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What is this?

need elaboration

important

Kombination löschen

Alle Zeilen die diesen Wert haben rausschmeißen

Anschauen um welche Effekte es hier genau geht

Kombination löschen

Anschauen um welche Effekte es hier genau geht

Checken:

1. Was sind logged differences?
2. Wie liest man eine Regression der Form "logged differences" auf "logged differences", und kann man das vergleichbar machen?

Für alle Variablen ansehen was genau gemessen wird, + Formeln heraussuchen. Im Idealfall können wir alles mit Employment per person kombinieren.

Anschaune ob es vergleichbar ist

Sind das besetze Jobs oder ausgeschriebene JObs?

Nochmal genauer anschauen

And I believe that this will be true and long as watchable media is still viable. People will always crave more and the "Impluse to go on watching" will always live in humans as it is their nature. The construction of the flow theory in television is not only true in media but in all of life. Humans are constantly questioning and wanting answers which is why this model works so well.

I don't think that there is an ideological metaphor for the power of television. I think that any media consumption on a screen is very powerful, especially in the modern age we have seen it tear families and people apart.

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

Learn more at Review Commons

Reply to the reviewers

Manuscript number: RC-2025-03064

Corresponding author(s): Massimo, Hilliard; Sean, Coakley

1. General Statements

We are grateful to the reviewers for taking time to review our manuscript and for providing such clear, insightful and actionable suggestions. The consensus between 4 independent reviewers that this story is of general interest to cell biologists, neurobiologists and clinical researchers is remarkable. In addition to our mechanistic insights into the regulation of GTPase activity, we think that the experimental systems we have developed will be of great value to study how GTPases their associated GAPs and GEFs function to maintain the nervous system, especially due to the demonstrated conservation of these molecules. We believe that our data provides a powerful and tractable model to study such molecules in a physiological context.

We agree with the reviewers' concerns and propose the following plan below to address them.

2. Description of the planned revisions

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

__Summary Stability of the PLM axon in C. elegans is maintained through interactions with the epidermis. Previous studies by this group found that loss of the tbc-10 Rab GTPase Activating Protein strongly enhanced the PLM axon break phenotype of unc-70/beta-spectrin mutants. TBC-10 is a GAP for RAB-35 and thus loss of rab-35 suppresses the tbc-10 phenotype. Of the two RAB-35 GEFs, loss of RME-4 partially suppressed the tbc-10 phenotype and FLCN-1 was not involved suggesting that there may be an additional GEF involved. Here Bonacossa-Pereira et al identify a point mutation in agef-1a (vd92) as a suppressor of tbc-10 PLM axon break phenotype (all experiments also have a dominant allele of unc-70) and confirm that point mutation is causative by replicating the mutation via genome editing (vd123). Rescue experiments demonstrate that AGEF-1a is required in the epidermis and not PLM as previous demonstrated with tbc-10 and unc-70. Rescue is dependent on a functional SEC7/GEF activity. AGEF-1a is a functional ortholog to human BIG2/ArfGEF2 as its expression fully rescues tbc-10. AGEF-1a functions upstream of RAB-35 as expression of activated RAB-35 can suppress loss of agef-1. AGEF-1a functions in parallel to RME-4 as the double has stronger suppression of tbc-10. AGEF-1a is an ARF GEF, however it functions independently of ARF-1.2 as loss of arf-1.2 does not suppress tbc-10. They demonstrate that AGEF-1a interacts with RAB-35 through colocalization experiments suggesting that AGEF-1a could directly activate RAB-35. Finally, they demonstrate that AGEF-1a regulates the localization of the LET-805 epidermal attached complex component as it restores localization in a tbc-10 mutant.

Major comments

The manuscript is well written and easy to understand.

The experiments are well done and controlled.

I enjoyed reading this paper. However...

Some of the claims are not supported by the data.__

__1) The claim that AGEF-1a directly interacts with RAB-35 was not demonstrated. The evidence provided to support a direct interaction are colocalization experiments in Figure 3. AGEF-1a does partially colocalize with RAB-35 in the epidermis. However, colocalization does not indicate a physical interaction direct or indirect. A simple fix would be to change the claim to that they partially colocalize. Optional, a physical interaction could be done with the split-GFP since they already have the AGEF-1 strain or they could perform co-IP experiments, though neither of those are proof of direct interactions.

__

We agree that the biochemical co-IP experiment could provide some answers, however, using a full length AGEF-1a would not only represent a significant technical challenge but will also not prove a direct interaction in a physiological context. To overcome this limitation, and to directly test their interaction in vivo, we propose to use a split-GFP approach as suggested by the reviewer. In this experiment, we will generate an endogenously tagged GFP1-10::rab-35 allele and combine it with the previously generated and available tagged agef-1a::GFP11x7. If AGEF-1 and RAB-35 closely interact, we should observe the reconstitution of full length GFP. It is possible that the endogenously tagged versions only provide a very weak GFP signal that will be difficult to detect. As an alternative approach, we will generate the same tagged molecules as overexpressed transgenes under epidermal-specific promoters (such as Pdpy-7). If the results are still negative, we agree to temper our claim that these molecules physically interact and rephrase the manuscript to reflect the new data.

• *

2) The claim that AGEF-1a facilitates RAB-35 activation is not supported. While it is likely that AGEF-1a facilitates RAB-35 activation based on the epistasis experiments as well as studies in mammalian cells there were no experiments to demonstrate that modulating AGEF-1a activity resulted in a change in RAB-35 activity. I would suggest tempering this claim to something along the line that the data are consistent with AGEF-1a regulating RAB-35 activity as shown in mammalian cells. An optional experiment would be to look at the colocalization of RAB-35 with a known effector in wild type and agef-1(vd92) with the expectation that there would be a higher level of colocalization in agef-1 mutants. Effector pull-down experiments or perhaps a cell based GEF assay could be used (PMID: 35196081).

We welcome this suggestion and acknowledge the limitations of these experiments. While we might be able to determine if AGEF-1 and RAB-35 physically interact in vivo with the experiments proposed above, screening for the relevant rab-35 effector in this context and/or doing effector pull-down/cell based GEF assays would be a significant technical challenge. We propose to temper our claim as suggested.

3) The claim that AGEF-1a functions independently of ARF-1.2 is not well supported. The fact that the ARF-1.2 mutant does not suppress tbc-10 suggests that ARF-1.2 may not be involved but does not eliminate the possibility that ARF-1.2 functions redundantly with ARF-5 or WARF-1/ARF-1.1. This can be resolved by toning down the claim. Alternatively, this can be tested by RNAi of arf-5 and warf-1 in tbc-10 and arf-1.2; tbc-10 mutants.

We agree that warf-1 and arf-5 could be functioning redundantly with arf-1.2. We have attempted to generate an AID::arf-5 allele to test the effect of cell-specific degradation, but homozygous AID::arf-5 animals were lethal. We have not yet examined warf-1. We believe the best way to test these two molecules is through RNAi knockdown, and we propose to do this experiment and adjust our interpretation and discussion according to the new data.

Minor comments

Figure 1C the CRISPR generated allele (vd123) is referred to as [S784L] and then in 1E vd92 is referred to as [S784L]. Perhaps it would be clearer if the allele name was used instead of the amino acid change.

We will reformat the manuscript to include the allele names instead of amino acid change.

Page 6 "We reasoned that if the S784L mutation we isolated causes a similar loss of the GTPase activation function, then SKIN::AGEF-1a[E608K] would not have the capacity to restore the rate of PLM axon breaks to background levels in agef-1[S784L]; tbc-10; vdSi2 animals." It was unclear to me whether you were testing if the S784L mutation could be disrupting a GEF independent function or might disrupt the nucleotide exchange activity as might be tested in a biochemical assay. There are many reasons this change could cause a loss of function phenotype (ie. Improper folding, mislocalization, etc.). The most clear explanation would be that you were testing if GEF function was required for rescue rather than testing if the S784L mutation disrupted GEF activity.

Indeed, this experiment reveals that reducing the activation of the AGEF-1 target phenocopies the effect of S784L and does not further enhance the effect of S784L. However, it does not answer if, specifically, the GEF function is affected by S784L. We propose to rewrite the quoted sentence as follows: "We asked whether the GEF function is required for axonal damage. If that is the case, then SKIN::AGEF-1a[E608K] overexpression should phenocopy the effect of AGEF-1a[S784L]."

• *

Page 13. It was unclear how testing if AGEF-1, RME-4, ARF-5 and RAB-35 form complexes in vivo (I assume you are suggesting colocalize based on figure 3 interpretation) would resolve how AGEF-1 was regulating RAB-35.

We apologize that our phrasing was not clear. We will rewrite this section to better reflect the following idea. Given literature data showing an allosteric interaction between RME-4/DENND1 and ARF-5/Arf5, and our own data showing that AGEF-1 regulates RAB-35, we believe these molecules could form a complex. Considering that we do not have data to support this notion, mostly due to the inability to test the effect of ARF-5, we will present this possibility in the discussion section.

__**Cross-commenting**

I agree with the comments made by the other reviewers and I stand by my own as well. I will echo that it is important to know the nature of their agef-1 allele.

Reviewer #1 (Significance (Required)):

Bonacossa-Pereira et al identify AGEF-1 as a regulator of axon integrity that functions in a pathway with RAB-35 in the epidermis is an exciting finding. As pointed out in the discussion, mutations in the human ortholog cause neurodevelopmental defects which leads to obvious characterization of BIG2/ArfGEF2 in neurons while this study indicates that this protein can have cell non-autonomous roles in regulating neurons. These findings could have important implications for understanding the etiology of these defects that would be of interest to neurobiologists and clinical researchers.

The finding of this paper would also be of interest to cell biologists and particularly those studying the roles of Rab and Arf GTPases in membrane trafficking, such as myself. The idea that AGEF-1 might function as a Rab35 GEF is provocative and would generate a lot of interest and skepticism from the field. However, there is no data to support that AGEF-1 would be a direct regulator of Rab35 over the previously demonstrated cross regulation of Rab35 by Arf GTPases. Therefore, it would be fine to speculate in the discussion a direct interaction, but I would refrain from suggesting this as a model and elsewhere in the manuscript.

__

Although we agree that current evidence is not sufficient to support the model where AGEF-1 is a direct regulator of RAB-35, our data points to the direction where there is an important genetic relationship between these molecules in a physiological context in a living animal, with a defined phenotype relevant to the nervous system maintenance. We think that the proposed revision experiments will provide a better understanding of how AGEF-1 functions with RAB-35 and we agree with the suggestion to rephrase our manuscript to reflect the limitations of our results.

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

This interesting manuscript reports the outcome of a fruitful C. elegans genetic screen with a complex but clever design. Through it, the authors identify AGEF-1 as a GEF that likely regulates the active state of the GTPase RAB-35 in the skin to protect touch receptor axons from mechanical breakage.

Major points: 1. Based on localization experiments, the authors claim "AGEF-1a interacts with RAB-35 in the epidermis" (Results heading) and state "these data demonstrate that AGEF-1a interacts with a subset of RAB-35 molecules in the epidermis." In general, localization studies cannot be used to conclude physical interaction (with some exceptions such as single-molecule kinetics). In this case, the data in my view do not even make a compelling argument for co-localization. There is a lot of AGEF-1 and RAB-35 signal everywhere and it may not be meaningful that the signals sometimes overlap. A more quantitative approach with controls would be needed to conclude meaningful co-localization. Importantly, this would still not demonstrate interaction.__

We thank the reviewer for the comment. Indeed, co-localization does prove a physical interaction, and we appreciate the concern about our imaging data not making a compelling argument. To address this notion, we plan to perform an experiment using a more robust, quantitative and physiologically relevant strategy. We will generate an endogenously tagged mScarlet3::rab-35 allele for precise endogenous localization. In addition, as a positive control, we will generate an endogenous rme-4::GFP11x7 allele to cell-specifically demonstrate the level of colocalization of RME-4 with mScarlet3::RAB-35 within the epidermis. To address the possible interaction between AGEF-1a and RAB-35 we will leverage a split-GFP approach to assess their interaction in vivo, in the context relevant to the phenotypes we observed (see reply to reviewer #1 point 1).

__2. The effect of the AGEF-1(S784L) mutation is not clear to me. Naively, as the S784L mutation lies in the auto-inhibitory domain, I would have expected AGEF-1 to become constitutively active, not inactive as the authors seem to suggest. Is the idea that it is constitutively auto-inhibited? The main evidence for a loss of function effect seems to be that a putative dominant negative mutation AGEF-1(E608K) does not further supress axon breakage when co-expressed in trans to AGEF(S784L), but in my view this only shows that, once the defect is suppressed, it cannot be suppressed any further. Defining the nature of the S784L allele is important. Some suggestions, although the authors may come up with different approaches: use of an inducible or cell-specific depletion system like AID/TIR1, Cre/lox, or FLP/FRT to circumvent the lethality of agef-1(0) and reveal what a true loss-of-function looks like; testing if deletion of the auto-inhibitory domain phenocopies S784L to test if this mutation impairs autoinhibition.

__

This is an very insightful comment. To address this point, we will follow the reviewer's suggestion and deplete AGEF-1 cell-specifically in the epidermis using the auxin-inducible degron system. Specifically, we will generate an agef-1::AID allele to degrade this molecule in a spatially and temporally controlled fashion, which will allow to circumvent the lethality of agef-1(0) and determine whether the S784L allele mimics the depletion of AGEF-1.

Although it would be interesting to further dissect the effect of this mutation on AGEF-1 activity, we believe that this falls outside of the scope of this manuscript. As an alternative, we propose to elaborate more in the discussion the implications of the possible roles for the S784L mutation to clarify our model of its function. Our data supports a model in which this mutation reduces AGEF-1 function leading to a reduction in the activity of its downstream target GTPases. It is possible that this is due to AGEF-1 becoming constitutively autoinhibited, or that this mutation affects the structure of the molecule in a way that it reduces its affinity towards its downstream effectors.

Minor points: 1. I am not able to see the "vesicle-like structures with a clear luminal space" or RAB-35 being "notably enriched at the membrane near the epidermal furrow" in Fig. 3. The "3D surface rendering" in Fig. 3e is grossly oversampled and should not be included.

We will rectify this section and include new super-resolved images using Airyscan confocal microscopy. We hope these will yield a better-quality representation of these concepts. __ 2. As the agef-1a isoform is specifically referenced throughout, please describe the different agef-1 isoforms somewhere to save readers from having to look this up.__

Yes, we will include a description of the isoforms. In C. elegans there are two: AGEF-1a which has been confirmed by cDNA and AGEF-1b which is predicted and partially confirmed by cDNA. The mutation we isolated exclusively affects AGEF-1a.

3. The authors include an interesting speculation in the Discussion: "Future investigations of BIG2-associated neurological disorders should consider... hyper-activity of BIG2 as a driver of neuropathology." If the authors have the tools to test the effect of hyperactive BIG2 in this system, it could be an exciting addition.

This is an exciting idea that we would like to keep in the Discussion. The biology of BIG2 activity regulation is a nascent field of research and we believe that to accurately generate and characterise a hyperactive BIG2 would be beyond the scope of this manuscript.

__ On a personal note, since GEFs act oppositely to GTPase Activating Proteins (GAPs), I had to stop and re-read carefully whenever the authors referred to a GEF "activating" a GTPase. I understand their meaning (i.e., putting the GTPase in its active GTP-bound state, not activating its GTPase function) but I wanted to point out this potential confusion in case there is a way to better define terms in the Introduction or change word choice. I realize this may be a standard jargon in the field.__

Indeed, this is confusing nomenclature and a difficult concept to deliver in an accurate and succinct manner. We propose to include a clearer, more didactic explanation of their function. In a simple explanation, GTPases perform cellular functions when bound to GTP. GAPs terminate GTPase activity by catalysing GTP hydrolysis, generating GDP. GEFs initiate GTPase activity by catalysing the release of GDP and allowing GTP binding.

__ Please check the correct nomenclature for CRISPR/Cas9.__

We will rectify where appropriate.

__6. p.7 "these molecules act in synergy", consider replacing with "redundantly".

__

We will rectify where appropriate.

__Reviewer #2 (Significance (Required)):

The significance of this story is to show that GEF-GTPases pairing can be highly context-dependent. Previous studies have identified GEFs that pair with RAB-35 and GTPases that pair with AGEF-1, but the authors find that these factors have at best a modest role in the context of skin-axon interactions. Instead, the authors suggest a novel GTPase-GEF pairing of RAB-35 with AGEF-1 and provide evidence that this relationship is conserved in the human homolog of AGEF-1. These results suggest that GTPase-GEF pairings depend not only on chemical affinity but also cellular context.

The main strength of the study is its clever genetics. For the screen, the authors looked for suppressors of a synthetic defect in axon integrity caused in part by elevated activity of RAB-35 due to loss of its GAP TBC-10. It is satisfying that this screen isolated a mutation in a GEF that in principle could counterbalance the loss of a GAP.

The main weakness of the study is the lack of direct evidence for an AGEF-1/RAB-35 interaction. While not necessary for publication, the inclusion of biochemical data to support the role of AGEF-1 as a GEF for RAB-35 and the effect of the S784L mutation on this activity would strongly elevate the study. The genetic data for this interaction are consistent with the model but not conclusive, and in my view the colocalization data are not compelling. Nevertheless this is a solid genetic story with a clever screen.__

__ __We appreciate the feedback and are grateful for the positive comments on the significance of our study. As explained in the significance section related to Reviewer 1, if we find evidence of a direct interaction between AGEF-1 and RAB-35 in the proposed new experiments, we will include it in the manuscript; alternatively, we will present it as a possibility in the discussion section, as suggested. We agree that a more nuanced understanding of the effect of the S784L is interesting and that our colocalization data can be improved, and we have proposed experiments to address these concerns.

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

This paper investigates the mechanism by which molecular pathways in the skin protect the processes of nerves that innervate them from damage. The authors previously showed that spectrin and the small GTPase RAB-35 act in the epidermis of C. elegans to protect mechanosensory axons from breaking. In this paper they used a suppression screen to identify another gene involved in this process, an ARF-GEF called AGEF-1. Partial loss-of-function mutations in agef-1 suppress the axon-breakage phenotype of spectrin mutations, and genetic experiments by the authors are consistent with the possibility that AGEF-1 could act directly as an exchange factor for RAB-35. Consistent with this model, they show that AGEF-1 and RAB-35 colocalise in the skin.

Major comments: The experiments in this paper are well-designed and well-controlled, and the interpretations of the results are all reasonable. On the other hand, I don't think the authors' hypothesis that AGEF-1 acts directly as an exchange factor for RAB-35, or that these two proteins directly interact, is definitively proven. This is not an issue of the authors overinterpreting their data--the paper is very carefully and thoughtfully written. However, the most interesting and counterintuitive finding--that an ARF-GEF could also be a RAB-GEF--might be strengthened with more experiments (for example, could they more directly show protein-protein interaction through co-IP or mass spec?).__

We thank the reviewer for the suggestion. We propose to further investigate the notion that AGEF-1a might be a direct interactor of RAB-35 using a split-GFP approach to assess whether these molecules closely interact, in vivo, in the physiological context that is relevant for the maintenance of the touch sensing neurons (please see reply to reviewer #1 major point 1 and reviewer #2 major point 1 for more details).

Minor comments: There are also two places where the fact that null mutations are lethal (for agef-1 and arf-5) prevented the authors from addressing the effect of agef-1 loss of function in the skin, and addressing whether ARF-5 could be an AGEF-1 target, respectively. In principle, they could have tried to make a CRISPR line in which these genes could be cell-specifically deleted in the skin (using a dpy-7-driven recombinase). I don't think either of these experiments are essential, but if it is feasible to make these lines it would tie up a couple of loose ends.

We agree to explore the roles of agef-1 and arf-5 loss-of-function. We propose to tissue-specifically degrade agef-1 using an auxin-inducible degradation strategy (please see reviewer #2 major point 2 reply for more details). For arf-5, we propose knocking-down its function using RNAi to overcome lethality (please see reviewer #1 major point 3 reply for more details).

__Reviewer #3 (Significance (Required)):

Overall I think this is an interesting paper on a topic of general interest. The most interesting finding is that an exchange factor for an ARF (a small GRPase involved in vesicle coating/uncoating) could also be an exchange factor for a RAB (a small GTPase involved in vesicle tethering). The evidence presented is suggestive and intriguing, though as noted above not completely definitive. In summary, I think it is an interesting paper in its current form, and anything it could do to more firmly establish a direct interaction between AGEF-1 and RAB-35 would increase its impact and importance.

__

We thank the reviewer for the positive evaluation of the significance of our study.

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

Summary: In this study Bonacossa-Pereira et al. identify AGEF-1a, an Arf-GEF, as a factor that functions in the epidermis through RAB-35 to regulate axonal integrity of the PLM mechanosensory neurons in C. elegans. Specifically, epidermal attachment sites are regulated by these genes form the epidermis and compromising these attachment sites results in axonal degeneration. The study provides some evidence that that RAB-35 and AGEF-1 at least partially colocalize in the skin. Finally, the authors provide evidence that the human orthologue BIG2 is capable of functionally replacing AGEF-1a in C. elegans. Overall, the experiments are well designed and the paper is clear and succinct. The conclusions are supported by the findings and provide an important extension of the author's findings a few back, when they identified the role of rab-35 in mediating the epidermal-neuronal attachment sites.

Major comments: 1. AGEF-1/BIG2 are known to regulate other GTPases such as ARF-5 or ARF-2. The authors exclude a non-redundant function for ARF-2, but are unable to establish a role for ARF-5 because of the lethality associated with the mutation. Alternative approaches, such as cell specific knock out or knock down experiment. In addition, studies to test potentially physical interaction such as pull-down assays, co-IP experiments and FRET could be used to test whether AGEF-can bind RAB-35 or ARF-5.__

We thank the reviewer for this suggestion. We propose addressing these concerns using a tissue-specific degradation for AGEF-1a (please see reviewer #1 major point 2 for details). To establish a role for ARF-5 we propose to do an RNAi mediated knock-down to overcome lethality (please see reviewer #1 major point 3 for details). Finally, we plan to use a split-GFP approach to test the physical interaction between agef-1a and rab-35 in vivo (please see reviewer #1 major point 1 for details)

__ Phenotypic readout has been limited to only axon breaks. It may be interesting to also test other aspects such as axonal deformities including swellings and vesiculation in other parts of the nervous system. Moreover, behavioral or functional experiments such as response to gentle touch or synaptic integrity could be informative.__

We have not observed any obvious touch receptor neurons axonal phenotypes other than axonal breaks in these mutants, and we will include a statement that reflects this concept. In relation to the behavior, we have not tested it as the results will be difficult to interpret for two reasons: first, the breaks are not always bilateral and one neuron is sufficient to provide mechanical response; second, the mixed identity of the PLM neurite allows it to retain some function despite being severed. However, if deemed essential, we will perform these experiments.

__ Overexpression constructs such as SKIN::RAB-35[Q69L], SKIN::BIG2, SKIN::AGEF-1a[E608K] in extrachromosomal transgenes could lead to non-physiological localization or effects. Single copy expression using MosSCI or CRISPR insertions are generally considered better approaches (other than endogenous reporters) to provide accurate insights at the physiological level. While the authors tacitly acknowledge this by conducting the experiments in a rab-35 mutant background and very low transgene concentration, at the very least this caveat regarding the localization should be discussed.__

This is an important remark, and we appreciate the comment. We acknowledge that experiments using extrachromosomal arrays have inherent caveats, especially for localization studies. To address the RAB-35 localization concern we plan to repeat the localization studies using an endogenously tagged RAB-35 using CRISPR to overcome the possible artifacts caused by extrachromosomal array driven expression (please see reviewer #1 point 1 for more details). For the cell-specific rescues or dominant-negative constructs expression, we believe that using extrachromosomal arrays is sufficient, since this allows us to compare genetically identical transgenic vs non-transgenic siblings of independent lines. Moreover, given these constructs are already driven by a tissue-specific promoter that is inherently stronger than their respective endogenous promoters, even a single-copy insertion would have the same caveats.

__4. The study does not address clearly whether AGEF-1a acts in parallel to spectrin or upstream/ downstream to it. Epistasis experiments could help to figure out the signaling pathway involved.

__

Indeed, this is a concept that we need to communicate more clearly. We have data showing that a mutation in agef-1 does not cause axonal damage on its own, and that it has no effect on the axonal damage caused by unc-70 dominant negative mutation alone. We only detect an effect of agef-1 when tbc-10 is mutated together with unc-70 (Fig. 1a of manuscript). Together, these data indicate that agef-1 functions upstream of rab-35, thus acting in parallel to unc-70 (see schematic below) to ensure the mechanical stability of neuron epidermal attachment. We plan to include this data and the following schematic as a supplement to better convey the idea and discuss the results appropriately.

__ The finding that BIG2 rescues the mutant defect is an important finding and rightfully finds its place in the abstract. I wonder whether a reference to the human diseases caused by loss of BIG2 in the abstract and introduction would not increase interest/impact for the study, rather than burying this potentially interesting connection in the discussion.

__

We appreciate the reviewer's comment, and welcome the suggestion. We propose to include relevant background about BIG2-related human diseases in the abstract and introduction as suggested and expand the discussion regarding BIG2 mutations.

__Minor comments:

1. Some explanation about how mutating the autoinhibitory domain could impact the catalytic activity of a GEF might be helpful.__

2. *

We acknowledge that this notion was not well communicated. We propose to elaborate more about why we think a mutation in the autoinhibitory domain might be affecting the GEF activity and we plan to do further experiments to dissect how this might be happening. Please see reviewer #2 major point 2 for a more detailed explanation.

__ The paper refers to rme-4(b1001) as a null allele while wormbase refers to the same as a missense allele. It would be more accurate to refer rme-4(b1001) as a strong loss of function or putative null.__

We agree and will refer to b1001 as a strong loss-of-function.

__ The paper does not clearly discuss limitations of the hypomorphic agef-1[S784L] and that the observed phenotypes in this hypomorph might underestimate the complete role of AGEF-1a.__

• *

We thank the reviewer for this suggestion. We propose to elaborate more on these limitations, especially considering the possible new results from the experiments suggested in reply to reviewer #2 major comment point 2.

__ In figure 1, where there really only one extrachromosomal transgenic line for some of the construct tested? __

• *

For the Pdpy-7::AGEF-1a lines we have scored 3 transgenic lines (data not included) and only one yielded a full rescue. For all extrachromosomal lines presented, we tested 3 independent transgenic lines. For brevity, we only included the result for the positive rescues (1 for BIG2 and 1 for AGEF-1a), except for the Pmec-4 lines, of which none rescued the phenotype (data included in Table S2). We will update Table S2 to include all the lines tested.

__ The concentrations of transgenes vary in different transgenes. Is there a rationale behind this? __

Yes, we have attempted multiple concentrations of injections for each transgene and there was some variability for each construct injected, thus we only included the ones where we observed an effect. As mentioned in point 4 above, we will update Table S2 to include details of all lines tested.

__ In Fig.1e: I may be useful to also show the "WT" phenotype, i.e. the strong defects to get a visual comparison for the degree of rescue. __

• *

We think this suggestion will help the readers. We will include this as a representative dashed line showing the WT phenotype.

__Reviewer #4 (Significance (Required)):

The study has identified AGEF-1a as a regulator of axonal maintenance, functioning to protect neurons against mechanical stress by acting through RAB-35. Additionally, this epidermal GEF, AGEF-1a is functionally conserved as its human orthologue BIG2 can replace AGEF-1a in C. elegans for axonal protection. Important points here are that the findings extend prior work by the authors of non-autonomous mechanism that regulates epidermal-neuronal attachment. In my humble opinion, the human disease connection, in particular with regard to the unexplained neuronal phenotypes in patients could be better developed in the manuscript. It may also increase impact/interest of a wonderful story that right now reads a bit 'wormy'.__

This is an important remark and we are grateful for the positive comments. The fact that human BIG2 is also conserved in C. elegans points to a fundamental role of this molecule in multicellular life, and it provides a tractable model to investigate the function of this molecule in a physiological context. We welcome the suggestion to elaborate more the connection with the unexplained neuronal phenotypes in patients and use a more accessible language to convey our findings to a wider audience.

3. Description of the revisions that have already been incorporated in the transferred manuscript

N/A

4. Description of analyses that authors prefer not to carry out

__Reviewer #1 __

"...studies to test potentially physical interaction such as pull-down assays, co-IP experiments and FRET could be used to test whether AGEF-can bind RAB-35 or ARF-5."

While pull-down assays, co-IP and FRET would reveal whether AGEF-1a can form a complex with RAB-35, we believe that using a full length AGEF-1a would not only represent a significant technical challenge but will also not prove a direct interaction in a physiological context.

"...An optional experiment would be to look at the colocalization of RAB-35 with a known effector in wild type and agef-1(vd92) with the expectation that there would be a higher level of colocalization in agef-1 mutants. Effector pull-down experiments or perhaps a cell based GEF assay could be used (PMID: 35196081)."

We think that screening for the relevant rab-35 effector in this context and/or doing effector pull-down/cell based GEF assays would be a significant technical challenge. We propose to address this concern by tempering our claim as suggested by the reviewer.

"...It may be interesting to also test other aspects such as axonal deformities including swellings and vesiculation in other parts of the nervous system. Moreover, behavioral or functional experiments such as response to gentle touch or synaptic integrity could be informative."

As indicated above in major point 2 of reviewer 4, these are interesting ideas that might answer how the function of these neurons might be affected. However, in addition to the challenges indicated above, they will not provide further insights into how their integrity is maintained. We believe these will fall outside the scope of the manuscript, but if deemed essential we will perform behavioral analysis.

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Referee #4

Evidence, reproducibility and clarity

Summary:

In this study Bonacossa-Pereira et al. identify AGEF-1a, an Arf-GEF, as a factor that functions in the epidermis through RAB-35 to regulate axonal integrity of the PLM mechanosensory neurons in C. elegans. Specifically, epidermal attachment sites are regulated by these genes form the epidermis and compromising these attachment sites results in axonal degeneration. The study provides some evidence that that RAB-35 and AGEF-1 at least partially colocalize in the skin. Finally, the authors provide evidence that the human orthologue BIG2 is capable of functionally replacing AGEF-1a in C. elegans. Overall, the experiments are well designed and the paper is clear and succinct. The conclusions are supported by the findings and provide an important extension of the author's findings a few back, when they identified the role of rab-35 in mediating the epidermal-neuronal attachment sites.

Major comments:

1. AGEF-1/BIG2 are known to regulate other GTPases such as ARF-5 or ARF-2. The authors exclude a non-redundant function for ARF-2, but are unable to establish a role for ARF-5 because of the lethality associated with the mutation. Alternative approaches, such as cell specific knock out or knock down experiment. In addition, studies to test potentially physical interaction such as pull-down assays, co-IP experiments and FRET could be used to test whether AGEF-can bind RAB-35 or ARF-5.
2. Phenotypic readout has been limited to only axon breaks. It may be interesting to also test other aspects such as axonal deformities including swellings and vesiculation in other parts of the nervous system. Moreover, behavioral or functional experiments such as response to gentle touch orsynaptic integrity could be informative.
3. Overexpression constructs such as SKIN::RAB-35[Q69L], SKIN::BIG2, SKIN::AGEF-1a[E608K] in extrachromosomal transgenes could lead to non-physiological localization or effects. Single copy expression using MosSCI or CRISPR insertions are generally considered better approaches (other than endogenous reporters) to provide accurate insights at the physiological level. While the authors tacitly acknowledge this by conducting the experiments in a rab-35 mutant background and very low transgene concentration, at the very least this caveat regarding the localization should be discussed.
4. The study does not address clearly whether AGEF-1a acts in parallel to spectrin or upstream/ downstream to it. Epistasis experiments could help to figure out the signaling pathway involved.
5. The finding that BIG2 rescues the mutant defect is an important finding and rightfully finds its place in the abstract. I wonder whether a reference to the human diseases caused by loss of BIG2 in the abstract and introduction would not increase interest/impact for the study, rather than burying this potentially interesting connection in the discussion.

Minor comments:

1. Some explanation about how mutating the autoinhibitory domain could impact the catalytic activity of a GEF might be helpful.
2. The paper refers to rme-4(b1001) as a null allele while wormbase refers to the same as a missense allele. It would be more accurate to refer rme-4(b1001) as a strong loss of function or putative null.
3. The paper does not clearly discuss limitations of the hypomorphic agef-1[S784L] and that the observed phenotypes in this hypomorph might underestimate the complete role of AGEF-1a.
4. In figure 1, where there really only one extrachromosomal transgenic line for some of the construct tested?
5. The concentrations of transgenes vary in different transgenes. Is there a rationale behind this?
6. In Fig.1e: I may be useful to also show the "WT" phenotype, i.e. the strong defects to get a visual comparison for the degree of rescue.

Significance

The study has identified AGEF-1a as a regulator of axonal maintenance, functioning to protect neurons against mechanical stress by acting through RAB-35. Additionally, this epidermal GEF, AGEF-1a is functionally conserved as its human orthologue BIG2 can replace AGEF-1a in C. elegans for axonal protection. Important points here are that the findings extend prior work by the authors of non-autonomous mechanism that regulates epidermal-neuronal attachment. In my humble opinion, the human disease connection, in particular with regard to the unexplained neuronal phenotypes in patients could be better developed in the manuscript. It may also increase impact/interest of a wonderful story that right now reads a bit 'wormy'.

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Referee #3

Evidence, reproducibility and clarity

This paper investigates the mechanism by which molecular pathways in the skin protect the processes of nerves that innervate them from damage. The authors previously showed that spectrin and the small GTPase RAB-35 act in the epidermis of C. elegans to protect mechanosensory axons from breaking. In this paper they used a suppression screen to identify another gene involved in this process, an ARF-GEF called AGEF-1. Partial loss-of-function mutations in agef-1 suppress the axon-breakage phenotype of spectrin mutations, and genetic experiments by the authors are consistent with the possibility that AGEF-1 could act directly as an exchange factor for RAB-35. Consistent with this model, they show that AGEF-1 and RAB-35 colocalise in the skin.

Major comments: The experiments in this paper are well-designed and well-controlled, and the interpretations of the results are all reasonable. On the other hand, I don't think the authors' hypothesis that AGEF-1 acts directly as an exchange factor for RAB-35, or that these two proteins directly interact, is definitively proven. This is not an issue of the authors overinterpreting their data--the paper is very carefully and thoughtfully written. However, the most interesting and counterintuitive finding--that an ARF-GEF could also be a RAB-GEF--might be strengthened with more experiments (for example, could they more directly show protein-protein interaction through co-IP or mass spec?).

Minor comments: There are also two places where the fact that null mutations are lethal (for agef-1 and arf-5) prevented the authors from addressing the effect of agef-1 loss of function in the skin, and addressing whether ARF-5 could be an AGEF-1 target, respectively. In principle, they could have tried to make a CRISPR line in which these genes could be cell-specifically deleted in the skin (using a dpy-7-driven recombinase). I don't think either of these experiments are essential, but if it is feasible to make these lines it would tie up a couple of loose ends.

Significance

Overall I think this is an interesting paper on a topic of general interest. The most interesting finding is that an exchange factor for an ARF (a small GRPase involved in vesicle coating/uncoating) could also be an exchange factor for a RAB (a small GTPase involved in vesicle tethering). The evidence presented is suggestive and intriguing, though as noted above not completely definitive. In summary, I think it is an interesting paper in its current form, and anything it could do to more firmly establish a direct interaction between AGEF-1 and RAB-35 would increase its impact and importance.

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Referee #2

Evidence, reproducibility and clarity

This interesting manuscript reports the outcome of a fruitful C. elegans genetic screen with a complex but clever design. Through it, the authors identify AGEF-1 as a GEF that likely regulates the active state of the GTPase RAB-35 in the skin to protect touch receptor axons from mechanical breakage.

Major points:

1. Based on localization experiments, the authors claim "AGEF-1a interacts with RAB-35 in the epidermis" (Results heading) and state "these data demonstrate that AGEF-1a interacts with a subset of RAB-35 molecules in the epidermis." In general, localization studies cannot be used to conclude physical interaction (with some exceptions such as single-molecule kinetics). In this case, the data in my view do not even make a compelling argument for co-localization. There is a lot of AGEF-1 and RAB-35 signal everywhere and it may not be meaningful that the signals sometimes overlap. A more quantitative approach with controls would be needed to conclude meaningful co-localization. Importantly, this would still not demonstrate interaction.
2. The effect of the AGEF-1(S784L) mutation is not clear to me. Naively, as the S784L mutation lies in the auto-inhibitory domain, I would have expected AGEF-1 to become constitutively active, not inactive as the authors seem to suggest. Is the idea that it is constitutively auto-inhibited? The main evidence for a loss of function effect seems to be that a putative dominant negative mutation AGEF-1(E608K) does not further supress axon breakage when co-expressed in trans to AGEF(S784L), but in my view this only shows that, once the defect is suppressed, it cannot be suppressed any further. Defining the nature of the S784L allele is important. Some suggestions, although the authors may come up with different approaches: use of an inducible or cell-specific depletion system like AID/TIR1, Cre/lox, or FLP/FRT to circumvent the lethality of agef-1(0) and reveal what a true loss-of-function looks like; testing if deletion of the auto-inhibitory domain phenocopies S784L to test if this mutation impairs autoinhibition.

Minor points:

1. I am not able to see the "vesicle-like structures with a clear luminal space" or RAB-35 being "notably enriched at the membrane near the epidermal furrow" in Fig. 3. The "3D surface rendering" in Fig. 3e is grossly oversampled and should not be included.
2. As the agef-1a isoform is specifically referenced throughout, please describe the different agef-1 isoforms somewhere to save readers from having to look this up.
3. The authors include an interesting speculation in the Discussion: "Future investigations of BIG2-associated neurological disorders should consider... hyper-activity of BIG2 as a driver of neuropathology." If the authors have the tools to test the effect of hyperactive BIG2 in this system, it could be an exciting addition.
4. On a personal note, since GEFs act oppositely to GTPase Activating Proteins (GAPs), I had to stop and re-read carefully whenever the authors referred to a GEF "activating" a GTPase. I understand their meaning (i.e., putting the GTPase in its active GTP-bound state, not activating its GTPase function) but I wanted to point out this potential confusion in case there is a way to better define terms in the Introduction or change word choice. I realize this may be a standard jargon in the field.
5. Please check the correct nomenclature for CRISPR/Cas9.
6. p.7 "these molecules act in synergy", consider replacing with "redundantly".

Significance

The significance of this story is to show that GEF-GTPases pairing can be highly context-dependent. Previous studies have identified GEFs that pair with RAB-35 and GTPases that pair with AGEF-1, but the authors find that these factors have at best a modest role in the context of skin-axon interactions. Instead, the authors suggest a novel GTPase-GEF pairing of RAB-35 with AGEF-1 and provide evidence that this relationship is conserved in the human homolog of AGEF-1. These results suggest that GTPase-GEF pairings depend not only on chemical affinity but also cellular context.

The main strength of the study is its clever genetics. For the screen, the authors looked for suppressors of a synthetic defect in axon integrity caused in part by elevated activity of RAB-35 due to loss of its GAP TBC-10. It is satisfying that this screen isolated a mutation in a GEF that in principle could counterbalance the loss of a GAP.

The main weakness of the study is the lack of direct evidence for an AGEF-1/RAB-35 interaction. While not necessary for publication, the inclusion of biochemical data to support the role of AGEF-1 as a GEF for RAB-35 and the effect of the S784L mutation on this activity would strongly elevate the study. The genetic data for this interaction are consistent with the model but not conclusive, and in my view the colocalization data are not compelling. Nevertheless this is a solid genetic story with a clever screen.

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Referee #1

Evidence, reproducibility and clarity

Summary

Stability of the PLM axon in C. elegans is maintained through interactions with the epidermis. Previous studies by this group found that loss of the tbc-10 Rab GTPase Activating Protein strongly enhanced the PLM axon break phenotype of unc-70/beta-spectrin mutants. TBC-10 is a GAP for RAB-35 and thus loss of rab-35 suppresses the tbc-10 phenotype. Of the two RAB-35 GEFs, loss of RME-4 partially suppressed the tbc-10 phenotype and FLCN-1 was not involved suggesting that there may be an additional GEF involved. Here Bonacossa-Pereira et al identify a point mutation in agef-1a (vd92) as a suppressor of tbc-10 PLM axon break phenotype (all experiments also have a dominant allele of unc-70) and confirm that point mutation is causative by replicating the mutation via genome editing (vd123). Rescue experiments demonstrate that AGEF-1a is required in the epidermis and not PLM as previous demonstrated with tbc-10 and unc-70. Rescue is dependent on a functional SEC7/GEF activity. AGEF-1a is a functional ortholog to human BIG2/ArfGEF2 as its expression fully rescues tbc-10. AGEF-1a functions upstream of RAB-35 as expression of activated RAB-35 can suppress loss of agef-1. AGEF-1a functions in parallel to RME-4 as the double has stronger suppression of tbc-10. AGEF-1a is an ARF GEF, however it functions independently of ARF-1.2 as loss of arf-1.2 does not suppress tbc-10. They demonstrate that AGEF-1a interacts with RAB-35 through colocalization experiments suggesting that AGEF-1a could directly activate RAB-35. Finally, they demonstrate that AGEF-1a regulates the localization of the LET-805 epidermal attached complex component as it restores localization in a tbc-10 mutant.

Major comments

The manuscript is well written and easy to understand.

The experiments are well done and controlled.

I enjoyed reading this paper. However...

Some of the claims are not supported by the data.

1. The claim that AGEF-1a directly interacts with RAB-35 was not demonstrated. The evidence provided to support a direct interaction are colocalization experiments in Figure 3. AGEF-1a does partially colocalize with RAB-35 in the epidermis. However, colocalization does not indicate a physical interaction direct or indirect. A simple fix would be to change the claim to that they partially colocalize. Optional, a physical interaction could be done with the split-GFP since they already have the AGEF-1 strain or they could perform co-IP experiments, though neither of those are proof of direct interactions.
2. The claim that AGEF-1a facilitates RAB-35 activation is not supported. While it is likely that AGEF-1a facilitates RAB-35 activation based on the epistasis experiments as well as studies in mammalian cells there were no experiments to demonstrate that modulating AGEF-1a activity resulted in a change in RAB-35 activity. I would suggest tempering this claim to something along the line that the data are consistent with AGEF-1a regulating RAB-35 activity as shown in mammalian cells. An optional experiment would be to look at the colocalization of RAB-35 with a known effector in wild type and agef-1(vd92) with the expectation that there would be a higher level of colocalization in agef-1 mutants. Effector pull-down experiments or perhaps a cell based GEF assay could be used (PMID: 35196081).
3. The claim that AGEF-1a functions independently of ARF-1.2 is not well supported. The fact that the ARF-1.2 mutant does not suppress tbc-10 suggests that ARF-1.2 may not be involved but does not eliminate the possibility that ARF-1.2 functions redundantly with ARF-5 or WARF-1/ARF-1.1. This can be resolved by toning down the claim. Alternatively, this can be tested by RNAi of arf-5 and warf-1 in tbc-10 and arf-1.2; tbc-10 mutants.

Minor comments

Figure 1C the CRISPR generated allele (vd123) is referred to as [S784L] and then in 1E vd92 is referred to as [S784L]. Perhaps it would be clearer if the allele name was used instead of the amino acid change.

Page 6 "We reasoned that if the S784L mutation we isolated causes a similar loss of the GTPase activation function, then SKIN::AGEF-1a[E608K] would not have the capacity to restore the rate of PLM axon breaks to background levels in agef-1[S784L]; tbc-10; vdSi2 animals." It was unclear to me whether you were testing if the S784L mutation could be disrupting a GEF independent function or might disrupt the nucleotide exchange activity as might be tested in a biochemical assay. There are many reasons this change could cause a loss of function phenotype (ie. Improper folding, mislocalization, etc.). The most clear explanation would be that you were testing if GEF function was required for rescue rather than testing if the S784L mutation disrupted GEF activity.

Page 13. It was unclear how testing if AGEF-1, RME-4, ARF-5 and RAB-35 form complexes in vivo (I assume you are suggesting colocalize based on figure 3 interpretation) would resolve how AGEF-1 was regulating RAB-35.

Cross-commenting

I agree with the comments made by the other reviewers and I stand by my own as well. I will echo that it is important to know the nature of their agef-1 allele.

Significance

Bonacossa-Pereira et al identify AGEF-1 as a regulator of axon integrity that functions in a pathway with RAB-35 in the epidermis is an exciting finding. As pointed out in the discussion, mutations in the human ortholog cause neurodevelopmental defects which leads to obvious characterization of BIG2/ArfGEF2 in neurons while this study indicates that this protein can have cell non-autonomous roles in regulating neurons. These findings could have important implications for understanding the etiology of these defects that would be of interest to neurobiologists and clinical researchers.

The finding of this paper would also be of interest to cell biologists and particularly those studying the roles of Rab and Arf GTPases in membrane trafficking, such as myself. The idea that AGEF-1 might function as a Rab35 GEF is provocative and would generate a lot of interest and skepticism from the field. However, there is no data to support that AGEF-1 would be a direct regulator of Rab35 over the previously demonstrated cross regulation of Rab35 by Arf GTPases. Therefore, it would be fine to speculate in the discussion a direct interaction, but I would refrain from suggesting this as a model and elsewhere in the manuscript.

mirroring interferes with pergos.net login

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I think that this statement is very important because now that we have recognized that it is a problem what are we doing to solve it? the answer is nothing. Content just keeps getting easier and shorter to consume and people keep consuming it

I think that this statement can now be applied with a modern lens and can speak to peoples current attention spans. Apps like Tik Tok and instagram have implemented ways of consuming hundreds of different types of content within just a few minutes of being on the app. I Truly Believe that cable television was the last time humans had a attention span that made it so they could sit through a whole broadcast on T.V

For some people, this unpredictable style of t.v has been something that people are fond of and keep coming back too. Fans of a series often try to search for and uncover outages from the show just because they crave more of that unpredictability that a certain show has. A stellar example of this would be the Comedy "The Office".

Why not sample a vector of size n, instead of sampling 4000 vectors of size 1 in a for loop?

This does not depend on i. You can do it outside of the for loop only once, instead of repeating the calculation 4000 times.

This is confusing. How are these probabilities? you mean parameters? but the parameters beta in the text were different: -2, 0, 2, 4.

Same as LP <- as.list(bp)? But why do you need the vector bp expressed as a list?

Not used subsequently.

I never used it, but I understand that you can use the categorical family of {brms} to fit multinomial models.

In the frequentist world, the package {mclogit} seems to fit this kind of models, and also the function multinom() from package {nnet}.

Hi! nice post, thank you! This is an interesting topic on which I worked a bit in the past trying to implement multinomial models in INLA using this trick. But I got lost in the details and computational problems and never got around to complete it.

To ne štima

psychology as a science, emphasizing that it relies on empirical evidence, measurable data. It highlights that psychology is not just about opinions or theories t’s grounded in systematic observation and experimentation.

Tukaj ne štima

DOCUMENT DE SYNTHÈSE : Les Politiques d'Accompagnement à la Parentalité en France

Source : Rapport d’information N° 1638, Assemblée Nationale, Délégation aux droits des femmes et à l’égalité des chances entre les hommes et les femmes, sur les politiques d’accompagnement à la parentalité, présenté par Mme Sarah Legrain et Mme Delphine Lingemann, enregistré le 24 juin 2025.

Synthèse Exécutive

Ce rapport de la Délégation aux droits des femmes et à l’égalité des chances entre les hommes et les femmes met en lumière les inégalités persistantes dans la répartition des charges domestiques et parentales en France, majoritairement assumées par les femmes.

Il révèle que la parentalité, loin d'être neutre en matière de genre, est une cause majeure des inégalités économiques, professionnelles et sociales entre les hommes et les femmes. La "pénalité parentale" affecte de manière significative la carrière et les revenus des femmes, tandis que les hommes en sont largement épargnés.

Les rapporteures identifient plusieurs axes clés pour favoriser une répartition plus égalitaire des tâches parentales et promouvoir une vision positive et égalitaire de la parentalité, formulant 44 recommandations pour y parvenir.

Ces recommandations couvrent l'éducation et l'information, la prise en compte de la parentalité au travail, l'accompagnement des parents dès le désir d'enfant, la refonte des systèmes de congés parentaux et des modes d'accueil, le soutien aux parents d'adolescents et l'accompagnement des familles monoparentales.

Thèmes Principaux et Idées Clés

1. La Charge Domestique et Parentale Inégalitaire : Un Frein à l'Égalité des Femmes

• Division Sexuée Persistante : Malgré une impression d'égalité, les femmes continuent d'assumer la majeure partie des responsabilités domestiques et parentales. En moyenne, elles réalisent 71% des tâches domestiques et 65% des tâches parentales du foyer. Cette division est profondément enracinée dans un héritage historique et des stéréotypes de genre tenaces.
• Stéréotypes de Genre : L'idée que "les mères savent mieux répondre aux besoins et attentes des enfants que les pères" est très présente chez les adultes (60% y adhèrent) et se perpétue chez les jeunes (54% des 18-24 ans). Ces stéréotypes contribuent à une dévalorisation sociale des tâches considérées comme féminines.
• "Double Journée" des Mères : L'arrivée des enfants aggrave cette inégalité. Pour les femmes, cela représente environ "cinq heures de travail supplémentaire", tandis que pour les hommes, cela "réduit leur temps domestique et parental de deux heures". Les femmes salariées cumulent travail professionnel, domestique et parental, totalisant "onze heures par jour contre moins de dix heures pour les hommes".
• Impact du Système de Congés : La différence de durée entre le congé maternité (16 semaines) et le congé paternité (28 jours) renforce la dynamique d'une "mère 'parent principal' et d'un père 'auxiliaire'". Le congé parental est également majoritairement pris par les mères (94% des cas), ce qui pénalise leur carrière.
• Difficultés des Modes d'Accueil : Le manque et la répartition inégale des places en crèche et chez les assistantes maternelles obligent souvent les mères à compenser les dysfonctionnements du système. "Près de 20% des parents n’obtiennent pas de mode d’accueil, plus de 160 000 ne reprennent pas le travail faute de solution de garde pour leur enfant", les mères étant la "variable d'ajustement".

2. Conséquences Lourdes pour les Mères : Coût Humain, Économique et Social

• Pénalité Parentale au Travail : La parentalité a un "impact négatif de la parentalité sur le parcours professionnel des femmes", alors qu'elle n'a "aucun effet ou presque sur l’évolution professionnelle des hommes". "90% des inégalités de revenu entre les femmes et les hommes sont directement dues à la 'pénalité parentale' que subissent les femmes". Dix ans après l'arrivée du premier enfant, le revenu moyen des femmes chute d'environ 38%.
• Discrimination : Plus de six femmes sur dix estiment qu’être mère est un frein à la carrière. 27% des femmes déclarant être discriminées au travail estiment que cette discrimination est liée à la grossesse ou au congé maternité.
• Vulnérabilité des Mères Solos : Les mères solos (82% des familles monoparentales) sont particulièrement touchées. Elles subissent une "triple pénalité croisée : leur genre, leur situation professionnelle […], leur situation familiale", les exposant aux emplois précaires et mal rémunérés, et augmentant leur risque de pauvreté. "Près d’une mère seule sur cinq est pauvre alors qu’elle a un emploi".
• Risque d'Épuisement et Santé Mentale : La charge disproportionnée entraîne un "risque réel d’épuisement pour les mères". L'isolement peut favoriser la dépression post-partum, qui touche environ 20% des femmes et est la "première cause de la mortalité maternelle dans l’année qui suit la naissance de l’enfant".
• Coût Économique Élevé : Outre la perte de revenus due aux congés maternité et à la réduction d'activité, la séparation a un "lourd coût pour les mères". Une femme séparée sur trois "bascule sous le seuil de pauvreté l’année de la séparation", son niveau de vie baissant d'environ 20% (contre 7% pour les hommes). 39% des enfants vivant en famille monoparentale sont en situation de pauvreté.

3. Propositions pour une Parentalité Égalitaire

Les rapporteures formulent 44 recommandations pour transformer les politiques d'accompagnement à la parentalité, axées sur l'égalité :

Éducation et Information :

• Mettre en place des "cours d’activités domestiques" à l'école ou au collège pour inculquer des compétences à tous les enfants.
• Lancer des "campagnes nationales contre les stéréotypes de genre" sur la parentalité.
• Adopter une "terminologie neutre" (ex: "école pré-élémentaire" au lieu d'"école maternelle", "prestation pour naissance et soin du mineur" au lieu de "congé maternité/paternité").
• Renforcer l'information des parents sur les dispositifs d'accompagnement.

Prise en Compte au Travail :

• Intégrer la parentalité dans la "responsabilité sociétale des entreprises (RSE)" et généraliser la "Charte de la parentalité" aux entreprises de plus de 50 salariés.
• Modifier le Code du travail pour inclure explicitement la parentalité dans les "négociations d’entreprises relatives à l’égalité professionnelle".
• Intégrer des critères sur la parentalité dans le futur "index égalité professionnelle".
• Accorder des "autorisations d’absence" (4 demi-journées/an) aux parents pour les moments clés de la scolarité de leurs enfants.
• Accompagnement dès le Désir d'Enfant et Post-Partum :
• Élargir les "consultations pré-conceptionnelles" au projet parental et permettre au second parent d'assister à tous les rendez-vous médicaux obligatoires de la grossesse.
• Consacrer une séance de préparation à la naissance au "projet parental".
• Renforcer le dispositif d'arrêt en cas d'interruption de grossesse et l'étendre aux interruptions volontaires, avec une autorisation d'absence pour le conjoint.
• Faciliter l'accès aux "consultations sur l’allaitement" et renforcer la "formation des praticiens sur la dépression post-partum".
• Prévoir une "consultation facultative et remboursée à 100% avec un psychologue" pour les mères dans les trois mois après la naissance.
• Étendre le "congé de 'proche aidant'" au second parent en soutien à la mère souffrant de dépression post-partum.
• Lutter contre l'isolement des mères en proposant aux parents "d’être mis en relation avec d’autres parents accueillant leur enfant au même moment".

Réforme des Congés et Modes d'Accueil :

• Congé Paternité : Porter "progressivement le congé paternité à seize semaines, soit à égalité avec le congé maternité". Huit semaines seraient obligatoires (4 à la naissance, 4 après le congé maternité de la mère) et huit facultatives et fractionnables. Cette mesure est un "levier clé pour l’égalité entre les parents" et répond à l'aspiration des pères à s'investir davantage.
• Congé Parental : Réformer le congé parental en "renforçant son attractivité financière sans réduire sa portée pour les ménages modestes", et réfléchir à une "reprise progressive" après le congé.
• Modes d'Accueil : Garantir la "lisibilité et la transparence" des modes de garde, "investir pour augmenter et harmoniser l’offre de crèches sur le territoire", et "revaloriser les métiers de la petite enfance".
• Soutien aux Parents d'Adolescents :
• Élargir les missions des "lieux d’accueil enfants-parents" pour qu’ils puissent "recevoir des adolescents".
• Mettre en place des "politiques publiques ciblant spécifiquement les parents d’adolescents".
• Renforcer l’offre en "pédopsychiatrie" et la "médecine scolaire" face à la dégradation de la santé mentale des jeunes.
• Lancer une "campagne d’information nationale sur la santé mentale des enfants et des adolescents".

Accompagnement des Familles Monoparentales :

• Repenser le "mode de calcul des pensions alimentaires" pour prendre en compte le coût réel de l’entretien d’un enfant et permettre au parent bénéficiaire de "défiscaliser la pension alimentaire".
• Instaurer un "abattement sur le montant de la pension alimentaire pris en compte dans les bases ressources des prestations familiales et des aides au logement, à hauteur de l’allocation de soutien familial (ASF)".
• "Déconjugaliser le versement de l’ASF" et "ouvrir les allocations logement (APL) aux deux parents" pour faciliter l’accueil des enfants.
• Ouvrir aux mères solos la "possibilité de transférer des droits de congés vers un proche de leur choix" et "doubler les jours 'enfant malade'".
• Étudier la création d'un "statut des familles monoparentales" avec des droits spécifiques.

Conclusion des Rapporteures

Les rapporteures affirment que malgré des évolutions, les mères restent le "parent principal", ce qui a des conséquences négatives sur leur santé et leur vie professionnelle.

Une "réforme ambitieuse du système des congés", en particulier du congé second parent, est un "moteur d'égalité" essentiel.

S'inspirant des modèles scandinaves et espagnols, la France peut avancer vers une parentalité égalitaire, non seulement pour l'émancipation des femmes, mais aussi comme réponse aux inquiétudes démographiques.

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Failures and mistakes are what shapes us as human beings. It teaches our brains in a way to know what and what not to do, it helps us develop and grow. Instead of being self-conscious about an error, we all should take it as a lesson for us to improve and become better not only as students, but as adults. These are skills and knowledge that will be with us for the rest of our lives.

Our brains are very powerful that's why a lot of people believe in manifestations. The more you think about something, it's more likely to take place because you are always subconsciously working towards what you want to happen.

When we know that intelligence can be improved/developed, that should encourage people to believe in themself and help people feel confident to learn and face challenges without fear.

Everyone reacts differently when confronted with personal setbacks. The approach you take in dealing with these challenges plays a crucial role in shaping your success. As for me, I live by the motto: "Step out of your comfort zone."

This comment reminded me of doing hard at work as working overtime means I were not good enough to handle my job. However, pressure makes diamond, every effort will deserved if you always try our best and expand our abilities.

It’s a really effective hook that drew me into the topic. At some point, each of us has experienced in failing, where praise from our parents or friends may have negatively impacted our mindset and learning

In fact, we can train our brain as others part of our body. It probably develops and cultivates after overcoming our setbacks

Does this mean that we will open the article from the link and log into our Hypothesis account directly from the article link we are given?

Try to do binary search in a natural way in the tree for recovering the classical approximation \(22/7\) for \(\pi\). Here you actually need to descend to more than deoth \(10\) in the tree!

The famous approximation \(355/113\) is above depth \(25\).

Begin by putting

$$ \texttt{left} = \frac{0}{1}\quad\texttt{mediant} = \frac{1}{1}\quad\texttt{right}=\frac{1}{0}. $$

If you move left, keep \(\texttt{left}\), put \(\texttt{right}\) equal to \(\texttt{mediant}\) and compute new \(\texttt{mediant}\). If you move right keep the \(\texttt{right}\) etc.

Engage in a workout inspired by your favourite 2005 Muffin The Mule episode

Squeak and scurry around the room like a mouse

Get enough sleep each night

Dance to calypso music

Fly around the room like a bird

This goes with #4.

This line right here goes with #5. I think tracking my happiness would affect me in a sense that I would get annoyed on how much I would have to keep inputting on a daily just to get a measurement on how I am feeling.

I think this make his argument stronger because he's challenging that can these apps that measure health off a metrics can make you feel happier.

I think that was great sarcasm. Lol.

This stood out to me the most cause it has so many levels of truth behind it.

This is was a turning point for the narrator. His confidence opposes with his unexpected fail.

I find that this is common amongst a lot of people. I can even say so for myself, when I wouldn’t do as well as I thought I did on a test or in a class I would get discouraged. It made me grow resentment towards a class I once enjoyed. That led me to procrastinate even more and made me dread doing the work for that class. Although failing can really mess with your head, I think this story points out the importance of determination despite all the discouragement

Nichols incorporates a little bit of vulnerability to connect with readers. She conveys her experience and emotions in a way that evokes empathy and relatability in the audience. Her vulnerability makes her essay eye catching and makes you want to read more to learn the outcome of her story.

This clearly illustrates how one thing can easily alter your view or feelings towards something. Whether that be your own self confidence, your love for said thing, or even a person/a place. Nichols really sets the tone for her essay, displaying disappointment and self doubt. This helps the audience relate to her work.

Failure affects not only grades but also identity and self-worth. Sometimes, failure does not allow people to really show who they are and turns them into being more isolated, which lets their voices be less heard.

The author feels exhausted after all the failures she is going through. Consistent failures lead to withdrawal. Failure should not limit people from what they are capable of.

I think this line shows that she lost hope in the grading system and she blames them rather than herself.

This shows how determined the writer is.

After failing twice, he lost his passion to write because of the disappointment he felt after failing.

Mickey Mouse and Pluto being mentioned in Beat The Parents

Seeing Angel sit atop the radiator looking down at the hungry Putzell

Renting a log cabin in Switzerland

Lady Penelope scratching my itchy neck

Leaving painted handprints on Alan's chest

Parker staying in Lady Penelope's room with us due to an outbreak of fleas in his own room

Lady Penelope and me getting comfortable in a soft white hotel bed

Lady Penelope looking after me when I have a bad cold

Mylo barking and growling in his sleep

Destination Moonbase Alpha - a one day event

Q: How do the unique backgrounds, cultural experiences, and moral/religious beliefs of actors contribute to the representation of these"subtexts" on stage?

Q: Playing off of the earlier line "We may not be told what to think but we are provided with the atmosphere in which to think," and this excerpt, how much of a reader's or viewer's understanding of a piece of literature or drama is their interpretation versus the creators intent? Is the meaning of x defined by the creator or the viewer?

Decorum- literary and dramatic propriety (merriam-webster dictionary)

This makes me think superman is a symbol for barriers, literally(the door) and figuratively (the barriers of illiteracy, & marginalization).

The passage shows how Native children were expected to fail in school but excelled in cultural knowledge outside the classroom. The narrator resisted these stereotypes by reading obsessively, using literacy as both empowerment and survival. Despite lacking role models, he became a writer, proving that Native voices belong in literature.

This passage shows how the fathers love for books shaped the narrator's identity. Despite limited money, he filled the home with books from pawn shops, thrift stores, and even gift shops, stacking them in chaotic piles and on homemade shelves. The narrator's own devotion to reading grew out of deep love and admiration for their father.

This line is showing that since he opened the comic book he is starting to see things differently.

This shows the struggle that they went through.

She uses repetition to show how hard she was trying to break the stereotype that Indian children were supposed to fail.

Although they were looked at as stupid, they could remember all the songs they were taught throughout their lives. This proves that they were not stupid, they just were not provided the same education as other children.

Being curious about other cultures will help with public speaking so that you can have a two way respect relationship with the audience

this situation would make me very stressed out because you would have to edit some of your speech so last minute without practicing beforehand. A good skill to also add would be to be able to work good under pressure

i have heard this which was wild to think about it because i have never thought about it before. it is so true though!

we can take account but some things are out of our hands, how is our speech going to make someone feel less hot? would you just mention it in the speech as a joke to make them feel seen and create more connection?

if you were making a speech to a group of individuals who spoke a different language maybe you would word your sentences differently so that you used more simpler words so more people would understand

this can also be situational, if someone is making a speech at a graduation they could be homing in on the negatives of school when a graduation is about celebration and growth

audience members are craving a connection with the speaker just as much as the speaker is craving a connection with the audience

this is a very useful guide, this will deter from "selfish" speeches, some speeches i notice the speaker is more talking for themselves rather than speaking for others to inspire and grow

i agree with this statement because our past is all that we know and whatever we have learned and experienced then will help us live now, thats why sometimes its easy to tell how someone was raised just after spending some time with them.

are they saying that they assumed the public speakers would be having an interactional conversation with their audience while their speech was being made?

not only does this account for public speaking but almost every aspect of your life, think about us as students and the majors that we picked, we had passion behind those majors which made us very determined in our work and it shows

i remember there was one assembly we had in elementary school where there was a man with a yo-yo and while he was giving his speech before the performance he was saying all of these little tidbits about very deep topics but i was left confused because there was no main message just a bunch of ideas lined up

what does this mean?

Realizing there could be some bumps in your argument is important so you can logically explain all sides and make sure you address the “holes” without letting them degrade your whole claim.

(< 0.005% of general population). Acquired nephrogenic DI may present in patients on long term lithium therapy, e.g., for bipolar disorder.

releases

(ADH) action. (Adding the word "action" covers ADH deficiency in pituitary DI as well as nephrogenic DI from vasopressin receptor (AVPR2) deficiency, and aquaporin (AQP2) deficiency.)

(aka, antidiuretic hormone, ADH)

with insulin in an "insulin tolerance test." (later in the chapter, this test is referred to in name, so the name should be given here.)

as discussed

that

change to: "so prolactin may be elevated in primary hypothyroidism if the lack of thyroid hormone feedback inhibition causes elevated TRH." (They didn't have thyroid yet, so the connection between low TH causing high TRH needs to be made here for them.)

(TRH)

Add: "Over-the-counter growth hormone may be used by body builders and athletes with the intention of increasing muscle mass; however the efficacy and long term effects are controversial." (Reword as you wish if you want to include this application- probably good for them to know people use this OTC, but I don't know if there is good data for its value.)

used

pituitary tumor

GH levels

glucose

as shown in the bottom image.

GH levels

glucose

explain what "TH" is on the graph. Maybe enlarge figure a bit to make it easier to see.

are

they require

comma within quotes

their

is this the prevalence in the US or world-wide?

period within quotes

GLUT4 translocation to the plasma membrane.

GH excess can cause high blood glucose (hyperglycemia), insulin resistance and diabetes mellitus.

glucose

glucose

glucose

Stimulates melanin synthesis and other functions

adrenocorticotropin hormone (ACTH)

remove semi-colon

Releases ACTH (adrenocorticotropin hormone/corticotropin), beta-endorphin, alpha-melanocyte stimulating hormone, and beta-lipotropin )

(termed releasing hormones (RH) or release inhibiting hormones (RIH))

(aka growth hormone release inhibiting hormone, GHRIH)

hormones (just to be consistent with naming - they are referred to as peptide hormones later)

thyrotropin releasing hormone (TRH)

It would be easier to see the text if you enlarge this figure.

To remember the difference between covalent network solids, atomic network solids, and amorphous solids think about in in dimensions, which they are.

3D= Diamond a three dimensional solid (covalent-atomic) 2D = Graphite, you draw 2D figures with a graphite pencil (covalent- molecular) 1D = No dimension like glass, no order

The counter argument and rebuttal shows that there is some merit to the counter argument but their can be alternatives with out just bashing the other side.

Metals conduct beaus of delocalized electrons. Band theory is similar to thinking about molecular orbitals the concept from chem 1. When two atoms bond their atomic orbital mix and form molecular orbitals. But in band theory it is on a much larger scale 10^23 atoms or avagadros number. Insulators have big gaps in the connection band and semi conductors have small gaps in the connective bands.

Making your mark when writing doesn’t always mean getting someone’s opinion to change by what you said but instead can be making them question their own thoughts and values. Getting someone to use their own empathy and respect also furthers your argument and what you are adding to the conversation.

Don’t necessarily need to be telling people what to do, this is an example of another circumstance where you would use this type of claim even though you aren’t convincing anyone to do anything but instead making them question for themself.

First off, a strand of time can be identified when May's mother says: "She has not been out since girlhood... Not out since girlhood... Where is she, it may be asked" (241). This quote by May's mother can be determined as a strand of time because it is emphasizing the disconnect May has with modern day. May's mother is suggesting that May has been both physically and emotionally confined during her girlhood, and her thoughts and experiences in the real world have not developed much past that. This quote highlights the uncertainty of May, and also supports the lingering effects that could still be haunting her of having to take care of her mother at a younger age. There is a pattern throughout this play of May constantly asking her mother if she needs any sort of assistance, such as propping up her pillow, which also underlines the time warp she is stuck in of having the natural instincts of always having to be there for her mother.

Moving forward, a strand of voice can be deciphered when May explains, "Still speak? Yes, some nights she does, when she fancies none can hear" (241). Although it is hard to determine if May is speaking about herself or her mother, there is an overall theme of insecurity and loss in terms of voice. Especially when May mentions how "she" speaks "some nights," it proposes that this voice is not consistently expressed and lacks confidence. This quote could also explain why May takes so many pauses when she speaks; she wants to make sure she voices herself assertively, as she may have struggled with this in the past or even her whole life.

Lastly, a binary of presence and absence is displayed in every moment of the play, but most importantly towards the end of the play when Amy (possibly an alter-ego of May) remarks: "I mean, Mother, that to say I observed nothing of any kind, strange, or otherwise. I saw nothing, heard nothing, of any kind. I was not there" (243). But then in response, Mrs. W (possibly an alter-ego of May's mother) rebutes: "Not there?... But I heard you respond... I heard you say Amen" (243). This conflict and misunderstanding between May and her mother presents how May is still present with the relationship she had with her, but is absent from realizing that no one is actually there conversing with her, that is why she finds herself in disagreement. May did not hear her mother because of inconvenience, she did not hear her because she does not exist anymore. There is also a binary between presence and absence when the lights fade into darkness, bringing the play to an end but leaving the audience with the presence of May's unresolved ideas.

We can not measure lattice energy as it is the energy released when gas ions can together to form a solid. It is calculated indirectly using Hess's Law applied to ionic solids. We break it down into key steps: Sublimation, ionization, bond dissociation, electron affinity, and formation. Remember these key steps

Relating to unit cells, the ionic structures depend on the cation vs anion size. For example, for CsCl it is primitive cubic, Cs+ in the center was Cl- on the corners. NaCl is FCC with Na+ in the 8 holes, Cl- ions are much bigger than Na+ so sodium fits into the holes left by chlorine. CaF2 is also FCC with tetrahedral holes Ca2+ fits into the gaps left by Flourine.

this is specific to transition of NaCl but keep the steps in mind

if the temperature and pressure is the same for two different moles of the same gas, the energy change from the phase transition is the same for both moles even if they were forced to change phases by different means

higher stability=lower energy

wouldn't dissociating an ionic compound result in the loss of some potential energy due to ions of like charges being farther apart therefore minimizing the repulsion between them? I know ultimately, transitioning to a gaseous phase is endothermic because the molecules gain kinetic energy, but is the loss of potential energy considered in the born haber cycle?

is this because the smaller the ions, the closer an ion is to another ion of a like charge? or because periodic trends associated with smaller atoms?

Claims apply to other types of writing then just argumentative, even scientists have to be persuasive sometimes.

In Crystal defects there are either imperfections that change crystal properties by swapping the atom (substitutional) or squeeze in an atom (interstitial). Example of substitutional: Brass. Cu + Zn. Copper atoms are replaced by zinc atoms, they are similar size so they substitute to make a stronger mental.

Example of Interstitial defects: Steel. Smaller carbon atoms squeeze into holes between iron atoms. This forms steel and makes it harder and stronger than iron alone.

Get side length from the density and geometry formulas to obtain the radius. Density to volume to edge length to radius

It is fascinating to me how big the empire's truly were. They had so many people and I personally think people don't realize how big they truly were.

This is so interesting to me how thousands of years ago they found it okay to do this so young and to take away the chances of a young man having a family. How did they plan to grow their family and have grandchildren? To continue on their name?

The difference in between the cultures is so drastic. The Chinese culture seems to be so much more knowledgeable and skilled.

This is very interesting to me how politics were still so important thousands of years ago. In todays world they seem to make or break friendships and so much more and apparently it did thousands of years ago to.

lmao

wow. true.

People are influenced by information from official channels, followed by their peers and personal connections rather than by larger institutions. As a result of public relations efforts, focus on building relationships, relationships with smaller influencers and voices within the community, aiming to get stories published in traditional news.

AI generates fakes information that causes a serious threat to public relations. Deepfakes allow people with harmful intentions to create fall stories that appear real, using the likeness in the voice of actual company leaders. This changes Public Relations from a profession that shares positive stories to one that constantly defends against high tech truths.

your skills will be more valued in the workspace

simply being a longtime loyal worker can mean that you have the aspects of this term though leader

even if you were to be someone who is the voice of an audiobook, there is such attention to detail to emphasize certain parts in certain ways in order to convey a specific message. The pace that you are speaking at etc

i reflect on myself and i think this is going to be the thing most struggle with because sometimes i have a difficult time expressing all of my thoughts in a non confusing way. i hope that there are some tips that can help me place everything together.

this sentence demonstrates the energy that needs to be brought behind our speeches that we make to be a true leader

even in the constitution it states that we the people have the right to "overthrow" the government with our freedom of speech. This just shows that its way more important than we realize

i love this take

i bet that even individuals who have to speak to the public a lot have signs of being nervous even after such a long time.

even feedback from the audience is good as well, they can possibly have feedback that a professional might not have just because they are seeing the speech at face value and not over analyzing.

a good public speaker is a good planner and good at analyzing. you need to find and solve the problem before the audience can even think about the problem.

what do they mean by "fine-tuning?"

I wish i was able to see videos of old speeches that occured back when no technology was available so that we could compare and contrasts the details of both

i am curious to know the specific tactics that public speakers have discovered and used to be more persuasive in their speech to their audience

a good example that some jobs although have to do repetitive public speaking to their students, there are times where your audience is a little bit different and you have to be able to navigate the best way to set up your speech so that the audience can absorb the material youre saying more.

This is the most nerve wracking type of public speaking that i could be apart of. I wonder if for some this would be the easiest

Although social media can be seen as a bad thing, im glad that there are sites like this that can help is connect rather then distract us from the real meaning things of life

I would have to say that she is most likely not wrong in her guess. there is a wave of uncontrollable high amounts of content.

.

we practice public speaking in culture of funerals, weddings and parties

i really need to hone on this aspect for my speech so that i can move my classmates in the correct way that will have the effect i want it to

very interesting that even the ones publishing these books about public speaking are following the guidelines too! it makes sense but its nice to see that

If anything it is more relevant now, its more of a rare thing to be able to have this ability and people are even more inspires by it then ever before

shows that public speaking has the potential to be a career

important

great advice

If you make do with something, you use or have it instead of something else that you do not have, although it is not as good.

figma 上的 "儀表板-設備(今日x所有設備)" 中的 gateway & 子設備資料如果很多的話，會有分頁嗎?