Reviewer #2 (Public review):

Summary:

The manuscript analyzes and attempts to discriminate genetic requirements for DNA damage-induced cell cycle checkpoint induction, maintenance, and adaptation in budding yeast bearing one or two unrepairable DNA double strand breaks using auxin-induced degradation (AID) of key DNA damage response (DDR) factors. The study paid particular attention to solving a puzzle regarding how yeasts bearing two unrepaired DNA breaks fail to engage in "adaptation" whereas those with a single unrepairable break eventually resume cell cycling after a prolonged (up to 12 h) G2 arrest.

The key findings are: 1. Genetic requirements for the entry and the maintenance of DDC are separable. For instance, Dun1 is partially required for the entry but not the DDC maintenance whereas Chk1 is only required for maintenance. 2. Cells with two unrepairable breaks respond to DDR only up to a certain time (~12-15 h post damage) and beyond this point, depend on spindle assembly checkpoint (SAC) and mitotic exit network (MEN) to halt cell cycling. 3. The authors also propose an interesting concept that the location of DNA breaks and their distance to centromeres are important factors dictating the effect of SAC/MEN on the duration of cell cycle arrest after prolonged arrest (and cells become "deaf" to persistent arrest signals) and yeast's adaptability following DNA damage. The results provide most compelling evidence to date on the role of SAC/MEN in DNA damage response and cell cycle arrest albeit its impact might be limited to the handful of model systems due to the vastly different centromeric elements and far larger chromosome sizes in metazoan cells. The study albeit briefly discussed the basis of transitions from entry, maintenance, and adaptation ( ex. changes in centromeric architectures), it does not offer detailed explanations or a testable hypothesis to this topic.

Overall, the conclusion of the study is well supported by the elegant set of genetic experimental data and employed multiple readouts on DDC factor depletion on checkpoint integrity and cell cycle status. Although the study simply measures Rad53 phosphorylation as the primary metric to assess checkpoint status, it successfully demonstrated how the signaling is modified through the different stages and that eventually cells become recalcitrant to DDC signaling after a prolonged arrest. The results are clear, and rigorously tested and carefully interpreted with good discussion on the possible limitations. The revision provided detailed responses to the reviewers' comments and addressed a few key concerns, one of which is universally raised by the reviewers on the full functionality of AID tagged DDC factors, by simply expressing excess Rad9-AID to restore more normal looking checkpoint response. It will be interesting if the excess expression of other DDC factors could overcome suboptimal checkpoints in cells after 24 h post damage.

Reviewer #3 (Public review):

Summary:

The DNA damage checkpoint (DDC) inhibits the metaphase-anaphase transition to repair various types of DNA damage, including DNA double strand breaks (DSBs). One irreparable DSB can maintain the DDC for 12-15 hours in yeast, after which the cells resume the cell cycle. If there are two DSBs, the DDC is maintained for at least 24 hours. In this study, the authors take advantage of this tighter DDC to investigate whether the best-known proteins involved in establishing the DDC are also responsible for its long-term maintenance during irreparable DSBs. They do this by cleverly degrading such proteins after DSB formation. They show that most, but not all, DDC proteins maintain the cell cycle block. Interestingly, DDC proteins become dispensable after 15 hours and the block is then maintained by spindle assembly checkpoint (SAC) proteins.

Strengths:

The authors have engineered a tight yeast system to study DDC shutdown after irreparable DSBs and used it to address whether checkpoint proteins (DDC and SAC) contribute to the long-term maintenance of DSB-mediated G2/M block. The different roles of Ddc2, Chk1 and Dun1 are interesting, while the fact that SAC overtakes DDC after 15 hours is intriguing and highlights how DSBs near and far from centromeres can have a profound impact on cell adaptation to DSBs. In their revision, the authors have now improved the Rad9-AID methodology to place Rad9 in the context of DDC adaptation, as well as widening the association between adaptation and proximity to centromeres.

Weaknesses:

Some of the results they present essentially confirm their own previous findings, albeit with a tighter strain design for long-term arrest. Conclusions about the maintenance of G2/M in several mutant combinations could have been strengthened by adding simple microscopy experiments with DAPI staining. No clear mechanism for how depletion of Bub2, but not Bfa1, can relieve the G2/M (metaphase) block is given.

Author response:

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

Reviewer #1 (Recommendations For The Authors):

To hopefully contribute to more strongly support the conclusions drawn by the authors, I am including a series of concerns regarding the manuscript, as well as some suggestions that could be useful to address these issues:

(1) The main results of this study derive from the use of auxin-inducible degron (AID)-tagged proteins. Despite the great advantages of the AID strategy to conditionally deplete proteins, the AID tag can affect the normal function of a protein. In fact, some of the AID-labeled DDC components generated in this work are shown to be hypomorphic. Hence, the manuscript would have benefited from the additional confirmation of some of the observations using a different way to eliminate the proteins (e.g., temperature-sensitive mutants).

Most ts mutants are also hypomorphic; hence we don’t see there is much advantage to their use. The addition of the AID to these proteins alone does not interfere with the ability to sustain checkpoint arrest as demonstrated in Figure S1. Instead we found that by overexpressing Rad9-AID we could demonstrate that inactivating Rad9 after 15 h behaved the same way as the inactivation of Ddc2, significantly strengthening our finding that the DDC checkpoint becomes dispensable while the SAC takes over. 

(2) In cells depleted of Rad53-AID, the deletion of CHK1 stimulates an earlier release from a mitotic arrest induced by two DSBs (Figures 2D and 3C). Likewise, the authors claim that a faster escape from the cell cycle block can also be observed when upstream factors such as Ddc2, Rad9, or Rad24 are depleted in the absence of CHK1 (Figures 2A-C and Figures 3D-F). However, this earlier release from the cell cycle arrest, if at all, is only slightly noticeable in a Rad9-AID background (Figures 2B and 3E). In this sense, it is also worth pointing out that Rad9-AID chk1Δ (Figure 3E) and Rad24-AID chk1Δ (Figure 3F) cells were only evaluated up to 7 h, while in all other instances, cells were followed for 9 h, which hinders a fair assessment of the differences in the release from the cell cycle arrest.

As noted above, we have now been able to examine Rad9 over the long-time frame.

(3) Although only 25% of the cells depleted for Dun1 remained in G2/M arrest 7 h following the induction of two DSBs, it is shocking that Rad53 was nonetheless still phosphorylated after the cells had escaped the cell cycle blockage (Figure 4A).

This persistence of Rad53 phosphorylation is also seen with the inactivation of Mad2, allowing escape in spite of continued Rad53 phosphorylation.

(4) Generation of Rad9-AID2 and Rad24-AID2 strains did not fully restore the function of these proteins, since most cells had adapted 24 h after induction of two DSBs (Figure S1C). Nonetheless, Rad9-AID2 and Rad24-AID2 are still likely more stable than their AID counterparts, and hence the authors could have instead used the AID2 proteins for the experiments in Figure 2 to better evaluate the role of Rad9 and Rad24 in the maintenance of the DDC-dependent arrest.

We note again that we have found a way to study Rad9 up to 24 h. 

(5) Deletion of BFA1 has been shown to promote the escape from a cell cycle arrest triggered by telomere uncapping (Wang et al. 2000, Hu et al. 2001, Valerio-Santiago et al. 2013). Likewise, while cells carrying the cdc5-T238A allele cannot adapt to a checkpoint arrest induced by one irreparable DSB, BFA1 deletion rescues the adaptation defect of this mutant CDC5 allele (Rawal et al., 2016). The authors show how, using AID-degrons of Bfa1 and Bub2, that only Bub2, but not Bfa1, is required to maintain a prolonged cell cycle arrest after the induction of two DSBs. To reinforce this point, and as shown for mad2Δ cells (Figure S6A), the authors could perform a complete time course using both the Bfa1-AID and a bfa1Δ mutant to demonstrate that they do indeed show the same behavior in terms of the adaptation to a two DSB-induced cell cycle arrest.

We thank the reviewer for noting these other instances where bfa1D promoted an escape from arrest. We tested a 2-DSB bfa1 deletion, data has been added to Figure S9E-F. We did not observe a difference in the percentage of cells escaping arrest between the 2-DSB bfa1 deletion and the 2-DSB BFA1-AID strains.

(6) Bypass or adaptation of a checkpoint-induced cell cycle arrest in S. cerevisiae often leads to cells entering a new cell cycle without doing cytokinesis and, hence, to the accumulation of rebudded cells. However, the experiments shown in the manuscript only account for G1 or budded cells with either one or two nuclei. Do any of the mutants show cytokinesis problems and subsequent rebudding of the cells? If so, this should have been also noted and quantified in the corresponding assays.

In the cases we have studied we have not seen instances where the cells re-bud without completing mitosis (at least as assessed by the formation of budded cells with two distinct DAPI staining masses). In the morphological assays we have done, we score the continuation of the cell cycle by the appearance of multiple buds, G1, and small budded cells. In our adaptation assays when cells escaped G2/M arrest they formed microcolonies indicating no short-term deficiency in cell division.

(7) The location of the DSB relative to the centromere of a chromosome seems to be a factor that determines the capacity of the SAC to sustain a prolonged cell cycle arrest. The authors discuss the possibility that the DSB could somehow affect the structure of the kinetochore. Did they evaluate whether Mad1 or Mad2 were more actively recruited to kinetochores in those strains that more strongly trigger the SAC after induction of the DSBs?

We have not attempted to follow Mad1/2 recruitment. ChIP-seq could be used to monitor Mad1/2 localization at the 16 centromeres in response to DSBs and the spread of g-H2AX across the centromere. Our previous data showed that g-H2AX could spread across the centromere region and could create a change that would be detected by Mad1/2.  This change does not, however, affect the mitotic behavior of a strain in which the H2A genes have been modified to the possibly phosphomimetic H2A-S129E allele.

(8) The authors could speculate in the discussion about the reasons that could explain why the DDC is required for the maintenance of checkpoint arrest at early stages but then becomes dispensable for the preservation of a prolonged cell DNA DSB-induced cycle arrest, which is instead sustained at later stages by the SAC.

Our suggestion is that cells would have adapted, but modification of the centromere region engages SAC.

Finally, some minor issues are:

(1) The lines in the graphs that display the results from adaptation assays (e.g., Figures 1B and 1E) or cell and nuclear morphology (e.g., Figures 1D and 1G) are too thick. This makes it sometimes difficult to distinguish the actual percentages of cells in each category, particularly in the experiments monitoring nuclear division.

Fixed

(2) While both the adaptation assay and the analysis of nuclear division in Figures 1E and 1G, respectively, show a complete DDC-dependent arrest at 4h, the Western blot in Figure 1F suggests that Rad53 is not phosphorylated at that time point. Do these figures represent independent experiments? Ideally, the analysis of cell budding and nuclear division, which is performed in liquid cultures, and the Western blot displaying Rad53 phosphorylation should correspond to the same experiment.

Cell budding in liquid cultures and adaptation assays were performed in triplicate with 3 biological replicates and the collective results are shown in each graph showing the percentage of large-budded cells. Western blot samples were collected in each liquid culture experiment. The western blot in 1G is a representative western blot.

(3) It is somewhat confusing that the blots for the proteins are not displayed in the same order in Figures 2A (Rad53 at the top) and 2B or 2C (Rad53 in the middle).

Fixed.  We place Rad53 – the relevant protein - at the top.

Reviewer #2 (Recommendations For The Authors):

(1) Yeast with the two breaks responds to DNA damage checkpoint (DDC) until sometimes 4-15 h post DNA damage. Since the auxin-induced degradation does not completely deplete all the tagged proteins in cells, the results should be more carefully considered and not to interpret if the checkpoint entry or maintenance depends on each target protein's ability to induce Rad53 phosphorylation. It should be theoretically possible if checkpoint maintenance requires only a modest amount of checkpoint factors especially because the experiments involve the induction of one or two DSBs. The low levels of DDC factors may be insufficient for Rad53 activation but could still be effective for cell cycle arrest. Indeed, the Haber group showed that the mating type switch did not induce Rad53 phosphorylation but still invoked detectable DNA damage response. To test such possibilities, the authors might consider employing yet another marker for DDC such as H2A or Chk1 phosphorylation besides Rad53 autophosphorylation. Alternatively, the authors might check if auxin-induced depletion also disrupts break-induced foci formation for checkpoint maintenance or their enrichment at DNA breaks using ChIP assays at various points post-damage.

DAPI staining of Ddc2-AID cells show that when IAA is added 4 h after DSB induction (Figure S3A), cells escape G2/M arrest as evidenced by the increase in large-budded cells with 2 DAPI signals, small budded cells, and G1 cells. Overexpression of Ddc2 can sustain the checkpoint past 24 h, but without SAC proteins like Mad2 they will eventually adapt (Figure S6B).

That Rad9-AID or Rad24-AID in the absence of added auxin (but in the presence of TIR1) is unable to sustain arrest suggests to us that low levels of Rad9 or Rad24 are not sufficient to maintain arrest.  As the reviewer notes, normal MAT switching doesn’t cause Rad53 phosphorylation or arrest, though early damage-induced events such as H2A phosphorylation do occur.  But our point is that Rad9 or Ddc2 is needed to maintain arrest only up to a certain point, after which they become superfluous and a different checkpoint arrest is imposed. At that point apparently a low level of these proteins plays no obvious role.

(2) It is interesting that DDC no longer responds to the damage signaling after 15 h of DSB-induced prolonged checkpoint arrest after two DNA double-strand breaks. Is this also applicable to other adaptation mutants? The results might improve the broad impact of the current conclusions. It is also possible that the transition from DDC to SPC depends on simply the changes in signaling or in part due to the molecular changes in the status of DNA breaks or its flanking regions. Indeed, the proposed model suggests that the spreading of H2A phosphorylation to centromeric regions induces SAC and thus mitotic arrest. The authors could measure H2A phosphorylation near the centromere using ChIP assays at various intervals post-DNA damage. It is particularly interesting if depletion of Ddc2 at 15 h post DNA damage does not alter the level of H2A phosphorylation at or near centromere.

Our previous data have suggested that the involvement of the SAC in prolonging DSB-induced arrest involved post-translational modification of centromeric chromatin such as the Mec1- and Tel1-dependent phosphorylation of the histone H2A (Dotiwala). In budding yeast there is also a similar DSB-induced modification of histone H2B (Lee et al.). To ask if there is an intrinsic activation of the SAC if the regions around centromeres were modified by checkpoint kinase phosphorylation, we examined cell cycle progression in strains in which histone H2A or histone H2B was mutated to their putative phosphomimetic forms (H2A-S129E and H2B-T129E).  As shown in Figure S11, there was no effect on the growth rate of these strains, or of the double mutant, suggesting that cells did not experience a delay in entering mitosis because of these modifications. We note that although histone H2A-S129E is recognized by an antibody specific for the phosphorylation of histone H2A-S129, the mutation to S129E may not be fully phosphomimetic. 

(3) It is puzzling why Rad9-AID or Rad24-AID are proficient for DDC establishment but cannot sustain permanent arrest in the two break cells. It appears Rad53 phosphorylation for DDC is weaker in cells expressing Rad9-AID or Rad24-AID according to Fig.2B and C even though their protein level before IAA treatment is still robust. This might also explain why the results of depleting Rad53 and Rad9 are very different. It also raises concern if the effect of Rad24 depletion on checkpoint maintenance is in part due to the weaker checkpoint establishment. It might be necessary to use the AID2 system to redo Rad24 depletion to exclude such a possibility.

We believe that the AID mutants are very sensitive to the low level of IAA present in yeast.  The instability of the protein is entirely dependent on the TIR1 SCF factor, so the proteins themselves are not intrinsically defective; they are just subject to degradation.  Overexpressing Rad9 allowed us to evaluate its role at late time points. 

(4) It is intriguing that the switch from DDC to SAC might take place at around 12 h when yeasts with a single unrepairable break ignore DDC and resume cell cycling (so-called "adaptation"). Since 4h and 15h are far apart and the transition point from DDC to SAC likely takes place between these two points, it will be very helpful to analyze and compare cell cycle exit after 24 h by treating IAA at multiple points between 4-15h.

When we add IAA to Mad2-AID and Mad1-AID 4 h after DSB induction, cells remain arrested for up to 12 h after DSB induction. At 15 h cells begin to exit checkpoint arrest indicating that the handoff of checkpoint arrest must occur between 12 to 15 h after DSB induction. If we degraded DNA damage checkpoint proteins at any point before Mad2, Mad1, and Bub2 begin to contribute to checkpoint arrest, then arrested cells will likely adapt in a similar manner to when IAA was added 4 h after DSB induction.

(5) Some of the Western blot quality is poor. For instance, in Figure 6C, Mad1-AID level after IAA addition is not compelling especially because the TIR level (the loading control) is also very low.

In Figure 6C, while the relative levels of TIR1 are similar in the IAA treated and untreated samples, there is no detectable amount of Mad1-AID in the IAA treated samples indicating that Mad1-AID was successful degraded with the AID system.

(6) Fig. 8 is complex. It might be helpful to define the different types of arrows in the figure. The legend also has a spelling error, Rad23 should be Rad24.

We’ve defined what each arrow means in the legend and corrected the spelling error in the figure legend.

Reviewer #3 (Recommendations For The Authors):

Major concerns:

Much of the manuscript states that two unrepairable DSBs lead to a long and severe G2/M arrest. Two main cytological approaches are used to make this statement: bud size and number on plates after micromanipulation (microcolony assay), and cell and nuclear morphology in liquid cultures. While the latter gives a clear pattern that can be assigned to a G2/M block as expected by DDC, i.e. metaphase-like mononucleated cells with large buds, the former can only tell whether cells eventually reach a second S phase (large budded cells on the plate can be in a proper G2/M arrest, but can also be in an anaphase block or even in the ensuing G1). The authors always performed the microcolony assay, but there are several cases where the much more informative budding/DAPI assay is missing. These include Dun1-aid and others, but more importantly chk1D and its combinations with DDC proteins. Incidentally, for the microcolony assay, it is more accurate to label the y-axis of the corresponding graphs (and in the figure legends and main text) with something like "large budded cells"; "G2/M arrested cells" is misleading.

Figures have been updated to more accurately reflect what we are measuring.

The results obtained with the Bfa1/Bub2 partner are intriguing. These two proteins form a complex whose canonical function is to prevent exit from mitosis until the spindle is properly aligned, acting in a distinct subpathway within the SAC that blocks MEN rather than anaphase onset. The data presented by the authors suggest that, on the one hand, both SAC subpathways work together to block the cell cycle. However, why does canonical SAC (Mad1/Mad2) inactivation not lead to a transition from G2/M (metaphase-like) arrested cells to anaphase-like arrest maintained by Bfa1-Bub2? Since Bfa1-Bub2 is a target of DDC, is it possible that DDC knockdown also inactivates this checkpoint, allowing adaptation? On the other hand, can the authors provide more data to confirm and strengthen their claim of a Bfa1-independent Bub2 role in prolonged arrest? Perhaps long-term protein localization and PTM changes. Bub2-independent roles for Bfa1 have been reported, but not vice versa, to the best of my knowledge.

In the mitotic exit network Bfa1/Bub2 prime activation of the pathway by bringing Tem1 to spindle pole bodies. Phosphorylation of Bfa1 causes Tem1 to be released and phosphorylate Cdc5 to trigger exit by MEN. It has been shown that DNA damage, in a cdc13-1 ts mutant, phosphorylates Bfa1 in a Rad53 and Dun1 dependent manner. This phosphorylation of Bfa1 could release Tem1 and prime cells to exit checkpoint arrest when cells pass through anaphase. Looking at Tem1 localization to spindle pole bodies and interactions with Bfa1/Bub2 in response to DNA damage might give insight into why cells don’t experience an anaphase-like arrest when they are released by either deactivation of the DNA damage checkpoint or SAC.

We have previously shown that a deletion of bub2 in a 1-DSB background shortens DSB-induced checkpoint arrest. Deletion of bfa1 in a 2-DSB background showed ~80-70% of cells stuck in a large-budded state as measured through an adaptation assay tracking the morphology of G1 cells on a YP-Gal plate and DAPI staining. Deletion or degradation of bfa1 might not release cells from arrest because the Mad2/Mad1 prevent cells from transitioning into anaphase. Our DAPI data for Bub2-AID shows an increase in cells with 2 DAPI signals (transition into anaphase) and small budded cells indicating that degradation of Bub2 is releasing cells into anaphase and allowing cells to complete mitosis.

Further suggestions:

It would be richer if authors could provide more than one experimental replicate in some panels (e.g., S1A,B; S4A; and S6B).

S1C confirms that Rad9-AID and Rad24-AID will adapt by 24 h even with the point mutant TIR1(F74G) which has lower basal degradation than TIR1. S4A has been updated with additional experimental replicates. The 48 h timepoint after DSB induction was to show the importance of Mad2 even when Ddc2 is overexpressed.

Figure 1: Rearrange figure panels when they are first mentioned in the text. For example, it makes more sense to have the plate adaptation assay as panel B for both 1-DSB and 2-DSB strains, budding plus DAPI as panel C, and Rad53 as panel D.

These figures have been rearranged in the order that they are mentioned in the paper.

Figure 5: Correct Ph-5-IAA in the Rad53 WBs (it should be 5-Ph-IAA).

This has been corrected.

Figure S2: The straight line under the "+IAA" text box is misleading. I think it should also cover the "-2" time point, right? Also, check the figure legend. Information is missing and does not correspond to the figure layout.

This has been corrected.

Figure S3: Perhaps "Cell cycle profile as determined by budding and DAPI staining" is a better and more accurate legend title.

The legend title has been updated to “Cell cycle profile as determined by budding and DAPI staining in Ddc2-AID and Rad53-AID mutants ± IAA 4 h after galactose.”

Figure S5: Detection of both Rad53 and Ddc2 in the same blot could lead to misinterpretation as hyperphosphorylated Rad53 appears to coincide with Ddc2 migration.

Figure S5A-B are representative western blots where Rad53 was probed to show activation of the DNA damage checkpoint by Rad53 phosphorylation. When measuring the relative abundance of Ddc2 we did not probe all blots for Rad53.

Table S1: Include the post-hoc test used for comparisons after ANOVA.

A Sidak post-hoc test was used in PRISM for the one-way ANOVA test. PRISM listed the Sidak post-hoc test as the recommended test to correct for multiple comparisons. A column has been added to S. Table 1 to show which post-hoc test was used.

Page 10, line 4: The putative additive effect of chk1 knockout with Dun1 depletion should also be compared to chk1 alone (in Figure 3A).

We address the additive effect of chk1 knockout with Dun1-AID depletion in a later section on Page 11, line 6. Since we had not explored possible effects from downstream targets of Rad53 for prolonging checkpoint arrest when Rad53 was depleted, we did not mention the effect of the chk1 knockout on Dun1 depletion.

Page 14, second paragraph, line 4: "Figure 6A-D", is it not?

Figure S6A is measuring checkpoint arrest in a deletion of mad2 in a 2-DSB strain. Figure 6A-D shows how degradation of Mad2-AID and Mad1-AID after the handoff of arrest causes cells to exit the checkpoint in a Rad53 independent manner.

I feel like one of the goals of trolling is to manipulate emotions by introducing and using fake content, which then often results in frustration or chaos on this social media pages which can create attention. Trolling does its part in truly undermining genuine conversation between users and it can also turn these social media pages and their comment sections into hostile enviroments.

One example of Punishment via Trolling that comes to mind is during 2021, Texas opened an abortion reporting site. However, TikTokers from Texas started broadcasting to the fans to overload the website with thousands of fake messages, complete with local zip codes. In this way, people were encouraged to troll in the name of punishing anti-abortion services.

The long term emotional benefits ofpet oeneership became evident in the lower PTSD scores among pet owners over time. This suggests that pets may serve as a critical source of emotional support during the long-term recovery phase. Pets provide companionship, a sense of routine, and an emotional outlet, all of which contribute to reducing feelings of isolation and anxiety. The act of caring for a pet may also help restore a sense of purpose and normalcy in a post-disaster context. Moreover, interacting with pets has been shown to increase levels of oxytocin, a hormone associated with reducing stress and promoting feelings of well-being. In the context of prolonged recovery, where survivors may struggle with ongoing challenges like housing instability, financial difficulties, and rebuilding their lives, pets can offer consistency and unconditional love, which may buffer against the development of long-term psychological disorders like PTSD. This effect highlights the potential role of animal-assisted therapy or pet ownership as part of mental health interventions in disaster recovery programs.

The psychological. trauma and shock are so overwhelming that specific factors like pet ownership may not show a measurable impact on mental health outcomes. One potential explanation for the lack of a significant association between pet ownership and PTSD scores one month after the disaster is that all evacuees, regardless of whether they owned pets, were dealing with a similar level of acute stress and disruption. The chaos of the evacuation, uncertainty about future, and loss of property or loved ones likely overshadowed the nuanced emotional benefits that pets might offer. Furthermore, the conditions in evacuation shelters could have been so challenging for both humans and animals that any potential comfort a pet could provide was mitigated by the logistical difficulties of caring for them in such an environment. The delay in showing a positive effect on mental health suggests that pets may play a more substantial role in long-term recovery rather than in the immediate aftermath of a disaster.

The fact that pet acceptance was left to the discretion of individual shelter managers reveals the absence of a national or regional policy for handling animals in emergency situations. This lack of standardized protocol created a highly inconsistent experience for evacuees. In some shelters, animals were allowed but kept in separate areas, while in others, pets were outright refused. This inconsistency led to frustration, as many pet owners felt forced to make difficult choices between their own safety and their responsibility to their pets. The emotional toll on evacuees was exacerbated by the uncertainty of not knowing if their shelter would accept pets, causing additional anxiety during an already traumatic situation. This scenario highlights the need for comprehensive disaster planning that explicitly addresses pet welfare, ensuring uniformity and reducing the stress on pet owners during evacuations.

It's important to summarize other views before presenting your own for two reasons: one is to understand other perspectives and be in a position to give your "say," and the other is that it's expected as a way of strengthening your arguments in an academic conversation.

eLife Assessment

This important study examines the role of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Solid experimental results, including genome editing and chromatin profiling methods (ChIP-seq and Hi-C), provide a comprehensive picture of the critical role MORC plays in shaping parasite chromatin. Depletion of MORC results in a lethal collapse of heterochromatin and parasite death, nominating the factor as a new target of antimalarial therapies.

Reviewer #1 (Public review):

Summary:

The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.

Strengths:

The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.

Weaknesses:

Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation. Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

Author response:

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

Reviewer #1 (Public Review):

Summary: The authors investigated the function of Microrchidia (MORC) proteins in the human malaria parasite Plasmodium falciparum. Recognizing MORC's implication in DNA compaction and gene silencing across diverse species, the study aimed to explore the influence of PfMORC on transcriptional regulation, life cycle progression and survival of the malaria parasite. Depletion of PfMORC leads to the collapse of heterochromatin and thus to the killing of the parasite. The potential regulatory role of PfMORC in the survival of the parasite suggests that it may be central to the development of new antimalarial strategies.

Strengths: The application of the cutting-edge CRISPR/Cas9 genome editing tool, combined with other molecular and genomic approaches, provides a robust methodology. Comprehensive ChIP-seq experiments indicate PfMORC's interaction with sub-telomeric areas and genes tied to antigenic variation, suggesting its pivotal role in stage transition. The incorporation of Hi-C studies is noteworthy, enabling the visualization of changes in chromatin conformation in response to PfMORC knockdown.

We greatly appreciate the overall positive feedback and cognisense of our efforts. Our application of CRISPR/Cas9 genome editing tools coupled with complementary cellular and functional approaches shed light on the importance of _Pf_MORC in maintaining chromatin structural integrity in the parasite and highlights this protein as a promising target for novel therapeutic intervention.

Weaknesses: Although disruption of PfMORC affects chromatin architecture and stage-specific gene expression, determining a direct cause-effect relationship requires further investigation.

Our conclusions were made on the basis of multiple, unbiased molecular and functional genomic assays that point to the relevance of the _Pf_MORC protein in maintaining the parasite’s chromatin landscape. Although we do not claim to have precise evidence on the step-by-step pathway to which _Pf_MORC is involved, we bring forth first-hand evidence of its role in heterochromatin binding, gene-regulation and its association with major TFs as well as chromatin remodeling and modifying enzymes. We however agree with the comment regarding the lack of direct effects of _Pf_MORC KD and have since provided additional evidence by performing ChIP-seq experiments against H3K9me3 and H3K9ac during KD. Our new results are presented in Fig. 5. We showed that the level of H3K9me3 decreased significantly during _Pf_MORC KD.

Furthermore, while numerous interacting partners have been identified, their validation is critical and understanding their role in directing MORC to its targets or in influencing the chromatin compaction activities of MORC is essential for further clarification. In addition, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

Validation of the identified interacting partners is indeed critical and essential to understanding their role in directing MORC to its targets. Our protein pull down experiments have been done using several biological replicates. Several of the interacting partners have also been identified and published by other labs and collaborators. To confirm our results, we completed a direct comparison of our work with previous published work. Results have now been incorporated into the revised manuscript to confirm the identified interacting partners and the accuracy of the data we obtained in our experiment. Molecular validation of novel proteins identified in our protein pull down requires generation of tagged lines and may take a few more years but will be submitted for publication in a follow up manuscript.

Reviewer #2 (Public Review):

Summary: This paper, titled "Regulation of Chromatin Accessibility and Transcriptional Repression by PfMORC Protein in Plasmodium falciparum," delves into the PfMORC protein's role during the intra-erythrocytic cycle of the malaria parasite, P. falciparum. Le Roch et al. examined PfMORC's interactions with proteins, its genomic distribution in different parasite life stages (rings, trophozoites, schizonts), and the transcriptome's response to PfMORC depletion. They conducted a chromatin conformation capture on PfMORC-depleted parasites and observed significant alterations. Furthermore, they demonstrated that PfMORC depletion is lethal to the parasite.

Strengths: This study significantly advances our understanding of PfMORC's role in establishing heterochromatin. The direct consequences of the PfMORC depletion are addressed using chromatin conformation capture.

We appreciate the Reviewer’s comments and reflection on the importance of our work.

Weaknesses: The study only partially addressed the direct effects of PfMORC depletion on other heterochromatin markers.

Here again, we agree with the reviewer’s comment and have performed additional experiments to delve deeper into the multifaceted roles of _Pf_MORC. We have performed additional ChIP-sequencing analysis on _Pf_MORC depleted conditions focusing on known heterochromatin and euchromatin markers H3K9me3 and H3K9ac respectively. We hope our new results presented in figure 5 will shed light on the more direct implications of _Pf_MORC on heterochromatin and gene silencing.

Reviewer #1 (Recommendations For The Authors):

Suggestions for improved or additional experiments, data or analyses.

• Why does MORC, which was used in the pull-down, seem to be only minimally enriched in the volcano plot, while a series of proteins (marked in red) and AP2 (highlighted in green) are enriched with log2 fold changes exceeding 15?

We apologize for the confusion. MORC was detected with the highest number of peptides (97 and 113) and spectra (1041 and 1177) confirming the efficiency of our pull-down. However, considering the relatively large size of the MORC protein (295kDa) and it weak detection in the control (5 and 7 peptides; 16 and 43 spectra), the Log2 FoldChange and Z-statistic after normalization are minimal compared to smaller proteins that were not identified in the control samples.

Additionally, can you explain why these proteins appear to be enriched at the same fold? 

We can postulate that these proteins form a complex with a ratio of 1:1. Two of these three proteins are described to interact with MORC in several publications, supporting a strong interaction between them.

Variations in the interactome could result from the washing buffer's stringency.

We agree that the IP conditions could affect the detection of the interactome as well as the parasite stage used. As indicated below, the overlap with previous publications and the presence of AP2 TFs and chromatin remodelers strongly support our results.

It would be highly appropriate for the authors, similar to the co-submitted article (Maneesh Kumar Singh et al.), to present their mass spectrometry data in relation to previous purifications in Plasmodium (Bryant et al. 2020; Subudhi et al. 2023; Hillier et al. 2019) and also in Toxoplasma (Farhat et al. 2020). It would be good if authors could also put their results into perspective in light of the following pre-prints:

We agree with the reviewer’s comment. In this revised manuscript, we compared our IP-MS data to previous published manuscripts. Key proteins including the AP2-P (PF3D7_1107800) and HDAC1 were indeed identified in several experiments validating our initial findings of the formation of large complexes with MORC. However, it’s important to highlight that the MORC protein was not used as the bait protein in previously published papers, and thus some discrepancies can be observed.

Given the tendency of MORCs to form multiple complexes with AP2 factors, have you explored whether specific AP2s are conserved between Plasmodium and Toxoplasma, within the phylum?

P. falciparum encodes for 27 putative AP2s, while T. gondii has over 60 AP2s, making direct comparison challenging. Some Plasmodium AP2s have multiple counterparts in T. gondii and typically conservation is limited to the AP2 binding domains. Attempts to identify sequence homology among AP2s and the regions of conservation have been performed (PMID: 30959972, PMID: 30959972, PMID: 16040597). Although this information would provide interesting insight, we believe exploring this topic at this time would diverge from our primary objectives. It would be more appropriate to address this in future studies.

Could this conservation be identified either through phylogenetic means or by using tools such as AlphaFold, especially considering not just the AP2 domains but also any existing ACDC domains?

Although this may reveal important information regarding the association between MORC proteins and AP2 domains, we believe investigating the conservation between AP2 across apicomplexan parasites may prove too challenging and is beyond the scope of this work.

Most of the genes are depicted without their immediate surroundings (Fig. 2d and Fig S2c, d). For instance, the promoter region of AP2g is not shown (Fig. 2d). It is therefore very challenging to determine the presence or absence of MORC upstream or downstream; considering that this factor, which can create DNA loop protrusions, might bind at a distance from the genes in question.

All gene coverage plots, including AP2-G, show 500 bp up- and downstream of the displayed gene. We have modified our figure legends to make sure that this information is provided.

Upon examining Figure S3, it is evident that the authors have indicated a decline in PfMORC expression, represented as percentages over two unique time frames. The methodology behind this quantification remains ambiguous. It's essential for the authors to specify whether normalization was done using a loading control. As a benchmark, Singh et al. (2021) in their Figure 4 transparently used GAPDH as a loading control and included an untreated sample in their western blot analysis.

We thank the Reviewer for bringing this to our attention. Our initial quantification was performed using ImageJ. To address the Reviewer’s comment, we have reperformed the experiment. Our quantitative analysis was performed through Bio-Rad ImageLab software using aldolase expression as a loading control (50% of the MORC loading). This information has now been incorporated into the supplementary figures (Figure S3).

There's a striking observation that, despite significant degradation of PfMORC (as depicted in Figures S1 and S3), only the upper band in the western blot diminishes. This inconsistency needs addressing, as it can raise questions about the interpretation of the results.

We agree with the reviewer's comment. We experienced some challenges upon performing a Western Blot on such a large protein (295kDa). Our initial attempts required long exposure that may have highlighted non-specific signals of smaller proteins. To address the reviewer’s comment, we have performed the experiment one more time and made necessary changes to our WB protocol. Our new result better reflects the expected down regulation of _Pf_MORC. These changes have been incorporated to our manuscript and Fig S3.

Recommendations for improving the writing and presentation.

MORC KD quantification and consistency with previous findings (Figure S3): When comparing their results with those from another study (Singh et al. 2021), it's critical to ensure that the experimental conditions, especially the methodology for KD and the quantification of protein levels, are similar. If not, a direct comparison might be misleading.

We greatly appreciate the suggestions and have made efforts to redesign the MORC KD quantifications according to the reviewer’s recommendations.

While the manuscript mentions the level of KD, it does not delve into the functional consequences of such a decrease in protein levels. It would be of interest to understand how this level of KD affects the parasite's biology, especially in the context of the paper's main findings.

We have addressed this question by looking at the changes in chromatin structure in WT versus KD parasites upon atc removal. We have also validated this initial result by designing an additional ChIP-seq experiment against histone marks in WT versus KD parasites upon atc removal. Our findings showed a significant downregulation in H3K9me coverage in heterochromatin regions, specifically in genes associated with antigenic variation and invasion genes. These findings suggest that PfMORC regulates at least partially gene silencing and chromatin arrangements. The manuscript has been edited accordingly. 

Concluding page 5, the authors present an interpretation of their findings that suggests a multi-faceted role of PfMORC in regulating stage-specific gene families, particularly the gametocyte-related genes and merozoite surface proteins. While the narrative they present is intriguing, several concerns arise:

Over-reliance on correlation: The authors draw a direct line between the levels of PfMORC binding and the function of these genes in the parasite's life cycle. However, a mere correlation between PfMORC binding and stage-specific gene activity does not necessarily imply causation. They would need to provide experimental evidence showing that manipulation of PfMORC levels directly impacts these genes' expression.

We agree with the reviewer's comment. We have however partially addressed this issue by comparing our ChIP-seq, RNA-seq and Hi-C experiments. We concluded that several of the transcriptional changes observed were due to an indirect effect of PfMORC KD and were most likely induced by a cell cycle arrest and partial collapse of the chromatin structure. The collapse of the heterochromatin structure was validated using our Hi-C experiment. To further address additional concerns the review’s had, we have included additional ChIP-seq experiments targeting histone marks to confirm our initial hypothesis. Result of this additional experiment has been incorporated in the revised version of the manuscript.

Ambiguity surrounding "low levels" and "high levels": The terms "low levels" and "high levels" of PfMORC binding are qualitative and could be subject to interpretation. Without quantification or a clear benchmark, these descriptions remain vague.

We agree with the reviewers that the terms "low levels" and "high levels" of PfMORC binding are qualitative and could be subject to interpretation. We have however quantified our change in DNA binding using normalized reads (RPKM). In trophozoite and schizont stages, most of the genes contain a mean of <0.5 RPKM normalized reads per nucleotide of Pf_MORC binding within their promoter region, whereas antigenic gene families such as _var and rifin contain ~1.5 and 0.5 normalized reads, respectively (Fig. 2b). Similar results are also obtained for the gametocyte-specific transcription factor AP2-G  that contains levels of Pf_MORC binding similar to what is observed in _var genes (Fig. 2c and S2c, d).

Shift in Binding Sites: The observed minor switch in PfMORC binding sites from gene bodies to intergenic and promoter regions is mentioned, but without context on how these shifts impact gene expression or any comparative analysis with other proteins showing similar shifts. The claim that this shift implicates PfMORC as an "insulator" is a leap without direct evidence.

We apologize for the confusion. We  have compared our ChIP-seq with RNA seq results at different time points of the cell cycle and demonstrated that the shift observed has an effect in gene expression. We have edit the manuscript to clarify these results.

Overextension of PfMORC's Role: The authors suggest that PfMORC moves to the regulatory regions around the TSS to guide RNA Polymerase and transcription factors. This is a substantial claim and would require additional experiments to validate. Simply observing binding in a region is insufficient to assign a specific functional role, especially one as critical as guiding RNA Polymerase. Historically, the MORC family has been primarily linked with gene silencing across Apicomplexan, plants, and metazoans. On page 7, the authors noted a minimal overlap between the ChIP-seq and RNA-seq signals (Fig. 4e). They also acknowledged that the pronounced gene expression shifts at schizont stages result from a combination of direct and indirect impacts of PfMORC degradation, which could cause cell cycle arrest and potential heterochromatin disintegration, rather than just decreased PfMORC binding. Therefore, the authors should adjust their conclusions in the manuscript to more accurately represent the multifaceted functions of MORC in the parasite.

We agree with the reviewer's comment and have edited the manuscript accordingly.  

DISCUSSION:

The authors concluded that "Using a combination of ChIP-seq, protein knock down, RNA-seq and Hi-C experiments, we have demonstrated that the MORC protein is essential for the tight regulation of gene expression through chromatin compaction, preventing access to gene promoters from TFs and the general transcriptional machinery in a stage specific manner."

Again, the assertion that MORC protein is essential for tight regulation of gene expression, based purely on correlational data (e.g., ChIP-seq showing binding doesn't prove functionality), assumes causality which might not be fully substantiated. The phrase "preventing access to gene promoters from TFs and the general transcriptional machinery in a stage-specific manner" needs also validation. Asserting that MORC is essential for this function might oversimplify the process and overlook other critical contributors.

We agree with the reviewer’s comments and the conclusion has since been edited accordingly.

The discussion is quite poor. It would be pertinent to put MORC in perspective within the broader picture of regulatory mechanisms of chromatin state at telomeres and var genes. For instance, how do SIR2 and HDAC1 (associated with MORC) divide the task of deacetylation? Or the contribution of HP1 and other non-coding RNAs.

We agree with the reviewer’s suggestion. However, in order to put MORC in perspective within a broader picture, we would need to measure changes in localization of several molecular components regulating heterochromatin in WT versus KD condition. This will require access to several molecular tools and specific antibodies that we do not currently have. We have addressed these issues in our discussion.  

Minor corrections to the text and figures.

Figure 1d: Could you provide the ID for each AP2 directly on the volcano plot? While some IDs are referenced in the manuscript, visual representation in the plot would facilitate a clearer understanding of their enrichment levels.

ID for unknown AP2 proteins have been added on the volcano plot.

I recommend presenting Figure S2b as a panel within a primary figure. This change would offer readers a more quantitative understanding of the distinct differences between developmental stages. Notably, there seems to be a limited number of genes in common when considering the total, and there is an apparent lack of enrichment in the ring stage.

This has been done.

The captions are very minimally detailed. An effort must be made to better describe the panels as well as which statistical tests were used. 

We have improved the figure legends and add the number of biological replicates as well as the statistic used in each figure legend.

Figure 1A: The protein diagram with its domains does not take scale into account.

The figure has been modified.

Reviewer #2 (Recommendations For The Authors):

(1) The study lacks a direct link between PfMORC's inferred function and the state of heterochromatin in the genome post-depletion.

We agree with the reviewer's comment and have included additional ChIP-seq experiments to measure changes in histone marks in PfMORC depleted parasite line. We show a significant decrease in histone H3K9me3 marks in PfMORC KD condition.

Conducting ChIP-seq on well-known heterochromatin markers such as H3K9me3, HP1, or H3K36me2/3 could shed light on the consequences of PfMORC depletion on global heterochromatin and its boundaries.

With no access to an anti-HP1 antibody with reasonable affinity, we have not been able to study the impact of MORC KD on HP1 but have successfully observed the impact on H3K9me3 marks. These results have been added to the revised manuscript in (Fig. 5).

(2) The authors should conduct a more comprehensive analysis of PfMORC's genomic localization, comparing it to ApiAP2 binding (interacting proteins) and histone modifications. This would provide valuable insights.

We have performed a more comprehensive genome wide analysis of MORC binding through ChIP-seq on WT and MORC-KD conditions. Our results show that Pf_MORC localizes to heterochromatin with significant overlap with H3K9-trimethylation (H3K9me3) marks, at or near _var gene regions. When downregulated, level of H3K9me3 was detected at a lower level, validating a possible role of _Pf_MORC in gene repression. Regarding the comparison with AP2 binding, our proteomics datasets have shown extensive MORC binding with several AP2 proteins.

(3) RNA-seq data reveals that only a few genes are affected after 24 hours of PfMORC depletion, with an equivalent number of up-regulated and down-regulated genes. The reasons behind down-regulation resulting from a heterochromatin marker depletion are not clearly established.

We agree with the reviewer’s comment. At this stage (24 hours), _Pf_MORC depletion is limited and the effects at the transcriptional level are quite restricted. Furthermore, it is highly probable that down-regulated genes are most likely due to an indirect effect of a cell cycle arrest. We have edited the manuscript to address this comment. 

The relationship between this data and the partial depletion of PfMORC needs further discussion.

We agree with the reviewers and have improved our discussion in the revised version of the manuscript.

(4) The authors did not compare their ChIP-seq data with the genes found downregulated in the RNA-seq data. Examining the correlation between these datasets would enhance the study.

We apologize for the confusion. We have compared ChIP-seq and RNA-seq data and identified a very limited number of overlapping genes indicating that most of the changes observed in gene expression are in fact most likely indirect due to a cell cycle arrest and a collapse of the chromatin. We have edited the manuscript to clarify this issue.

(5) The discussion section is relatively concise and does not fully address the complexity of the data, warranting further exploration.

We have improved the discussion section in the revised version of the manuscript.

A consequentialist perspective that aligns politcally with the protest could believe that any harm done by the trolling would be outweighted by the impact the protest had, therefor believing it to be ethical. A Kantian perspective believe that acting disingenuously or maliciously, regardless of reason, is always unethical.

This was a really cool study/experiment I enjoyed reading it. I've never really heard about heterogeneity before so this was new for me to learn about!

peergos mirror

TLS

I have heard that it takes two to tango. When one party keeps trying to cause a riot, others will ignore him and he will lose interest in causing a riot. But is this really useful on the Internet? Some people are constantly venting their negative emotions in life through the Internet and spreading negative and pessimistic comments. Our disregard may make them more rampant, so there needs to be specific regulations to reduce the occurrence of such behavior.

Amanda Mireles is using hypothesis for students to get their own video clips and to annotate them for other students.

Students took scenes from THE BIG BANG and used literature and academic references to relate to the scene

I am watcing hypothesis annial awards and using hypothesis to gather our collective wisdom.

I have just placed this text into that seminar. Hypothesis is a modrn digital example of how ancient Druids were recorded by Celtic Monks to keep wisdom. A monk wrote down what a Druid said. They wrote the material in large letters with large gaps between the lines. When the monk died, a new monk woiuld take over the wisdom keeping. The new wisdom keeper wrote IN BETWEEN THE LINES and ON THE SIDE LINES. These were called glossaries becase they wrote in GREEN INK. Glas is the Irish word for Green.

militaristic discourse can connect countries across national borders

military masculinity

othering connected to militarism, enacted through it and created by it

What causes women to be treated unfairly in society? We can always see women being criticized online, from their appearance to their figure to their personality, and in many dirty jokes, women's privacy is the most ridiculed. However, people on the Internet have magnified this misogyny by taking advantage of the "anonymous nature" of the Internet.

Is consciousness the only thing that enables real beings and AI to have emotions? Or can you still be conscious and remain emotionless?

This shows that the cat might have been cared for and it was not just a wild cat that lingers around.

By using this choice of words the author is making the connection that the cat isn't just a thing that's there. It's a thing that goes where the person goes.

This shows the challenges and hardships the cat Pangur Ban went through his life journeys.

Like Equiano wrote about earlier he writes about how slaves that came together on the ships were still torn from their families and sold. Unlike Equiano, Cinqué was taken from his family that remained in Africa while he was taken to North America.

limits of MILITARISATION as it excludes crucial contexts

Reflects Abigail's eagerness for Americas independence from British rule. She explicitly asks John to consider women in the legislative process, indicating an early demand for women's inclusion in lawmaking. She also critiques traditional views on marriage and the power held by husbands over their wives. Abagail presents a bold statement about the potential for men to abuse power, highlighting the need for checks and balances Her warning implies that women are ready to rise against oppression, similar to the colonies fight against British rule .The closing assertion is a direct demand for inclusion and rights, emphasizing the importance of representation in governance. Abigail Adams' letter is foundational text in the discourse about women's rights and representation during the American Revolution. The themes highlighted through this annotation process illustrate her advocacy for gender equality ,critique of existing power structures, and demand for women's voices in the political arena. This letter not only reflects the historical context of the time but also resonates with contemporary discussions about gender rights and representation.

no worries

refelctors moon

I can see this, I know that you can pay to be at the top of a google page. I think that skews results to.

I wasn't aware that there was a way in which you should use Google. It makes sense, because Google is a search engine made to help everyone, it doesn't always know exactly what someone needs when searching.

UX unicorn exist for a reason and a really important one as shown in the article

I couldn't agree more with this quote! The more and more people begin to understand this the better the World Wide Web will be.

This is a really important statistic to understand. When making apps or softwares, creators need to be aware of disabilities and how they affect a lot of people. Having this understanding will help them to make sure their creations are accessible.

ok - now the connection is explicit!

The question remains, though: how much desubjectification is necessary?

whew - spirituality and business/entrepreneurship. Interesting...

(in a previous year, a student shared the following link -- memes about spirituality -- which definitely connects with entrepreneurship discourses too!)

https://cajspirituality.com/2018/04/09/warning-7-hilarious-spiritual-memes-guaranteed-to-make-your-soul-smile/

both western zen and entrepreneurial practice =

DING! --This relates explicitly to the claims made about “liquid society” in the “Unconventional entrepreneurship” article. It also relates implicitly to the claims made about entrepreneurship in the article, “‘Don't forget to like, share and subscribe’: Digital autopreneurs in a neoliberal world.” But whereas this latter article discusses “figures” that do not appear perfectly content in their management of their entrepreneurial selves, Saari and Harni seem to suggest mindfulness and a ‘spiritual’ relationship to one’s environment as a transcendent (or tactical) move that empowers entrepreneurial, flexible individuals in their journeys toward self-fulfilment. Clearly, there are many links between entrepreneurial adult education and the “world-affirming” insights it offers and the themes of the previous weeks we’ve examined in this course.

somewhat redressed by last week's content...

And how does this incessant happiness gel with the zen-like attitude of the following:

ok... but the zen-like peace gets displaced by the entrepreneurial ethos that says you always have to be working (in order to produce your own happiness):

meta-cognition!

context...

mindfulness as emancipating...

ends off with a strangely positive-sounding note...

...

how do you think this accords with entrepreneurial thinking? Clearly, one ought not be wrapped up in the past (either failures or successes), but what about a future-focus? A fixed mindset is clearly bad, but is a growth mindset also (especially if the pressure to "grow" and "develop" takes one away from a present focus)?

to limn = to depict/describe (from the root word "illuminate")

this isn't the original "zen"

**

linking the first week to grit etc.

Wow - this sounds, at first, like it's antithetical to entrepreneurial undertakings. Shouldn't one want to achieve something and invest oneself wholly in it?

indeed - this is a summation of the relevance of the last number of weeks, connected under the rubric of "mindset."

FYI - cognitive relates to thinking, affective relates to emotions, and conative relates to behaviour, specifically the inclination to act 'on purpose'. It's an effort or a drive to do something, but this isn't entirely rational...

what a link - liquid selves in an entrepreneurial context = zen-like...

...

zen is awfully implied here - not at all explicit.

whew!

**

!!

now we're getting somewhere!

interesting - how do you relate this to the entrepreneurial quest that motivates entrepreneurial personalities -- to turn the impossible (dream) into the real? To not be held by the bounds of the ordinary and others' expectations but to transcend them and become extraordinary...

thus, we get things like this:

does this sound wise (or good/meaningful) to you?

Your response will locate you relative to spirituality/mindfulness/mediation as a lens for self-actualization ...

...

background...

...

...

aha

*

*

this seems like a critique rather than something to be celebrated...

!

part of their overall argument

(fancy) theoretical framing...

Yowza!!

even if we're not always working (at our jobs), we're always working on ourselves ...

...

interesting

context

(see my last comment)

...

ok

ok!

connection to other article!

interesting

!

!

ok!

*

Interestingly [NSFW language alert!], as Tim Desmond argued in his 2019 book “How to stay human in a fucked-up world: Mindfulness practices for real life,” one can be “too” compassionate: “The challenge of staying human in a fucked-up world comes down to how we respond to the immensity of suffering that confronts us from every direction. [one has] to find a way to take care of the compassion in me so that I don’t end up overwhelmed. If I can’t I’ll find myself in despair.” One must be mindful in order to not drown in anger or bitterness.

food for thought - seeing one's own emotions and detaching from them is posited as good (but doesn't this assume that those emotions get one into trouble?) -- The idea is that over-identifying with feelings is bad. But does this apply to "good emotions" (like compassion)?

good examples, but maybe a little too manichean -- compassion = a good emotion and greed/aggresiveness = a bad emotion. Can you have a well-functioning mix of the two?

...

*

in other words...

possible route #1

(as another article put it, this is an attempt to offer ‘the wisdom and the heart of Buddhist meditation without the Buddhism’)

the basis for the aforementioned hope

bringing the different parts of the literature review together...

the hope...

implications...

nice term.

to review...

one of the reasons we've been focusing on affect just as much as effect, recently...

hah! easier said than done

widening the aperture!

interesting. What do you think of this (and how this idea connects to this course in general, or this week in particular)?

that's a tall order!

interesting. akin to re-framing

(link to experiencing failure too)

! with great power comes great responsibility...

to recap...

...

and as the very first reading of the year stated, we're all living in an environment of perpetual uncertainty now, having to all be entrepreneurs and assess risk and make our own opportunities...

quite frankly, this has been one of the "hidden agendas" of this course - how do we think about thinking (often by thinking about ourselves first)

!

mindfulness ought to help us eliminate cognitive biases and see things the way they are (not the way we want them to be, or to be trapped in vicious circles of judgement)

attitude

(with the idea that mindfulness can help us with this task)

connecting the axioms to the definition

"flow"

good - starting out with a definition (kind of ... more of a prefatory comment)

attention

intention (including the entrepreneurial type...)

entrepreneurial traits/actions can be bad (for the entrepreneurial agent, and for others) ... but mindfulness can avert that (or re-center one while on an entrepreneurial journey)

an attitude of non-judgement

more background, linking it to the need of all entrepreneurs to be creative ... creating opportunities, creating change...

link to EQ!

I appreciate Tim Desmond's observation that mindfulness is a very conscious exercise of relating to the world (in a way that many think meditation is not): Mindfulness does not mean taking deep breaths, sitting on a cushion, watching your thoughts and feelings with disinterest (like you’re watching a boring TV show). RATHER, mindfulness = a way of relating to the world (and specifically to suffering) that contains compassion, joy, equanimity, and wisdom. It is precisely the quality that allows us to stay human in fucked-up situations – to stay open, caring, and able to relate.

meditation...

d'uh

implications of mindfulness - connecting the internal to the external

a better definition

The purpose - bringing mindfulness into the entrepreneurial equation (how does it help with discovering opportunities...)

the point is that one ought to aspire to ...

d'uh

!

whoa! Big question. And something to consider for yourselves...

Theoretically, your highest aspiration should (hopefully) coincide with what we would imagine others also seek to fulfill ...

It basically boils down to your version of the "good life" (not necessarily good for others, or even only good for you, but what makes a life good or what makes one's life filled with well-being? What does it take to "be well"? Being good doesn't necessarily mean "doing" good...

super important

Crow, Belknap, and Cheyenne Reservations - wyola

badlands

1.) Reparations, romanticized, loyal evangelists, exploitative, colonized, history, slavery, emancipation, property, psychological violence, obedience, narrative, industry. 2.) Short history of rum and sugar industry, slavery and exploitation in the Caribbean on the basis of skin color and production of rum. Modern impact of colonizer influence follows, which focuses on its effects in the corporate aspects, being that people of color are not allowed into the higher levels of rum companies, financial inequity, and lack of Caribbean representation in positions of power in outsider-owned spirit companies operating in the Caribbean.

This is representative of the argument, as it lists the acts committed against the people, the reason why current "reparation" actions being taken are not sufficient, and what the people want.

FINAL THOUGHTS: This article touched a lot on India's relationship with the US, China, and Russia, specially after the US had withdrawn from Afghanistan. India has developed a growing connection with the US, and there are lots of concerns about China's influence in the region. Some questions I had throughout the reading include, how can India leverage its economic strength to navigate its relationships with other powers? Additionally, are there potential risks of increased US and India cooperation?

Historical support from Russia is crucial for India.

India’s being quiet about it's concern.

Afghanistan’s situation could heavily increase tensions between India and China.

So this pushed India closer to the US.

Russia has a growing rivalry with India and its allies.

Hurts India's diplomatic efforts.

India's commitment to Kabul is very different than Russia's approach.

Russia's shifting alliances complicate India's plan.

Cautiously increasing its influence in Afghanistan.

Does this mean India’s actions are warranted given the stakes and concern they had?

Pakistan’s role could boost China’s strategy.

China’s increased involvement could destabilize the states.

US's withdrawal creates new challenges for India.

Shifting dynamics.

India's strategic outlook.

Shift in focus for India.

A more strategic evolution in India’s approach to its neighbors.

Increased terrorism.

Historical irritants in US and India relations.

A weakened US and Pakistan relationship could shift the power dynamics more favorably for India.

A need for India to recalibrate its expectations of US support.

What specific dangers are they anticipating?

How will it change relations with China?

Sense of ownership for artifacts of Troy

Thesis

n the U.S., freedom of expression is highly protected under the First Amendment, with only limited exceptions. Two key cases illustrate this broad interpretation:

Texas v. Johnson (1989): Gregory Lee Johnson burned an American flag in protest during a political demonstration, violating a Texas law that prohibited flag desecration. The U.S. Supreme Court ruled in Johnson's favor, holding that his act of flag burning was a form of "symbolic speech" protected by the First Amendment. The Court emphasized that the government cannot prohibit expression simply because society finds it offensive. United States v. Eichman (1990): After Texas v. Johnson, Congress passed the Flag Protection Act to criminalize flag desecration on a federal level, attempting to override the Court's decision. In U.S. v. Eichman, the Supreme Court again ruled in favor of free speech, declaring the federal law unconstitutional. The Court reaffirmed that flag desecration, as a form of protest, is protected by the First Amendment, regardless of its symbolism. These cases underscore the Court's strong stance on protecting freedom of expression, even when it involves controversial or offensive acts like flag burning.

explicitly states his position

reasons and evidence for how Honore came to see work as a blessing

Honore takes a position in response to another position: his father's.

search: "sd-ndn" ipfs

to: https://hyp.is/xCrXKoyREe-zZCOJFZ39mg/www.journaltocs.ac.uk/index.php?action=tocs&journalID=18837

searched in vein for the acroym IPFS

or Inter Planetary File System

which is an SD-NDN

a transformative approach in network architectures poised to deliver substantial benefits. By addressing the limitations inherent in traditional host-centric network architectures, SD-NDN offers improvements in network performance, scalability, and efficiency. The paper commences with an overview of named data networking (NDN) and software-defined networking (SDN), the two fundamental building blocks of SD-NDN. It then explores the specifics of integrating NDN with SDN, illustrating examples of various SD-NDN models. These models are designed to leverage SDN for NDN routing, caching, and forwarding. The paper concludes by proposing potential strategies for further integration of SDN and NDN and some open research questions. These proposed strategies aim to stimulate further exploration and innovation in the field of SD-NDN.

subjunctive time

this section discusses how we can inject communication errors into the game. The form used creates a tradeoff between more signals (expressivity) and accuracy (expected no of correct messages)

if stems have a hierarchy via phonemic similarity and there is a rich morphology, learning words may be greatly simplified by a learning a few systematic rules. Idealy we need to learn a lexicon with just one base form and apply a group action to get all the forms. In this case we only need to memorize a small lexicon.

Here we may be considering a form of compositionality, where semantics are established by preferential selection of languages with related patterns of phonemes. What is missing is that these patterns are not grounded in the structural features if the state being expressed..

decoding the state is mandated on decoding the each signal in the sequence

I agree with this statement because if you know there's something you don't want anybody to take advantage of or see, the best thing you should do is think about what you post on the internet . If you accidentally post something that wasn't meant to be seen by other users, the chances of people knowing and probably sharing it with numerous people is very high. The internet is not always trust worthy.

The very essens

all you need is naming mutbl a What's in a name?

everything is possible if you know how to name things

nd immutable data

all in th way you name things

Its both interesting and a great source of ethos to be able to admit and address this problem to the general public.

Is this because of the tenth amendment?

Were people generally excited about a way out of this/this crucial change?

Bradley states that it is likely that we “share our general morality with a common man.” Eliot, however, suggests that it is not simply these values that we share, but rather a confinement within them.

This notion is reflected in the title: “What the thunder said” becomes ironic. We don’t care what the thunder had to say; instead, we interpret it according to our own perspectives. The presumable thunder echoes its teaching—“da”— to gods, humans, and demons, each of whom constrains it to their singular interpretation: to give (“datta”), to control oneself (“dayahadvam”), and to sympathize (damyata). Thus, it appears that the teaching itself is less significant than its interpretation. What if “da” were intended to encompass all three meanings? Yet, different creatures choose to confine it within just one.

Eliot goes further than that and crafts a world in which “da” means none of these interpretations. The first pronoun following each of the “da” gives insight into this irony of the values we established. The first “datta” – to give – is dominated by the pronoun “we.” By definition, “give” is “to freely transfer the possession to.” The collective pronoun “we” evokes a sense of shared responsibility, suggesting that giving is a communal activity rather than a purely selfless act. With a simple use of “we” Eliot hints at how noble intentions – this “freely” – turn into transactional relationships and the concept of giving becomes confined within the bounds of reciprocity.

As we switch to “dayahadvam,” however, Eliot introduces a pronoun “I.” I represents a personal agency, where individual confinement leaves no room for compassion to others. Eliot reinforces this idea with a double repetition of “each.” Without compassion and connection with others we are isolated – “each” in their own prison, where thinking of escape we further confine us within this trap. The use of “I” reveals the limitations of self-centered thinking, suggesting that without genuine connection and compassion, we are ultimately confined, entrapped, and isolated from one another.

Finally, as we transition to “damyata,” the pronoun shifts again, this time to the third-person “the boat.” This transition signifies a move from internal self-control, which damyata is supposed to signify, to a search for external control. The boat, an object, thus, becomes a metaphor for the external forces we seek to exert control over our lives. In this way, the use of the third person reflects a desire to externalize control rather than cultivating it from within. This search, however, never culminates with a discovery; instead, Eliot uses a hypothetical tense, where “the heart would have responded gaily” if it was “invited” into these “controlling hands.” The hypothetical tense suggests an absence of such an external force that could have provided stability from the outside.

Through these pronouns, thus, it appears that Eliot suggests that it is not the values that connect us; instead it is the entrapment within the interpretations of these values, obscuring their true significance, that we share. The act of giving becomes transactional, compassion is separated by barriers of isolation, and self-control turns into an unfulfilled quest for external authority. Consequently, the teachings of connection only serve to highlight the constraints we ourselves have imposed, trapping us in this prison of distorted interpretations.

for - definition - physiocrats - Steve Keen - economy - history - economic flow as biomimicry of body's circulation system

definition - physiocrat - During the 18th and 19th century, a group of mostly French "economists" led by Francois Quesnay, physician to the King of France at the time, performed some of the first autopsies of the time. - Autopsies were banned for the longest time for religious reasons - When Quesnay performed autopsies, he discovered networks of tubes in the circulation system and this led him to surmise a network of circulation in another field, economics - Quesnay advised the king, hence the name physiocrat - So modern economics has its roots in biology - it was a case of biomimicry!

It's impossible to write an algorithm that can determine, given an arbitrary program and input, whether the program will halt (stop running) or run forever.

dus bij functioneel level gaat het om valure chains die bestaan uit; 1. kwaliteit, 2. innovatie 3. klant responsiveness 4. efficientie