition, listening to one's speech which just before has been articulated may, potentially strategically even, enhance inner speech and "mouthing" in the participants, hereby thus engaging the production mechanism. Similarly, during production, the participants already hear their own voice (which serves as input in the subsequent listening condition). Taken together, both similarities or differences between speaking and listening connectivity may have been due to or influenced by these order effects, and the fact that the different speech behaviors are to some extent present in both conditions.

This is a valid point raised by the reviewer. By listening to their own previously produced speech, our participants might have anticipated and predicted the sentences easier. However, during designing our experiment, we tried to lower the chance of this anticipation by several steps. First, participants were measured in separate sessions for speech production and perception tasks. There were always several days' intervals between performing these two conditions. Secondly, our questions were mainly about a common/general topic. Consequently, participants may not remember their answers completely.

Importantly, using the same stimulus material for speaking and listening guaranteed that there was no difference in the low-level features of the material for both conditions that could have affected the results of our statistical comparison.

Due to bone conduction, hearing one’s unaltered own speech from a recording may seem foreign and could lead to unwanted emotional reactions e.g. embarrassment, so participants were asked whether they heard their own voice in a recording already (e.g. from a self-recorded voice-message in WhatsApp) which most of them confirmed. Participants were also informed that they were going to hear themselves during the measurement to further reduce unwanted psychophysiological responses.

(5) The ability of the authors to analyze the spatiotemporal dynamics during continuous speech is a potentially important feat of this study, given that one of the reasons that speech production is much less investigated compared to perception concerns motor and movement artifacts due to articulation (e.g., Strijkers et al., 2010). Two questions did spring to mind when reading the authors' articulation artifact correction procedure: If I understood correctly, the approach comes from Abbasi et al. (2021) and is based on signal space projection (SSP) as used for eye movement corrections, which the authors successfully applied to speech production. However, in that study, it concerned the repeated production of three syllables, while here it concerns continuous speech of full words embedded in discourse. The articulation and muscular variance will be much higher in the current study compared to three syllables (or compared to eye movements which produce much more stable movement potentials compared to an entire discourse). Given this, I can imagine that corrections of the signal in the speaking condition were likely substantial and one may wonder (1) how much signal relevant to speech production behavior is lost?; (2) similar corrections are not necessary for perception, so how would this marked difference in signal processing affect the comparability between the modalities?

One of the results of our previous study (Abbasi et al., 2021) was that the artefact correction was not specific to individual syllables but generalised across syllables. Also, the repeated production of syllables was associated with substantial movements of the articulators mimicking those observed during naturalistic speaking. We therefore believe that the artefact rejection is effective during speaking. We also checked this by investigating speech related coherence in brain parcels in spatial proximity to the articulators. In our previous study we also show that the correction method retains neural activity to a very large degree. We are therefore confident that speaking and listening conditions can be compared and that the loss of true signals from correcting the speaking data will be minor.

References:

• Abbasi, O., Steingräber, N., & Gross, J. (2021). Correcting MEG artifacts caused by overt speech. Frontiers in Neuroscience, 15, 682419.

• D'Mello, A. M., Gabrieli, J. D., & Nee, D. E. (2020). Evidence for hierarchical cognitive control in the human cerebellum. Current Biology, 30(10), 1881-1892.

• Fairs, A., Michelas, A., Dufour, S., & Strijkers, K. (2021). The same ultra-rapid parallel brain dynamics underpin the production and perception of speech. Cerebral Cortex Communications, 2(3), tgab040.

• Floegel, M., Fuchs, S., & Kell, C. A. (2020). Differential contributions of the two cerebral hemispheres to temporal and spectral speech feedback control. Nature Communications, 11(1), 2839.

• Giglio, L., Ostarek, M., Sharoh, D., & Hagoort, P. (2024). Diverging neural dynamics for syntactic structure building in naturalistic speaking and listening. Proceedings of the National Academy of Sciences, 121(11), e2310766121.

• Heinks‐Maldonado, T. H., Mathalon, D. H., Gray, M., & Ford, J. M. (2005). Fine‐tuning of auditory cortex during speech production. Psychophysiology, 42(2), 180-190.

• Price, C. J. (2010). The anatomy of language: a review of 100 fMRI studies published in 2009. Annals of the new York Academy of Sciences, 1191(1), 62-88.

• Runnqvist, E., Chanoine, V., Strijkers, K., Pattamadilok, C., Bonnard, M., Nazarian, B., ... & Alario, F. X. (2021). Cerebellar and cortical correlates of internal and external speech error monitoring. Cerebral Cortex Communications, 2(2), tgab038.

• Runnqvist, E. (2023). Self-monitoring: The neurocognitive basis of error monitoring in language production. In Language production (pp. 168-190). Routledge.

• Stockert, A., Schwartze, M., Poeppel, D., Anwander, A., & Kotz, S. A. (2021). Temporo-cerebellar connectivity underlies timing constraints in audition. Elife, 10, e67303.

• Strijkers, K., Costa, A., & Thierry, G. (2010). Tracking lexical access in speech production: electrophysiological correlates of word frequency and cognate effects. Cerebral cortex, 20(4), 912-928.

• Todorović, S., Anton, J. L., Sein, J., Nazarian, B., Chanoine, V., Rauchbauer, B., ... & Runnqvist, E. (2023). Cortico-cerebellar monitoring of speech sequence production. Neurobiology of Language, 1-21.

Reviewer #2 (Public Review):

Summary:

The authors re-analyse MEG data from a speech production and perception study and extend their previous Granger causality analysis to a larger number of cortical-cortical and in particular cortical-subcortical connections. Regions of interest were defined by means of a meta-analysis using Neurosynth.org and connectivity patterns were determined by calculating directed influence asymmetry indices from the Granger causality analysis results for each pair of brain regions. Abbasi et al. report feedforward signals communicated via fast rhythms and feedback signals via slow rhythms below 40 Hz, particularly during speaking. The authors highlight one of these connections between the right cerebellum lobule VI and auditory association area A5, where in addition the connection strength correlates negatively with the strength of speech tracking in the theta band during speaking (significant before multiple comparison correction). Results are interpreted within a framework of active inference by minimising prediction errors.

While I find investigating the role of cortical-subcortical connections in speech production and perception interesting and relevant to the field, I am not yet convinced that the methods employed are fully suitable to this endeavour or that the results provide sufficient evidence to make the strong claim of dissociation of bottom-up and top-down information flow during speaking in distinct frequency bands.

Strengths:

The investigation of electrophysiological cortical-subcortical connections in speech production and perception is interesting and relevant to the field. The authors analyse a valuable dataset, where they spent a considerable amount of effort to correct for speech production-related artefacts. Overall, the manuscript is well-written and clearly structured.

Weaknesses:

The description of the multivariate Granger causality analysis did not allow me to fully grasp how the analysis was performed and I hence struggled to evaluate its appropriateness. Knowing that (1) filtered Granger causality is prone to false positives and (2) recent work demonstrates that significant Granger causality can simply arise from frequency-specific activity being present in the source but not the target area without functional relevance for communication (Schneider et al. 2021) raises doubts about the validity of the results, in particular with respect to their frequency specificity. These doubts are reinforced by what I perceive as an overemphasis on results that support the assumption of specific frequencies for feedforward and top-down connections, while findings not aligning with this hypothesis appear to be underreported. Furthermore, the authors report some main findings that I found difficult to reconcile with the data presented in the figures. Overall, I feel the conclusions with respect to frequency-specific bottom-up and top-down information flow need to be moderated and that some of the reported findings need to be checked and if necessary corrected.

Major points

(1) I think more details on the multivariate GC approach are needed. I found the reference to Schaum et al., 2021 not sufficient to understand what has been done in this paper. Some questions that remained for me are:

(i) Does multivariate here refer to the use of the authors' three components per parcel or to the conditioning on the remaining twelve sources? I think the latter is implied when citing Schaum et al., but I'm not sure this is what was done here?

If it was not: how can we account for spurious results based on indirect effects?

Yes, multivariate refers to the three components.

(ii) Did the authors check whether the GC of the course-target pairs was reliably above the bias level (as Schaum et. al. did for each condition separately)? If not, can they argue why they think that their results would still be valid? Does it make sense to compute DAIs on connections that were below the bias level? Should the data be re-analysed to take this concern into account?

We performed statistics on DAI and believe that this is a valid approach. We argue that random GC effects would not survive our cluster-corrected statistics.

(iii) You may consider citing the paper that introduced the non-parametric GC analysis (which Schaum et al. then went on to apply): Dhamala M, Rangarajan G, Ding M. Analyzing Information Flow in Brain Networks with Nonparametric Granger Causality. Neuroimage. 2008; 41(2):354-362. https://doi.org/10.1016/j.neuroimage.2008.02. 020

Thanks, we will add this reference in the revised version.

(2) GC has been discouraged for filtered data as it gives rise to false positives due to phase distortions and the ineffectiveness of filtering in the information-theoretic setting as reducing the power of a signal does not reduce the information contained in it (Florin et al., 2010; Barnett and Seth, 2011; Weber et al. 2017; Pinzuti et al., 2020 - who also suggest an approach that would circumvent those filter-related issues). With this in mind, I am wondering whether the strong frequency-specific claims in this work still hold.

This must be a misunderstanding. We are aware of the problem with GC on filtered data. But GC was here computed on broadband data and not in individual frequency bands.

(3) I found it difficult to reconcile some statements in the manuscript with the data presented in the figures:

(i) Most notably, the considerable number of feedforward connections from A5 and STS that project to areas further up the hierarchy at slower rhythms (e.g. L-A5 to R-PEF, R-Crus2, L CB6 L-Tha, L-FOP and L-STS to R-PEF, L-FOP, L-TOPJ or R-A5 as well as R-STS both to R-Crus2, L-CB6, L-Th) contradict the authors' main message that 'feedback signals were communicated via slow rhythms below 40 Hz, whereas feedforward signals were communicated via faster rhythms'. I struggled to recognise a principled approach that determined which connections were highlighted and reported and which ones were not.

(ii) "Our analysis also revealed robust connectivity between the right cerebellum and the left parietal cortex, evident in both speaking and listening conditions, with stronger connectivity observed during speaking. Notably, Figure 4 depicts a prominent frequency peak in the alpha band, illustrating the specific frequency range through which information flows from the cerebellum to the parietal areas." There are two peaks discernible in Figure 4, one notably lower than the alpha band (rather theta or even delta), the other at around 30 Hz. Nevertheless, the authors report and discuss a peak in the alpha band.

(iii) In the abstract: "Notably, high-frequency connectivity was absent during the listening condition." and p.9 "In contrast with what we reported for the speaking condition, during listening, there is only a significant connectivity in low frequency to the left temporal area but not a reverse connection in the high frequencies."

While Fig. 4 shows significant connectivity from R-CB6 to A5 in the gamma frequency range for the speaking, but not for the listening condition, interpreting comparisons between two effects without directly comparing them is a common statistical mistake (Makin and Orban de Xivry). The spectrally-resolved connectivity in the two conditions actually look remarkably similar and I would thus refrain from highlighting this statement and indicate clearly that there were no significant differences between the two conditions.

(iv) "This result indicates that in low frequencies, the sensory-motor area and cerebellum predominantly transmit information, while in higher frequencies, they are more involved in receiving it."

I don't think that this statement holds in its generality: L-CB6 and R-3b both show strong output at high frequencies, particularly in the speaking condition. While they seem to transmit information mainly to areas outside A5 and STS these effects are strong and should be discussed.

We appreciate the reviewer's thoughtful comments. We acknowledge that not all connectivity patterns strictly adhere to the initial observation regarding feedback and feedforward communication. It's true that our primary focus was on interactions between brain regions known to be crucial for speech prediction, including auditory, somatosensory, and cerebellar areas. However, we also presented connectivity patterns across other regions to provide a more comprehensive picture of the speech network. We believe this broader perspective can be valuable for future research directions.

Regarding the reviewer's observation about the alpha band peak in Figure 4, we agree that a closer examination reveals the connectivity from right cerebellum to the left parietal is in a wider low frequency range. We will refrain from solely emphasizing the alpha band and acknowledge the potential contribution of lower frequencies to cerebellar-parietal communication.

We also appreciate the reviewer highlighting the need for a more nuanced interpretation of the listening condition connectivity compared to the speaking condition. The reviewer is correct in pointing out that while Figure 4 suggests a high-frequency connectivity from L-A5 to R-CB only in the speaking condition, a direct statistical comparison between conditions might not reveal a significant difference. We will revise the manuscript to clarify this point.

Finally, a closer examination of Figure 3 revealed that the light purple and dark green edges in the speaking condition for R-CB6 and L-3b suggest outgoing connections at low frequencies, while other colored edges indicate information reception at high frequencies. We acknowledge that exceptions to this directional pattern might exist and warrant further investigation in future studies.

(4) "However, definitive conclusions should be drawn with caution given recent studies raising concerns about the notion that top-down and bottom-up signals can only be transmitted via separate frequency channels (Ferro et al., 2021; Schneider et al., 2021; Vinck et al., 2023)."

I appreciate this note of caution and think it would be useful if it were spelled out to the reader why this is the case so that they would be better able to grasp the main concerns here. For example, Schneider et al. make a strong point that we expect to find Granger-causality with a peak in a specific frequency band for areas that are anatomically connected when the sending area shows stronger activity in that band than the receiving one, simply because of the coherence of a signal with its own linear projection onto the other area. The direction of a Granger causal connection would in that case only indicate that one area shows stronger activity than the other in the given frequency band. I am wondering to what degree the reported connectivity pattern can be traced back to regional differences in frequency-specific source strength or to differences in source strength across the two conditions.

This is indeed an important point. That is why we are discussing our results with great caution and specifically point the reader to the relevant literature. We are indeed thinking about a future study where we investigate this connectivity using other connectivity metrics and a detailed consideration of power.

Reviewer #3 (Public Review):

In the current paper, Abbasi et al. aimed to characterize and compare the patterns of functional connectivity across frequency bands (1 Hz - 90 Hz) between regions of a speech network derived from an online meta-analysis tool (Neurosynth.org) during speech production and perception. The authors present evidence for complex neural dynamics from which they highlight directional connectivity from the right cerebellum to left superior temporal areas in lower frequency bands (up to beta) and between the same regions in the opposite direction in the (lower) high gamma range (60-90 Hz). Abbasi et al. interpret their findings within the predictive coding framework, with the cerebellum and other "higher-order" (motor) regions transmitting top-down sensory predictions to "lower-order" (sensory) regions in the lower frequencies and prediction errors flowing in the opposite direction (i.e., bottom-up) from those sensory regions in the gamma band. They also report a negative correlation between the strength of this top-down functional connectivity and the alignment of superior temporal regions to the syllable rate of one's speech.

Strengths:

(1) The comprehensive characterization of functional connectivity during speaking and listening to speech may be valuable as a first step toward understanding the neural dynamics involved.

(2) The inclusion of subcortical regions and connectivity profiles up to 90Hz using MEG is interesting and relatively novel.

(3) The analysis pipeline is generally adequate for the exploratory nature of the work.

Weaknesses:

(1) The work is framed as a test of the predictive coding theory as it applies to speech production and perception, but the methodological approach is not suited to this endeavor.

We agree that we cannot provide definite evidence for predictive coding in speech production and perception and we believe that we do not make that claim in the manuscript. However, our results are largely consistent with what can be expected based on predictive coding theory.

(2) Because of their theoretical framework, the authors readily attribute roles or hierarchy to brain regions (e.g., higher- vs lower-order) and cognitive functions to observed connectivity patterns (e.g., feedforward vs feedback, predictions vs prediction errors) that cannot be determined from the data. Thus, many of the authors' claims are unsupported.

We will revise the manuscript to more clearly differentiate our results (e.g. directed Granger-Causality from A to B) from their interpretation (potentially indicating feedforward or feedback signals).

(3) The authors' theoretical stance seems to influence the presentation of the results, which may inadvertently misrepresent the (otherwise perfectly valid; cf. Abbasi et al., 2023) exploratory nature of the study. Thus, results about specific regions are often highlighted in figures (e.g., Figure 2 top row) and text without clear reasons.

Our connectograms reveal a multitude of results that we hope is interesting to the community. At the same time the wealth of findings poses a problem for describing them. We did not see a better way then to highlight specific connections of interest.

(4) Some of the key findings (e.g., connectivity in opposite directions in distinct frequency bands) feature in a previous publication and are, therefore, interesting but not novel.

We actually see this as a strength of the current manuscript. The computation of connectivity is here extended to a much larger sample of brain areas. It is reassuring to see that the previously reported results generalise to other brain areas.

(5) The quantitative comparison between speech production and perception is interesting but insufficiently motivated.

We thank the reviewer for this comment. We have addressed that in detail in response to the point (1&4) of reviewer 1.

(6) Details about the Neurosynth meta-analysis and subsequent selection of brain regions for the functional connectivity analyses are incomplete. Moreover, the use of the term 'Speech' in Neurosynth seems inappropriate (i.e., includes irrelevant works, yielding questionable results). The approach of using separate meta-analyses for 'Speech production' and 'Speech perception' taken by Abbasi et al. (2023) seems more principled. This approach would result, for example, in the inclusion of brain areas such as M1 and the BG that are relevant for speech production.

We agree that there are inherent limitations in automated meta-analysis tools such as Neurosynth. Papers are used in the meta-analysis that might not be directly relevant. However, Neurosynth has proven its usefulness over many years and has been used in many studies. We also agree that our selection of brain areas is not complete. But Granger Causality analysis of every pair of ROIs leads to complex results and we had to limit our selection of areas.

(7) The results involving subcortical regions are central to the paper, but no steps are taken to address the challenges involved in the analysis of subcortical activity using MEG. Additional methodological detail and analyses would be required to make these results more compelling. For example, it would be important to know what the coverage of the MEG system is, what head model was used for the source localization of cerebellar activity, and if specific preprocessing or additional analyses were performed to ensure that the localized subcortical activity (in particular) is valid.

There is a large body of evidence demonstrating that MEG can record signals from deep brain areas such as thalamus and cerebellum including Attal & Schwarz 2013, Andersen et al, Neuroimage 2020; Piastra et al., 2020; Schnitzler et al., 2009. These and other studies provide evidence that state-of-the-art recording (with multichannel SQUID systems) and analysis is sufficient to allow reconstruction of subcortical areas. However, spatial resolution is clearly reduced for these deep areas. We will add a statement in the revised manuscript to acknowledge this limitation.

(8) The results and methods are often detailed with important omissions (a speech-brain coupling analysis section is missing) and imprecisions (e.g., re: Figure 5; the Connectivity Analysis section is copy-pasted from their previous work), which makes it difficult to understand what is being examined and how. (It is also not good practice to refer the reader to previous publications for basic methodological details, for example, about the experimental paradigm and key analyses.) Conversely, some methodological details are given, e.g., the acquisition of EMG data, without further explanation of how those data were used in the current paper.

We will revise the relevant sections of the manuscript.

(9) The examination of gamma functional connectivity in the 60 - 90 Hz range could be better motivated. Although some citations involving short-range connectivity in these frequencies are given (e.g., within the visual system), a more compelling argument for looking at this frequency range for longer-range connectivity may be required.

Given previous evidence of connectivity in the gamma band we think that it would be a weakness to exclude this frequency band from analysis.

(10) The choice of source localization method (linearly constrained minimum variance) could be explained, particularly given that other methods (e.g. dynamic imaging of coherent sources) were specifically designed and might potentially be a better alternative for the types of analyses performed in the study.

Both LCMV and DICS are beamforming methods. We used LCMV because we wanted used Granger Causality which requires broadband signals. DICS would only provide frequency-specific band-limited signals.

(11) The mGC analysis needs to be more comprehensively detailed for the reader to be able to assess what is being reported and the strength of the evidence. Relatedly, first-level statistics (e.g., via estimation of the noise level) would make the mGC and DAI results more compelling.

We perform group-level cluster-based statistics on mGC while correcting for multiple comparisons across frequency bands and brain parcels and report only significant results. This is an established approach that is routinely used in this type of studies.

(12) Considering the exploratory nature of the study, it is essential for other researchers to continue investigating and validating the results presented in the current manuscript. Thus, it is concerning that data and scripts are not fully and openly available. Data need not be in its raw state to be shared and useful, which circumvents the stated data privacy concerns.

We acknowledge the reviewer's concern regarding the full availability of the dataset. Due to privacy limitations on the collected data, we are unable to share it publicly at this time. However, to promote transparency and enable further exploration, we have provided the script used for data analysis and an example dataset. This example dataset should provide a clear understanding of the data structure and variables used in the analysis. Additionally, we are happy to share the complete dataset upon request from research teams interested in performing in-depth secondary analyses.

Clara, pra ser sincera achei a introdução dela meio fraca... Muito bem, ela explica a comissão da Anistia, situando o leitor. Mas acho que falta um pequeno fechamento, dizendo do que se trata o artigo (na 1a frase da introdução isso aparece) e a importância de publicá-lo ou republicá-lo neste momento.

Atenção gente. Há negritos por demais neste texto! Havia começado a marcar de baixo para cima, assinalar 'grifo da autora'. Mas são tantos, que me parece inoperante repetir a cada vez. Uma ou duas vezes ela própria inseriu 'grifo nosso'. Talvez o melhor seja pegar o primeiro deles e fazer uma nota dizendo que todos os grifos neste texto advêm da pluma da autora ou são de autoria de Eneá...

Assinalar o grifo dela

Isso aqui faz parte da bio dela e não deve entrar no resumo do texto

to believe i sprokened to call dis an AESOP miracle just for a second; as if the Valdeez and Yuan had found Arabian Coffers spilled all over the Carribean Galactica ... or something

maybe didn't... and Shakespeare ties that same story of some kind of ... betrayal ... to his famous words and those words somehow in my mind directly link to the kiss of Judas. Another table, another era; and one less "mesa" ... but again here we are staring at what is supposed to be an obvious and clear "technical revolution" of the progress of democracy--one that's clearly been stalled and halted by things like "not amending the Constitution properly" (that's according to me, and that's before really seeing you) ... and then here even in a place where we can acknowledge that those laws are archaic and backwards and not "up to speed" with the current needs of a technocratic civilization, we still fail to do anything about "voting for ideas over people" something he once said was a victory--or about the world's software degrading to something less than even remotely "unclear" ... communication itself is clearly being lost, and it's clearly something to do with "censorship" and something to do with "a secret" and something to do with ...

If I could tell you, I really can't discerne what exactly it is that Dave says in his songs stands between "what we see" and "what we do."

[a chanukah miracle!]

To be standing here and having an actual well schooled and intelligent rabbi trying to rewrite the meaning of the word "anarchy" ... in order to promote it; to say it's something other than ... "anarchy" that's the crux of what I see--a world that just wants to skip ahead, to fast forward through all the work and the struggle of actually rebuilding or building something that works; to "assuming" the system that allowed for this place to be ... so poorly managed, that it must be everything it says or shows or makes believe it truly isn't capable of ... "emancipation" of something that doesn't like the word water, and doesn't like the idea of being masses, or massless; and on top of all that doesn't even want to put it's two cents in--other than to say, I'm with Ivan or I'm with Taylor or "long live Bianca" and through it all, words like "now that you're gone, I can finally step up and move along" ...

Back to the sky; I suppose ... is what it is that the "river" means to Taylor, and I just wonder in our song what's left here--if I was wrong to assume our system wouldn't have allowed the mass slavery of entire civilization and to believe that your "brains" must be simulated ... as in "not actually here" but rather there--or just to assume you wouldn't be so damned hubratically sure that whatever you are ... it's so far above and so far advanced of the "things you pretend to be here" that you can just disregard the mass enslavement of them or yourselves or whatever it truly is; to look around and show you in this place, it's clear we care more for animals than for each other, and it's surely obvious that in the grand scheme---were we not so wrong here--we might have been right about "being so much more advanced." It's that leap to something so much smarter and so much ... more capable; like growing fingers and exiting the womb, that's the kind of experience and the magical leap forward I envision the ascension and the singularity to be--except here we are,

NEGA-OIL: in a symphonic creschendo straight out of Carnegie's "Old Bailey" ...

` "Voilà! In view, a humble vaudevillian veteran cast vicariously as both victim and villain by the vicissitudes of Fate. This visage, no mere veneer of vanity, is a vestige of the vox populi, now vacant, vanished. However, this valorous visitation of a bygone vexation stands vivified, and has vowed to vanquish these venal and virulent vermin vanguarding vice and vouchsafing the violently vicious and voracious violation of volition!

The only verdict is vengeance; a vendetta, held as a votive not in vain, for the value and veracity of such shall one day vindicate the vigilant and the virtuous.

Verily, this vichyssoise of verbiage veers most verbose, so let me simply add that it's my very good honor to meet you and you may call me V." ` https://bookroo.com/quotes/v-for-vendetta

This article provides an overview of the issues related to missing data in research in higher education. The authors point out that much of the literature on handling missing data is in the fields of psychology and sociology and the majority of research in higher education does not mention methods for dealing with missing data.

The authors suggest that traditional methods of listwise deletion, pairwise deletion, imputation, and dummy-variable adjustment are all inadequate as they lead to biased results that underestimate the variance in parameters and standard errors.

They suggest that maximum likelihood (ML) and multiple imputation (MI) be used whenever possible to provide more accurate estimates of the population. If possible, Full-Information Maximum-Likelihood (FIML) procedures should be used if interaction variables are of interest. The authors recommend that all studies use MI as the default method of dealing with missing data if possible unless the context calls for a different approach.

They then complete all of these missing data procedures on an actual dataset from higher education, arriving at the conclusion that EM was the most appropriate approach due to their context, but that MI is usually the most appropriate approach, particularly if the data will be subject to regression analysis.

https://muse-jhu-edu.wv-o-ursus-proxy02.ursus.maine.edu/article/539119

Cox, B. E., McIntosh, K., Reason, R. D., & Terenzini, P. T. (2014). Working with missing data in higher education research: A primer and real-world example. The Review of Higher Education, 37(3), 377–402.

Introduzir um conectivo para iniciar o desenvolvimento

Ahmet Davutoğlu Türkiye'nin ekonomik gücünün en yüksek olduğu dönemde, Batı'ya ve doğuya jeopolitik açılımın gerçekleştiği en yoğun dönemde Türkiye'nin etki alanının genişlemesini kendi ideolojik görüşleri yönünde kullandı. Neo-Osmanlıcılık cumhuriyet Türkiye'sinde uygulanma alanı bulmayan ve altyapısı olmayan bir düşünceydi. Keza Osmanlı İmparatorluğunun son döneminde bile bu düşünce saraydan halka kadar birçok kişi tarafından tartışılmış fakat vazgeçilerek milliyetçi bir politika izlenmiştir. Ahmet Davutoğlu bir akademisyen olarak jeopolitik ile özel ilgilendi fakat tıpkı Ratzel'in düşüncelerini kendi çıkarları için kullanan düşünürler gibi o da farklı bir şey yapmadı. Ayrıca bugün Türkiye'de yaklaşık 12 milyon yabancı nüfusun bulunmasının temel aktörüdür. Suriye iç savaşından itibaren sığınmacıları özellikle kabul etmiş ve bunun devamında Türkiye'yi daha islamist bir çizgiye sokmayı hedefledi. Fakat neredeyse tüm dünyada bilinen önemli bir yanılgı var. Cumhuriyet kurulduğundan beri Türkler milliyetçi bir çizgide yetiştirildi ve sığınmacı fikri bunun da vesilesiyle oldukça zararlı bir sonuca sebep oldu. Türkler yeryüzünde müslümanlığa en büyük altın çağını yaşatmış bir millet olsa da günümüzde islam kurallarına sıcak bakmayan en büyük müslüman kitleyi temsil ederler (küçük bir azınlık grup hariç). Bu yüzden Suriye, Afganistan, Pakistan gibi ülkelerden gelen toplulukların yaptığı davranışlar ve temsil ettiği düşünceler toplumun genelinde çok ciddi rahatsızlıklara yol açtı. Türkiye kimlik bazında %90 müslüman bir nüfusa sahip olsa da şeriat isteyenlerin oranı %10'u bulmaz. Hatta Türkiye'de "aşırı sağ" günümüze kadar aşırı cumhuriyetçi, laik, militarist kesimin temsil ettiği grubu ifade ederken; islamcı, muhafazakar kesim daha merkez sağ hatta kimi zaman sol kesimi ifade etti. Günümüzde bu bir değişim içindedir. Fakat en kısa tabiriyle Davutoğlu 85 milyon içinde küçük bir azınlığa hitap eden bir politikayı Türkiye'nin dış politikası olarak sürdürdü ve başarısız bir siyasetçi olarak tarihe karıştı. *

Davutoğlu, "alan" bakımından genişleyen bir Türkiye değil "isamcı etkinin tüm topluma sirayet etmesi gayretiyle müslüman nüfusu ülke içine alıp ülke dışında da çeşitli tampon bölgeler yaratma" hevesinde olan yani pek Ratzel'e uymayan pratiğe sahip olan birisidir.

(Yabancı halkların Türkiye'ye gelmesi hakkında: Suriye iç savaşı sırasında gelen savaş mağdurlarının yanı sıra Türkiye çok uzun bir süre "açık sınır" politikası izleyerek milyonlarca resmi ve özellikle neredeyse tamamı gayrıresmi kişiyi hiçbir kayıt ve resmi prosedür olmadan kabul etti. Bu iki durumun karıştırılmaması oldukça önemlidir. Çünkü kayıt dışı birçok insan suç işlemiş, sınır dışı edilmiş ve kaçak yollarla tekrar Türkiye'ye girerek çalışmaya devam etmiştir. Keza "savaş mağduru" olarak nitelendirilen bazı kesimlerin dini bayramlarda Suriye'deki yakınlarını ziyaret edip sonra Türkiye'ye tekrar dönmeleri Türk halkının ciddi tepkisine yol açmıştır. Hatta modern Türkiye'de birçok kitlesel hastalığın yok edilmesinden sonra gelen kayıtsız birçok kişinin bazı hastalıkları tekrar yaymaya başladığı görüldü. Üstelik bu gelen kitle çok ciddi bir şeriat politikası yürütmeye başlayınca Türkiye'de çok ciddi bir göçmen karşıtlığı başladı. Bunun en temel sebebi "mantık" sorunudur. Türkiye 85 milyon bir ülke olarak genç bir nüfusa sahiptir. Dolayısıyla halkın önemli bir bölümü kayıtsız göçmen almanın, iş kollarına dahil edilmesinin mantıksız olduğunu ifade etmektedir (Bu oran iktidar partisini destekleyen kesimde dahi %80'e varan oranlara sahiptir). Özellikle 2016 da yaşanan darbe girişiminden sonra ekonomik açıdan kriz yaşayan Türkiye'nin son 5 yılda milyonlarca kayıtsız/kaçak nüfus alması tepkilerin yoğunluğunu daha da artırdı.) Şuan Türkiye'de ara sıra gündeme gelen fakat gelecekte Türkiye'nin en büyük problemi olacağı düşünülen ve araştırmaya ihtiyaç duyulan en önemli konu bu göç konusudur. Çünkü çok net ifade edilebilir ki Türkiye'de süre gelen göç hikayesi tarihte eşi benzeri görülmemiş bir süreçle işlemektedir. Batı dünyasında tüm prosedürlerle kayıt altına alınmış sığınmacılara karşı çıkılması ile Türkiye'de göçmenlere/kaçaklara karşı çıkılması kesinlikle aynı durum değildir. Dolayısıyla araştırmacılara tavsiyem şudur: Türkiye'deki göç sorununa tüm teorilerden, göç tarihinden, günümüzdeki eleştirilerden bağımsız bir şekilde, şahsına münhasır bir şekilde araştırma yapılmalı. Aksi taktirde ulaşacağınız sonuç pek de sağlıklı olmayacaktır.

Contrato de gestão é o instrumento jurídico apto a firmar parceria entre o Poder Público e a Organização Social.

Obs.:

1) A organização social é a única entidade privada que celebra contrato de gestão com o Poder Público;

2) Embora se aponte para a terminologia "contrato", o STF, na ADI 1.923, assentou que a natureza jurídica de tal relação jurídica é de convênio.

Bugün Türkiye'nin yer aldığı Anadolu da önemini ulaşımından ve su kaynaklarından alıyordu. Fakat bu duruma farklı bir bakış açısı ile bakılabilir: Su kaynakları ve ulaşım birer hayati organdır, bu doğru. Fakat bu organlara sahip olmak isteyen birçok devlet bu organizma (Anadolu) içerisinde birçok kez mücadeleye girdi ve hayatını kaybetti (yıkıldı). Dolayısıyla bu hayati organ olarak bahsedilen unsurlar aynı zamanda "elde tutulamayınca kronik bir hastalığa ve daha sonra ölüme neden olan birer organ" olarak da nitelendirilebilir mi? O zaman şu soru tarih sahnesinde birçok savaşın yaşanmasının coğrafi temelini oluşturabilir mi?: "Devletlerin çeşitli periyodlarda kendisini sağlıklı tutacak yeni organlara ihtiyacı vardır".

for - progress trap - AI music - critique - Folia Sound Studio - to - P2P Foundation - Michel Bauwens - Commons Transition Plan - Netarchical Capitalism - Predatory Capitalism

to - P2P Foundation - Michel Bauwens - Commons Transition Plan - Netarchical Capitalism - Predatory Capitalism https://hyp.is/o-Hp-DCAEe-8IYef613YKg/wiki.p2pfoundation.net/Commons_Transition_Plan

DOI: 10.1038/s41398-024-02902-2

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What is this?

这里的 \(\partial o / \partial h\) 的结果是符合矩阵求导的，得到的也是 \(W^{(2)\prime}\)

为什么是 \({\mathbf{W}^{(2)}}^\top \frac{\partial J}{\partial \mathbf{o}}\) 而不是 \(\frac{\partial J}{\partial \mathbf{o}}{\mathbf{W}^{(2)}}^\top\) 这个主要是因为前面也提到，prod运算符是指执行必要的操作，也就是说会自动根据需要进行换位和交换输入位置等，然后再进行相乘。然而这里的 \(\frac{\partial J}{\partial \mathbf{o}}\) 是h维向量，\(W^{(1)}\) 是 h❌d 维，所以（转置后）必须写到前面

下面这个求导法则在数学上是有点问题的

$$ W^{(2)}h = \begin{pmatrix} w^{(2)\prime}{1}\ w^{(2)\prime}{2} \ \vdots& \ w^{(2)\prime}{q} \ \end{pmatrix} \begin{pmatrix}h{1} \ h_{2} \\vdots\ h_{h}\end{pmatrix} = \begin{pmatrix} w^{(2)\prime}{1} h\ w^{(2)\prime}{2} h\ \vdots& \ w^{(2)\prime}_{q} h\ \end{pmatrix} $$

• \(w^{(2)}_{1}\) 为 \(W^{(2)}\) 每个输出单元对应的仿射变换的权重 h维 列向量，上面转置以适应矩阵乘法 \(o=W^{(2)}h\)

\(\frac{\partial (W^{(2)}h)}{\partial W^{(2)}}\) 这种矩阵求导不存在的，实际上是做什么呢？其每个分量对每个分量求导，即 \(\frac{\partial (w^{(2)\prime}{1}h)}{\partial w^{(2)}{1}}=h\) ，其他分量也得到 \(h\) 。

为了 \(W^{(2)}\) 能够与这个结果直接进行加减运算、更新梯度， 求导的结果可记为（注意这种求导依然是不存在的）

$$ \frac{\partial (W^{(2)}h)}{\partial W^{(2)}} = \begin{pmatrix}h^T \ h^T \ \vdots \ h^T\end{pmatrix} $$

W2-=eta * G ，其中eta为学习率，G为 \(h^{T}\) ，这样写没问题，因为有广播原则！

Venda e Dante, o casal ta arrebentando nos kills-of-the-week!! S2

O mestre sabe onde botar os mimicos pra pegar os jogadores

• Daí se incluir, entre os deveres do locatário, o de permitir a vistoria do imóvel pelo locador, o que não traduz turbação da posse. Mas é evidente que o locador poderia abusar do seu direito, marcando visitas repetidas, com curto prazo de intervalo, ou em horários inconvenientes, que perturbassem a privacidade, o descanso ou o lazer do locatário. Para se evitar este comportamento, que constrangeria o locatário, levando-o, até mesmo, a encerrar a locação, a lei condicionou a vistoria à combinação prévia do dia e da hora em que se realizará. Igual obrigação tem o locatário, quando o locador pretender alienar o imóvel, e, para isto, precisa mostrá-lo aos eventuais pretendentes. Esta disposição não constava da lei anterior, e, graças a esta omissão, felizmente agora suprida, muitos locatários dificultavam a venda, impedindo visitas dos candidatos. Se os locatários agora assim procederem, estarão cometendo infração legal e sujeitando-se à ação de despejo. Não havendo acordo entre locador e locatário, quanto aos dias e horários das visitas, caberá ao juiz designá-los, de acordo com seu prudente arbítrio, respeitando os costumes do lugar e as peculiaridades de cada caso concreto. (Sylvio Capanema de Souza, Lei do Inquilinato Comentada Artigo por Artigo, 10ª edição, Rio de Janeiro, Ed. Forense, p. 142).

Askeri coğrafya tezi çalışmam boyunca Almanya'nın zaman zaman bunu yaşadığını, kısmen Rusya'nın yaşadığını fakat itici güçler arasında hep Britanya'nın olduğunu tespit ettim. Coğrafya kader olmasa da ulusların yürüyeceği yolun en önemli belirleyicilerinden birisidir.

idea principal

Idea principal

tema: Modelos embrionarios

Tema: Las limitaciones de los modelos embrionarios

Idea principal

Esta es la idea principal del párrafo.

El tema de este párrafo seria :Limitaciones de los modelos embrionarios

Como se generan los modelos embrionarios

Como se generan los modelos embrionarios

Idea principal del párrafo

idea principal

Idea principal.

Idea principal

Tema: Limitaciones de los modelos embrionarios

O lobo da estepe conta a história de Harry Haller, um homem de 50 anos que acredita que sua integridade depende da vida solitária que leva em meio às palavras de Goethe e às partituras de Mozart; um intelectual tentando equilibrar-se à beira do abismo dos problemas sociais e individuais, ante os quais sua personalidade se torna cada vez mais ambivalente e, por fim, estilhaçada.

A primeira parte do livro é o pesadelo do lobo Haller, sua depressão e sua incapacidade de se comunicar que está na base da crueldade e da destruição. Na segunda, o lobo se humaniza, através da entrada em cena de Hermínia, que tenta reaproximá-lo do mundo, no caso uma comunidade simplória, com salas de baile poeirentas e bares pobres.

O lobo da estepe foi escrito quando Hesse tinha 50 anos, como seu personagem, e estava profundamente influenciado pela psicanálise. O estilo adotado, altamente revolucionário para a época, foi elogiado por Thomas Mann, para quem, como novela experimental, O lobo da estepe era tão genial quanto Ulisses , de James Joyce.

1. conjunto dos costumes e hábitos fundamentais, no âmbito do comportamento (instituições, afazeres etc.) e da cultura (valores, ideias ou crenças), característicos de uma determinada coletividade, época ou região. "o é. da Antiguidade grega, do povo brasileiro, dos nordestinos" 2. RETÓRICA parte da retórica clássica voltada para o estudo dos costumes sociais.

DOI: 10.1016/j.xcrm.2024.101620

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What is this?

DOI: 10.1016/j.immuni.2024.05.018

Resource: (BD Biosciences Cat# 553258, RRID:AB_394738)

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What is this?

DOI: 10.1016/j.immuni.2024.05.018

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What is this?

DOI: 10.1016/j.celrep.2024.114330

Resource: Fiji (RRID:SCR_002285)

Curator: @scibot

SciCrunch record: RRID:SCR_002285

What is this?

PREPARE and VACS: $50 million announced at COP28