Route Data: York, England. Date: May 1349. This is a very specific route (along the rivers).

Third Data Point: Dalkey and Drogheda, Ireland. Date: August 1348. This helps trace the route over the sea.

Second Data Point: London, England. Date: End of 1348. This is important for showing how the disease followed a river.

First Data Point: Weymouth, England. Date: Summer 1348.

This is a specific scientific detail that helps me understand why the rat theory might be wrong in places like Scandinavia. My map can visually test if the plague slowed down in colder areas.

This is a great piece of evidence for the human parasite side. It tells me to look for things in the old chronicles that don't mention rats, which strengthens the argument against the rat theory.

This supports the newer idea (the human parasite theory) that challenges the rat theory. I will use my maps to see which one looks more likely.

This clearly backs up the old idea (the rat-flea theory) that I need to test with my map. It gives me a clear route to follow.

This is exactly what my project aims to do. I can use this quote to show that comparing the plague's spread with trade routes is the right way to study this problem.

This point is well taken, and it is easy to make a mistake. Treating repeated measurements as if they were separate artificially inflates the sample size and may result in misleading p-values. It reminds me of the importance of study design: often, statistical mistakes reflect structural problems in how evidence was collected and analyzed.

A significant advantage of the unpaired t-test is that, for moderate sample sizes, it has high statistical power to compare two groups. That is efficient for clinical trials or public health studies where you’re comparing outcomes of treatment versus control groups. It creates familiarity in hypothesis testing, which simplifies the interpretation of results in different disciplines

This clarifies why normality is such a basic assumption. It’s not simply a default rule; parametric tests, such as the t-test, for instance, depend on the parameters of the distribution (mean and SD) to resemble the population. When the distribution is off, those numbers do not accurately reflect the data, which can be misleading for the results.

This analysis examined wage and rent data across 400 German cities and districts from 2014 to 2024. Rent burden calculations compare the median net income (Steuerklasse I, single person) with the average monthly rent for a standardized 50 m² apartment. Net income was calculated using a simplified progressive tax model with deduction rates of 30 percent (below €30,000), 35 percent (€30,000-60,000), and 40 percent (above €60,000), capturing both income tax and social insurance contributions typical for German employees.

Wage data come from the Federal Employment Agency (Bundesagentur für Arbeit) and show median monthly gross salaries for full-time employees. National wage trends use Destatis compensation data (Table 81000-0008). Inflation adjustments apply the standard Consumer Price Index (2016-2024: 25.58 percent). Real wage calculations use the exact compound method rather than simple subtraction to avoid overstatement across the eight-year period.

Rent data derives from the empirica Immobilienpreisindex, converted from cold to warm rent using a uniform 25 percent surcharge for utilities. All values represent asking rents for new contracts, not existing tenancies, which typically show lower costs due to tenant protections. The 30 percent affordability threshold follows standard economic guidelines, though German regulations don't mandate specific income-to-rent ratios.

For the apartment space analysis, profession-specific salaries are available only at federal state level. Cities like Frankfurt use Hessen averages, Munich uses Bayern averages, while city-states Berlin and Hamburg have exact data. The four professions shown (geriatric care, hospitality, IT/informatics, and electrical engineering) represent the top two winners and bottom two losers in wage growth during 2016-2024, providing a complete spectrum of German wage development.

In Berlin, a geriatric nurse earning the median salary could afford approximately 48 m² in 2016. By 2024, this figure had shrunk to 41 m², despite significant wage increases in the sector.

At the current growth rate of 0.31 percentage points per year, rent burdens are projected to reach 24.3% nationally by 2026. In Munich and Frankfurt, they have already surpassed 30%. The question is no longer whether Germany has a housing crisis, but how many more districts will cross the threshold before policymakers take action to address this pressing reality.

In Munich, the squeeze is even sharper. A median-paid engineer could afford around 50 m² in 2016; today it's closer to 47 m². // In 2016, 30% of an electrical engineer's income in Hamburg could rent 73m². By 2024, that same 30% only covered 60 m².

Care workers in Leipzig and Dresden gained 11-17 m² of living space. The same profession in Berlin lost 8 m². Location now matters more than salary.

Dresden care workers saw wages nearly double (+91.5%), yet gained only 11 m² of space. In Berlin, the same 60% wage increase resulted in 8 m² less space.

Across Germany, the ability to keep up with rent now depends on both wage growth and geography. In more affordable cities, rising wages in essential jobs have genuinely improved financial stability. However, in expensive metropolitan areas, even strong wage growth cannot keep pace with housing inflation.

The result is a new economic reality: for many professions, where you live matters more than how much your salary has increased. To understand what this means in everyday life, we translated these wage trajectories into square meters, illustrating how much living space different professions can still afford today compared to 2016.

Since 2016, the median monthly salary for geriatric nurses has risen 56% from €2,436 to €3,792. Although inflation ate away much of those gains, nurses today can still afford 24% more goods and services than eight years ago. This matters for housing: in cities where rents have remained stable or only moderately increased (Leipzig, Chemnitz, parts of Eastern Germany), these wage gains help essential workers keep up with local living costs. Despite receiving significant raises, care workers still struggle to afford to live in areas where their services are most needed. For instance, in Berlin, they lost 8 m² despite a 60% wage increase, while in Munich, they gained only 3 m² despite a 40% raise.

Rising rents affect everyone, but not everyone faces the housing market with the same financial stability. A closer look at wage development across different professions reveals a surprising pattern that challenges long-held beliefs about who is advancing and who is falling behind.

When considered as a whole, these patterns indicate that Germany's housing crisis is both urban and regional. It spreads from major cities into their commuter belts, while many smaller towns and rural districts still have relatively low rent burdens. But even this is only part of the story.

A similar trend is observed in Holzminden (16.0%) and other industrial districts, where diverse economies have maintained moderate rent burdens. Even in areas like Altenburger Land (14.2%) and Erzgebirgskreis (14.2%), affordability remains significantly higher than in major cities. These regions show little of the dramatic rent escalation seen in Munich, Berlin, or Frankfurt, and for many households, they continue to offer a financial buffer against rising living costs.

In 2024, the lowest rent burdens are found in regions like Salzgitter, where a single tenant spends only 14.7% of their net income on a 50 m² apartment. Other districts in the bottom group include Chemnitz (15.4%), Holzminden (16.0%), and Wolfsburg (16.3%), all of which are well below the 20% threshold. These areas highlight a combination of relatively stable rents and incomes that, while lower than in many western metropolitan regions, keep housing costs comfortably within affordable limits.

While these metropolitan spillovers raise housing costs, several districts—particularly in eastern Germany and industrial hubs—remain relatively affordable. This demonstrates that rent burdens vary widely across the country, and not all households experience the same financial pressure.

And then there are cities like Leipzig, which are still relatively affordable in absolute terms, but are changing rapidly. Rents in Leipzig jumped by 74% over the last decade, while wages increased by 49%. While the gap is smaller than in Berlin or Munich, the acceleration is noteworthy, indicating that affordability pressures are spreading beyond traditional hotspots.

These cities exemplify the core pattern of Germany's affordability issue: in metropolitan areas with the strongest labor markets, rent inflation is outpacing income growth.

For example, in Berlin, rents have surged by 91% since 2014, while nominal wages have only risen by 45%. In Munich, the situation is slightly better but still concerning: rents climbed by 53%, compared to wage growth of only 38% during the same period. In Frankfurt and Düsseldorf, the trend is similar: rent increases of 42% and 44% respectively, versus wage gains of 32% and 29%.

One of the steepest increases in rent burden shows up in Germany's major cities, where demand for housing has outpaced wage growth. In these metropolitan areas, even solid salary increases are often not enough to keep pace with rising rents.

In 2014, only 6 districts had rent burdens above this critical level, all clustered around Munich. By 2024, that number had more than quadrupled to 26 districts across the country. This fourfold increase reveals how the housing crisis has spread far beyond Germany's traditional hotspots.

The true extent of Germany's housing affordability crisis becomes clear when we focus on the 30% threshold, the point where housing costs begin to undermine financial stability.

By hovering over the maps, readers can view the rent-to-income ratios for each city or district and observe how their region has changed over the past ten years.

The two maps below highlight the shift in rent burden from 2014 to 2024. The 2014 map is predominantly lighter, reflecting that most regions remained well below the 30% threshold. In contrast, the 2024 map shows noticeably darker shades across large parts of Germany, signaling rising rent burdens and shrinking financial buffer for households.

Using district-level income and rent data, the researchers calculated the rent-to-income ratio for each region, creating a detailed map of housing affordability across the country and illustrating how this has evolved over the past decade.

The study shows that across Germany, rent burden has increased noticeably over the past decade. In 2014, single-person households spent an average of 20.6% of their net income on rent. By 2024, this number rose to 23.7%. A shift that steadily pushes households closer to the 30% threshold.

One of the clearest indicators of housing affordability is the rent burden, which is the percentage of a household's net income spent on rent. Economists generally consider 30% of net income to be the upper limit for a healthy financial budget. When rent exceeds this threshold, households have less flexibility for savings, unexpected expenses, or discretionary spending, even as nominal wages increase.

As a result, housing now takes up an increasing portion of household income, especially in urban areas where wage growth has not kept pace with rising costs. This shift makes housing one of the most visible pressure points of the cost-of-living crisis, highlighting the growing gap between people’s earnings and the cost of living.

Energy and food prices triggered the initial inflation shock, but while these costs have begun to stabilise, housing prices have remained persistently high. In most cities, rents continued to climb throughout the past decade, and although some rural areas saw brief pauses or slight declines, the overall trend is upward.

Instead, they gained only 1.3%, a fraction of what would be expected in a stable growth period. The culprit? The years 2020 to 2023. Those three years erased nearly all accumulated gains. By early 2024, real wages had fallen back to their 2016 level, effectively undoing almost a decade of progress.

What makes the recent stagnation particularly striking is that Germany performed well for most of the past two decades. Economists typically consider 1% real wage growth an indication of a balanced, expanding labor market. This suggests that workers should have experienced an increase of 8–10% in their real incomes between 2016 and 2024.

German wages rose 27 percent in eight years. Inflation absorbed 25.6 percent of that increase. As a result, the slight real increase of around 1.3 percent vanished as rents climbed far faster than broader prices. Berlin recorded a 91 percent rise. Leipzig reached 74 percent. Munich added another 53 percent.

A software developer in Berlin earned a net monthly salary of approximately €3,185 in 2016 and could rent about 98 m² of space. Today, that same role pays around €3,771, yet even with the extra income, the rental budget now covers only 61 m². The gain of nearly €600 comes with a loss of about 37 m². Most workers show this pattern across all the cities we reviewed, with space shrinking by around 10 m² in Munich and roughly 37 m² in Berlin.

For years, German media and policymakers pointed to strong nominal wage growth as evidence of a resilient labor market. However, the headline figure of +27% wages tells only half the story. Inflation rose in tandem, driven first by supply chain disruptions, then by the 2022 energy crisis, and finally by broad cost increases across everyday essentials. As a result, nearly all wage growth was offset, leaving workers with just 1.3% real improvement. The chart below reveals this alarming reality of wage stagnation in Germany.

In 2014, only six districts in Germany crossed the critical 30 percent rent burden level. By 2024, the number had grown to 26. Pressure no longer concentrates in major centres. It now spreads across the country.

*The correct sequence of Authors - Heyel Sodhi-Kalra, Shivangi Jha, MBBS, MPH, and Subba R. Digumarthy, MD. This will be reflected in the final Manuscript soon.**

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

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Reply to the reviewers

Authors’ reply (____Ono et al)

Review Commons Refereed Preprint #RC-2025-03137

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

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

We greatly appreciate the reviewer’s supportive comments. The reviewer has accurately recognized our new findings concerning the collaborative roles of condensin II and cohesin in establishing and maintaining interphase chromosome territories.

Major point:

They propose a functional 'handover' from condensin II to cohesin, for the organization of CTs at the M-to-G1 transition. However, the 'handover', i.e. difference in timing of executing their functions, was not experimentally substantiated. Ideally, they can deplete condensin II and cohesin at different times to prove the 'handover'. However, this would require the use of two different degron tags and go beyond the revision of this manuscript. At least, based on the literature, the authors should discuss why they think condensin II and cohesin should work at different timings in the CT organization.

We take this comment seriously, especially because Reviewer #2 also expressed the same concern.　

First of all, we must admit that the basic information underlying the “handover” idea was insufficiently explained in the original manuscript. Let us make it clear below:

• Condensin II bound to chromosomes and is enriched along their axes from anaphase through telophase (Ono et al., 2004; Hirota et al., 2004; Walther et al., 2018).
• In early G1, condensin II is diffusely distributed within the nucleus and does not bind tightly to chromatin, as shown by detergent extraction experiments (Ono et al., 2013).
• Cohesin starts binding to chromatin when the cell nucleus reassembles (i.e., during the cytokinesis stage shown in Fig. 1B), apparently replacing condensins I and II (Brunner et al., 2025).
• Condensin II progressively rebinds to chromatin from S through G2 phase (Ono et al., 2013). The cell cycle-dependent changes in chromosome-bound condensin II and cohesin summarized above are illustrated in Fig. 1A. We now realize that Fig. 1B in the original manuscript was inconsistent with Fig. 1A, creating unnecessary confusion, and we sincerely apologize for this. The fluorescence images shown in the original Fig. 1B were captured without detergent extraction prior to fixation, giving the misleading impression that condensin II remained bound to chromatin from cytokinesis through early G1. This was not our intention. To clarify this, we have repeated the experiment in the presence of detergent extraction and replaced the original Fig. 1B with a revised panel. Figs. 1A and 1B are now more consistent with each other. Accordingly, we have modified the correspsonding sentences as follows:

Although condensin II remains nuclear throughout interphase, its chromatin binding is weak in G1 and becomes robust from S phase through G2 (Ono et al., 2013). Cohesin, in contrast, replaces condensin II in early G1 (Fig. 1 B)(Abramo et al., 2019; Brunner et al., 2025), and establishes topologically associating domains (TADs) in the G1 nucleus (Schwarzer et al., 2017; Wutz et al., 2017)*. *

While there is a loose consensus in the field that condensin II is replaced by cohesin during the M-to-G1 transition, it remains controversial whether there is a short window during which neither condensin II nor cohesin binds to chromatin (Abramo et al., 2019), or whether there is a stage in which the two SMC protein complexes “co-occupy” chromatin (Brunner et al., 2025). Our images shown in the revised Fig. 1B cannot clearly distinguish between these two possibilities.

From a functional point of view, the results of our depletion experiments are more readily explained by the latter possibility. If this is the case, the “interplay” or “cooperation” rather than the “handover” may be a more appropriate term to describe the functional collaboration between condensin II and cohesin during the M-to-G1 transition. For this reason, we have avoided the use of the word “handover” in the revised manuscript. It should be emphasized, however, that given their distinct chromosome-binding kinetics, the cooperation of the two SMC complexes during the M-to-G1 transition is qualitatively different from that observed in G2. Therefore, the central conclusion of the present study remains unchanged.

For example, a sentence in Abstract has been changed as follows:

a functional interplay between condensin II and cohesin during the mitosis-to-G1 transition is critical for establishing chromosome territories (CTs) in the newly assembling nucleus.

While the reviewer suggested one experiment, it is clearly beyond the scope of the current study. It should also be noted that even if such a cell line were available, the proposed application of sequential depletion to cells progressing from mitosis to G1 phase would be technically challenging and unlikely to produce results that could be interpreted with confidence.

Other points:

Figure 2E: It seems that the chromosome length without IAA is shorter in Rad21-aid cells than H2-aid cells or H2-aid Rad21-aid cells. How can this be interpreted? This comment is well taken. A related comment was made by Reviewer #3 (Major comment #2). Given the substantial genetic manipulations applied to establish multiple cell lines used in the present study, it is, strictly speaking, not straightforward to compare the -IAA controls between different cell lines. Such variations are most prominently observed in Fig. 2E, although they can also be observed to lesser extent in other experiments (e.g., Fig. 3E). This issue is inherently associated with all studies using genetically manipulated cell lines and therefore cannot be completely avoided. For this reason, we focus on the differences between -IAA and +IAA within each cell line, rather than comparing the -IAA conditions across different cell lines. In this sense, a sentence in the original manuscript (lines 178-180) was misleading. In the revised manuscript, we have modified the corresponding and subsequent sentence as follows:

Although cohesin depletion had a marginal effect on the distance between the two site-specific probes (Fig.2, C and E), double depletion did not result in a significant change (Fig.2, D and E), consistent with the partial restoration of centromere dispersion (Fig. 1G).

• *

In addition, we have added a section entitled “Limitations of the study” at the end of the Discussion to address technical issues that are inevitably associated with the current approach.

Figure 3: Regarding the CT morphology, could they explain further the difference between 'elongated' and 'cloud-like (expanded)'? Is it possible to quantify the frequency of these morphologies? In the original manuscript, we provided data that quantitatively distinguished between the “elongated” and “cloud-like” phenotypes. Specifically, Fig. 2E shows that the distance between two specific loci (Cen 12 and 12q15) is increased in the elongated phenotype but not in the cloud-like phenotype. In addition, the cloud-like morphology was clearly deviated from circularity, as indicated by the circularity index (Fig. 3F). However, because circularity can also decrease in rod-shaped chromosomes, these datasets alone may not be sufficiently convincing, as the reviewer pointed out. We have now included an additional parameter, the aspect ratio, defined as the ratio of an object’s major axis to its minor axis (new Fig. 3F). While this intuitive parameter was altered upon condensin II depletion and double depletion, again, we acknowledge that it is not sufficient to convincingly distinguish between the elongated and cloud-like phenotypes proposed in the original manuscript. For these reasons, in the revised manuscript, we have toned down our statements regarding the differences in CT morphology between the two conditions. Nonetheless, together with the data from Figs. 1 and 2, it is that the Rabl configuration observed upon condensin II depletion is further exacerbated in the absence of cohesin. Accordingly, we have modified the main text and the cartoon (Fig 3H) to more accurately depict the observations summarized above.

Figure 5: How did they assign C, P and D3 for two chromosomes? The assignment seems obvious in some cases, but not in other cases (e.g. in the image of H2-AID#2 +IAA, two D3s can be connected to two Ps in the other way). They may have avoided line crossing between two C-P-D3 assignments, but can this be justified when the CT might be disorganized e.g. by condensin II depletion? This comment is well taken. As the reviewer suspected, we avoided line crossing between two sets of assignments. Whenever there was ambiguity, such images were excluded from the analysis. Because most chromosome territories derived from two homologous chromosomes are well separated even under the depleted conditions as shown in Fig. 6C, we did not encounter major difficulties in making assignments based on the criteria described above. We therefore remain confident that our conclusion is valid.

That said, we acknowledge that our assignments of the FISH images may not be entirely objective. We have added this point to the “Limitations of the study” section at the end of the Discussion.

Figure 6F: The mean is not indicated on the right-hand side graph, in contrast to other similar graphs. Is this an error? We apologize for having caused this confusion. First, we would like to clarify that the right panel of Fig. 6F should be interpreted together with the left panel, unlike the seemingly similar plots shown in Figs. 6G and 6H. In the left panel of Fig. 6F, the percentages of CTs that contact the nucleolus are shown in grey, whereas those that do not are shown in white. All CTs classified in the “non-contact” population (white) have a value of zero in the right panel, represented by the bars at 0 (i.e., each bar corresponds to a collection of dots having a zero value). In contrast, each CT in the “contact” population (grey) has a unique contact ratio value in the right panel. Because the right panel consists of two distinct groups, we reasoned that placing mean or median bars would not be appropriate. This was why no mean or median bars were shown in in the tight panel (The same is true for Fig. S5 A and B).

That said, for the reviewer’s reference, we have placed median bars in the right panel (see below). In the six cases of H2#2 (-/+IAA), Rad21#2 (-/+IAA), Double#2 (-IAA), and Double#3 (-IAA), the median bars are located at zero (note that in these cases the mean bars [black] completely overlap with the “bars” derived from the data points [blue and magenta]). In the two cases of Double#2 (+IAA) and Double#3 (+IAA), they are placed at values of ~0.15. Statistically significant differences between -IAA and +IAA are observed only in Double#2 and Double#3, as indicated by the P-value shown on the top of the panel. Thus, we are confident in our conclusion that CTs undergo severe deformation in the absence of both condensin II and cohesin.

Figure S1A: The two FACS profiles for Double-AID #3 Release-2 may be mixed up between -IAA and +IAA. The review is right. This inadvertent error has been corrected.

The method section explains that 'circularity' shows 'how closely the shape of an object approximates a perfect circle (with a value of 1 indicating a perfect circle), calculated from the segmented regions'. It would be helpful to provide further methodological details about it. We have added further explanations regarding the circularity in Materials and Methods together with a citation (two added sentences are underlined below):

To analyze the morphology of nuclei, CTs, and nucleoli, we measured “circularity,” a morphological index that quantifies how closely the shape of an object approximates a perfect circle (value =1). Circularity was defined as 4π x Area/Perimeter2, where both the area and perimeter of each segmented object were obtained using ImageJ. This index ranges from 0 to 1, with values closer to 1 representing more circular objects and lower values correspond to elongated or irregular shapes (Chen et al, 2017).

Chen, B., Y. Wang, S. Berretta and O. Ghita. 2017. Poly Aryl Ether Ketones (PAEKs) and carbon-reinforced PAEK powders for laser sintering. J Mater Sci 52:6004-6019.

Reviewer #1 (Significance (Required)):

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

See our reply above.

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

Summary:

Ono et al use a variety of imaging and genetic (AID) depletion approaches to examine the roles of condensin II and cohesin in the reformation of interphase genome architecture in human HCT16 cells. Consistent with previous literature, they find that condensin II is required for CENP-A dispersion in late mitosis/early G1. Using in situ FISH at the centromere/q arm of chromosome 12 they then establish that condensin II removal causes lengthwise elongation of chromosomes that, interestingly, can be suppressed by cohesin removal. To better understand changes in whole-chromosome morphology, they then use whole chromosome painting to examine chromosomes 18 and 19. In the absence of condensin II, cells effectively fail to reorganise their chromosomes from rod-like structures into spherical chromosome territories (which may explain why CENP-A dispersion is suppressed). Cohesin is not required for spherical CT formation, suggesting condensin II is the major initial driver of interphase genome structure. Double depletion results in complete disorganisation of chromatin, leading the authors to conclude that a typical cell cycle requires orderly 'handover' from the mitotic to interphase genome organising machinery. The authors then move on to G2 phase, where they use a variety of different FISH probes to assess alterations in chromosome structure at different scales. They thereby establish that perturbation of cohesin or condensin II influences local and longer range chromosome structure, respectively. The effects of condensin II depletion become apparent at a genomic distance of 20 Mb, but are negligible either below or above. The authors repeat the G1 depletion experiment in G2 and now find that condensin II and cohesin are individually dispensable for CT organisation, but that dual depletion causes CT collapse. This rather implies that there is cooperation rather than handover per se. Overall this study is a broadly informative multiscale investigation of the roles of SMC complexes in organising the genome of postmitotic cells, and solidifies a potential relationship between condensin II and cohesin in coordinating interphase genome structure. The deeper investigation of the roles of condensin II in establishing chromosome territories and intermediate range chromosome structure in particular is a valuable and important contribution, especially given our incomplete understanding of what functions this complex performs during interphase.

We sincerely appreciate the reviewer’s supportive comments. The reviewer has correctly acknowledged both the current gaps in our understanding of the role of condensin II in interphase chromosome organization and our new findings on the collaborative roles of condensin II and cohesin in establishing and maintaining interphase chromosome territories.

Major comments:

In general the claims and conclusions of the manuscript are well supported by multiscale FISH labelling. An important absent control is western blotting to confirm protein depletion levels. Currently only fluorescence is used as a readout for the efficiency of the AID depletion, and we know from prior literature that even small residual quantities of SMC complexes are quite effective in organising chromatin. I would consider a western blot a fairly straightforward and important technical control.

Let me explain why we used immunofluorescence measurements to evaluate the efficiency of depletion. In our current protocol for synchronizing at the M-to-G1 transition, ~60% of control and H2-depleted cells, and ~30% of Rad21-depleted and co-depleted cells, are successfully synchronized in G1 phase. The apparently lower synchronization efficiency in the latter two groups is attributable to the well-documented mitotic delay caused by cohesin depletion. From these synchronized populations, early G1 cells were selected based on their characteristic morphologies (see the legend of Fig. 1C). In this way, we analyzed an early G1 cell population that had completed mitosis without chromosome segregation defects. We acknowledge that this represents a technically challenging aspect of M-to-G1 synchronization in HCT116 cells, whose synchronization efficiency is limited compared with that of HeLa cells. Nevertheless, this approach constitutes the most practical strategy currently available. Hence, immunofluorescence provides the only feasible means to evaluate depletion efficiency under these conditions.

Although immunoblotting can, in principle, be applied to G2-arrested cell populations, we do not believe that information obtained from such experiments would affect the main conclusions of the current study. Please note that we carefully designed and performed all experiments with appropriate controls: H2 depletion, RAD21 depletion, and double depletion, with outcomes confirmed using independent cell lines (Double-AID#2 and Double-AID#3) whenever deemed necessary.

We fully acknowledge the technical limitations associated with the AID-mediated depletion techniques, which are now described in the section entitled “Limitations of the study” at the end of the Discussion. Nevertheless, we emphasize that these limitations do not compromise the validity of our findings.

I find the point on handover as a mechanism for maintaining CT architecture somewhat ambiguous, because the authors find that the dependence simply switches from condensin II to both condensin II and cohesin, between G1 and G2. To me this implies augmented cooperation rather than handover. I have two further suggestions, both of which I would strongly recommend but would consider desirable but 'optional' according to review commons guidelines.

First of all, we would like to clarify a possible misunderstanding regarding the phrase “handover as a mechanism for maintaining CT architecture somewhat ambiguous”. In the original manuscript, we proposed handover as a mechanism for establishing G1 chromosome territories, not for maintaining CTs.

That said, we take this comment very seriously, especially because Reviewer #1 also expressed the same concern. Please see our reply to Reviewer #1 (Major point).

In brief, we agree with the reviewer that the word “handover” may not be appropriate to describe the functional relationship between condensin II and cohesin during the M-to-G1 transition. In the revised manuscript, we have avoided the use of the word “handover”, replacing it with “interplay”. It should be emphasized, however, that given their distinct chromosome-binding kinetics, the cooperation of the two SMC complexes during the M-to-G1 transition is qualitatively different from that observed in G2. Therefore, the central conclusion of the present study remains unchanged.

For example, a sentence in Abstract has been changed as follows:

a functional interplay between condensin II and cohesin during the mitosis-to-G1 transition is critical for establishing chromosome territories (CTs) in the newly assembling nucleus.

Firstly, the depletions are performed at different stages of the cell cycle but have different outcomes. The authors suggest this is because handover is already complete, but an alternative possibility is that the phenotype is masked by other changes in chromosome structure (e.g. duplication/catenation). I would be very curious to see, for example, how the outcome of this experiment would change if the authors were to repeat the depletions in the presence of a topoisomerase II inhibitor.

The reviewer’s suggestion here is somewhat vague, and it is unclear to us what rationale underlies the proposed experiment or what meaningful outcomes could be anticipated. Does the reviewer suggest that we perform topo II inhibitor experiments both during the M-to-G1 transition and in G2 phase, and then compare the outcomes between the two conditions?

For the M-to-G1 transition, Hildebrand et at (2024) have already reported such experiments. They used a topo II inhibitor to provided evidence that mitotic chromatids are self-entangled and that the removal of these mitotic entanglements is required to establish a normal interphase nucleus. Our own preliminary experiments (not presented in the current manuscript) showed that ICRF treatment of cells undergoing the M-to-G1 transition did not affect post-mitotic centromere dispersion. The same treatment also had little effect on the suppression of centromere dispersion observed in condensin II-depleted cells.

Under G2-arrested condition, because chromosome territories are largely individualized, we would expect topo II inhibition to affect only the extent of sister catenation, which is not the focus of our current study. We anticipate that inhibiting topo II in G2 would have only a marginal, if any, effect on the maintenance of chromosome territories detectable by our current FISH approaches.

In any case, we consider the suggested experiment to be beyond the scope of the present manuscript, which focuses on the collaborative roles of condensin II and cohesin as revealed by multi-scale FISH analyses.

Secondly, if the author's claim of handover is correct then one (not exclusive) possibility is that there is a relationship between condensin II and cohesin loading onto chromatin. There does seem to be a modest co-dependence (e.g. fig S4 and S7), could the authors comment on this?

First of all, we wish to point out the reviewer’s confusion between the G2 experiments and the M-to-G1 experiments. Figs. S4 and S7 concern experiments using G2-arrested cells, not M-to-G1 cells in which a possible handover mechanism is discussed. Based on Fig. 1, in which the extent of depletion in M-to-G1 cells was tested, no evidence of “co-dependence” between H2 depletion and RAD21 depletion was observed.

That said, as the reviewer correctly points out, we acknowledge the presence of marginal yet statistically significant reductions in the RAD21 signal upon H2 depletion (and vice versa) in G2-arrested cells (Figs. S4 and S7).

Another control experiment here would be to treat fully WT cells with IAA and test whether non-AID labelled H2 or RAD21 dip in intensity. If they do not, then perhaps there's a causal relationship between condensin II and cohesin levels?

According to the reviewer’s suggestion, we tested whether IAA treatment causes an unintentional decreases in the H2 or RAD21 signals in G2-arrested cells, and found that it is not the case (see the attached figure below).

Thus, these data indicate that there is a modest functional interdependence between condensin II and cohesin in G2-arrested cells. For instance, condensin II depletion may modestly destabilize chromatin-bound cohesin (and vice versa). However, we note that these effects are minor and do not affect the overall conclusions of the study. In the revised manuscript, we have described these potentially interesting observations briefly as a note in the corresponding figure legends (Fig. S4).

I recognise this is something considered in Brunner et al 2025 (JCB), but in their case they depleted SMC4 (so all condensins are lost or at least dismantled). Might bear further investigation.

Methods:

Data and methods are described in reasonable detail, and a decent number of replicates/statistical analyses have been. Documentation of the cell lines used could be improved. The actual cell line is not mentioned once in the manuscript. Although it is referenced, I'd recommend including the identity of the cell line (HCT116) in the main text when the cells are introduced and also in the relevant supplementary tables. Will make it easier for readers to contextualise the findings.

We apologize for the omission of important information regarding the parental cell line used in the current study. The information has been added to Materials and Methods as well as the resource table.

Minor comments:

Overall the manuscript is well-written and well presented. In the introduction it is suggested that no experiment has established a causal relationship between human condensin II and chromosome territories, but this is not correct, Hoencamp et al 2021 (cell) observed loss of CTs after condensin II depletion. Although that manuscript did not investigate it in as much detail as the present study, the fundamental relationship was previously established, so I would encourage the authors to revise this statement.

We are somewhat puzzled by this comment. In the original manuscript, we explicitly cited Hoencamp et al (2021) in support of the following sentences:

• *

(Lines 78-83 in the original manuscript)

*Moreover, high-throughput chromosome conformation capture (Hi-C) analysis revealed that, under such conditions, chromosomes retain a parallel arrangement of their arms, reminiscent of the so-called Rabl configuration (Hoencamp et al., 2021). These findings indicate that the loss or impairment of condensin II during mitosis results in defects in post-mitotic chromosome organization. *

• *

That said, to make the sentences even more precise, we have made the following revision in the manuscript.

• *

(Lines 78- 82 in the revised manuscript)

*Moreover, high-throughput chromosome conformation capture (Hi-C) analysis revealed that, under such conditions, chromosomes retain a parallel arrangement of their arms, reminiscent of the so-called Rabl configuration (Hoencamp et al., 2021). These findings,together with cytological analyses of centromere distributions, indicate that the loss or impairment of condensin II during mitosis results in defects in post-mitotic chromosome organization. *

• *

The following statement was intended to explain our current understanding of the maintenance of chromosome territories. Because Hoencamp et al (2021) did not address the maintenance of CTs, we have kept this sentence unchanged.

• *

(Lines 100-102 in the original manuscript)

Despite these findings, there is currently no evidence that either condensin II, cohesin, or their combined action contributes to the maintenance of CT morphology in mammalian interphase cells (Cremer et al., 2020).

• *

• *

Reviewer #2 (Significance (Required)):

General assessment:

Strengths: the multiscale investigation of genome architecture at different stages of interphase allow the authors to present convincing and well-analysed data that provide meaningful insight into local and global chromosome organisation across different scales.

Limitations:

As suggested in major comments.

Advance:

Although the role of condensin II in generating chromosome territories, and the roles of cohesin in interphase genome architecture are established, the interplay of the complexes and the stage specific roles of condensin II have not been investigated in human cells to the level presented here. This study provides meaningful new insight in particular into the role of condensin II in global genome organisation during interphase, which is much less well understood compared to its participation in mitosis.

Audience:

Will contribute meaningfully and be of interest to the general community of researchers investigating genome organisation and function at all stages of the cell cycle. Primary audience will be cell biologists, geneticists and structural biochemists. Importance of genome organisation in cell/organismal biology is such that within this grouping it will probably be of general interest.

My expertise is in genome organization by SMCs and chromosome segregation.

We appreciate the reviewer’s supportive comments. As the reviewer fully acknowledges, this study is the first systematic survey of the collaborative role of condensin II and cohesin in establishing and maintaining interphase chromosome territories. In particular, multi-scale FISH analyses have enabled us to clarify how the two SMC protein complexes contribute to the maintenance of G2 chromosome territories through their actions at different genomic scales. As the reviewer notes, we believe that the current study will appeal to a broad readership in cell and chromosome biology. The limitations of the current study mentioned by the reviewer are addressed in our reply above.

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

Summary:

The manuscript “Condensin II collaborates with cohesin to establish and maintain interphase chromosome territories" investigates how condensin II and cohesin contribute to chromosome organization during the M-to-G1 transition and in G2 phase using published auxin-inducible degron (AID) cell lines which render the respective protein complexes nonfunctional after auxin addition. In this study, a novel degron cell line was established that enables the simultaneous depletion of both protein complexes, thereby facilitating the investigation of synergistic effects between the two SMC proteins. The chromosome architecture is studied using fluorescence in situ hybridization (FISH) and light microscopy. The authors reproduce a number of already published data and also show that double depletion causes during the M-to-G1 transition defects on chromosome territories, producing expanded, irregular shapes that obscure condensin II-specific phenotypes. Findings in G2 cells point to a new role of condensin II for chromosome conformation at a scale of ~20Mb. Although individual depletion has minimal effects on large-scale CT morphology in G2, combined loss of both complexes produces marked structural abnormalities, including irregular crescent-shaped CTs displaced toward the nucleolus and increased nucleolus-CT contact. The authors propose that condensin II and cohesin act sequentially and complementarily to ensure proper post-mitotic CT formation and maintain chromosome architecture across genomic scales.

We greatly appreciate the reviewer’s supportive comments. The reviewer has accurately recognized our new findings concerning the collaborative roles of condensin II and cohesin in the establishment and maintenance of interphase chromosome territories.

Concenrs about statistics:

• The authors provide the information on how many cells are analyzed but not the number of independent experiments. My concern is that there might variations in synchronization of the cell population and in the subsequent preparation (FISH) affecting the final result. We appreciate the reviewer’s important comment regarding the biological reproducibility of our experiments. As the reviewer correctly points out, variations in cell-cycle synchronization and FISH sample preparation can occur across experiments. To address this concern, we repeated the key experiments supporting our main conclusions (Figs. 3 and 6) two additional times, resulting in three independent biological replicas in total. All replicate experiments reproduced the major observations from the original analyses. These results further substantiated our original conclusion, despite the inevitable variability arising from cell synchronization or sample preparation in this type of experiments. In the revised manuscript, we have now explicitly indicated the number of biological replicates in the corresponding figures.

The analyses of chromosome-arm conformation shown in Fig. 5 were already performed in three independent rounds of experiments, as noted in the original submission. In addition, similar results were already obtained in other analyses reported in the manuscript. For example, centromere dispersion was quantified using an alternative centromere detection method (related to Fig. 1), and distances between specific chromosomal sites were measured using different locus-specific probes (related to Figs. 2 and 4). In both cases, the results were consistent with those presented in the manuscript.

• Statistically the authors analyze the effect of cells with induced degron vs. vehicle control (non-induced). However, the biologically relevant question is whether the data differ between cell lines when the degron system is induced. This is not tested here (cf. major concern 2 and 3). See our reply to major concerns 2 and 3.

• Some Journal ask for blinded analysis of the data which might make sense here as manual steps are involved in the data analysis (e.g. line 626 / 627the convex hull of the signals was manually delineated, line 635 / 636 Chromosome segmentation in FISH images was performed using individual thresholding). However personally I have no doubts on the correctness of the work. We thank the reviewer for pointing out that some steps in our data analysis were performed manually, such as delineating the convex hull of signals and segmenting chromosomes in FISH and IF images using individual thresholds. These manual steps were necessary because signal intensities vary among cells and chromosomes, making fully automated segmentation unreliable. To ensure objectivity, we confirmed that the results were consistent across two independently established double-depletion cell lines, which produced essentially identical findings. In addition, we repeated the key experiments underpinning our main conclusions (Figs. 3 and 6) two additional times, and the results were fully consistent with the original analyses. Therefore, we are confident that our current data analysis approach does not compromise the validity of our conclusions. Finally, we appreciate the reviewer’s kind remark that there is no doubt regarding the correctness of our work.

Major concerns:

• Degron induction appears to delay in Rad21-AID#1 and Double-AID#1 cells the transition from M to G1, as shown in Fig. S1. After auxin treatment, more cells exhibit a G2 phenotype than in an untreated population. What are the implications of this for the interpretation of the experiments? In our protocol shown in Fig. 1C, cells were released into mitosis after G2 arrest, and IAA was added 30 min after release. It is well established that cohesin depletion causes a prometaphase delay due to spindle checkpoint activation (e.g., Vass et al, 2003, Curr Biol; Toyoda and Yanagida, 2006, MBoC; Peters et al, 2008, Genes Dev), which explains why cells with 4C DNA content accumulated, as judged by FACS (Fig. S1). The same was true for doubly depleted cells. However, a fraction of cells that escaped this delay progressed through mitosis and enter the G1 phase of the next cell cycle. We selected these early G1 cells and used them for down-stream analyses. This experimental procedure was explicitly described in the legends of Fig. 1C and Fig. S1A as follows:

(Lines 934-937; Legend of Fig. 1C)

From the synchronized populations, early G1cells were selected based on their characteristic morphologies (i.e., pairs of small post-mitotic cells) and subjected to downstream analyses. Based on the measured nuclear sizes (Fig. S2 G), we confirmed that early G1 cells were appropriately selected.

(Lines 1114-1119; Legend of Fig. S1A)

In this protocol, ~60% of control and H2-depleted cells, and ~30% of Rad21-depleted and co-depleted cells, were successfully synchronized in G1 phase. The apparently lower synchronization efficiency in the latter two groups is attributable to the well documented mitotic delay caused by cohesin depletion (Hauf et al., 2005; Haarhuis et al., 2013; Perea-Resa et al., 2020). From these synchronized populations, early G1 cells were selected based on their characteristic morphologies (see the legend of Fig. 1 C).

• *

Thus, using this protocol, we analyzed an early G1 cell population that had completed mitosis without chromosome segregation defects. We acknowledge that this represents a technically challenging aspect of synchronizing cell-cycle progression from M to G1 in HCT116 cells, whose synchronization efficiency is limited compared with that of HeLa cells. Nevertheless, this approach constitutes the most practical strategy currently available.

• Line 178 "In contrast, cohesin depletion had a smaller effect on the distance between the two site-specific probes compared to condensin II depletion (Fig. 2, C and E)." The data in Fig. 2 E show both a significant effect of H2 and a significant effect of RAD21 depletion. Whether the absolute difference in effect size between the two conditions is truly relevant is difficult to determine, as the distribution of the respective control groups also appears to be different. This comment is well taken. Reviewer #1 has made a comment on the same issue. See our reply to Reviewer #1 (Other points, Figure 2E).

In brief, in the current study, we should focus on the differences between -IAA and +IAA within each cell line, rather than comparing the -IAA conditions across different cell lines. In this sense, a sentence in the original manuscript (lines 178-180) was misleading. In the revised manuscript, we have modified the corresponding and subsequent sentence as follows:

Although cohesin depletion had a marginal effect on the distance between the two site-specific probes (Fig.2, C and E), double depletion did not result in a significant change (Fig.2, D and E), consistent with the partial restoration of centromere dispersion (Fig. 1G).

• In Figures 3, S3 and related text in the manuscript I cannot follow the authors' argumentation, as H2 depletion alone leads to a significant increase in the CT area (Chr. 18, Chr. 19, Chr. 15). Similar to Fig. 2, the authors argue about the different magnitude of the effect (H2 depletion vs double depletion). Here, too, appropriate statistical tests or more suitable parameters describing the effect should be used. I also cannot fully follow the argumentation regarding chromosome elongation, as double depletion in Chr. 18 and Chr. 19 also leads to a significantly reduced circularity. Therefore, the schematic drawing Fig. 3 H (double depletion) seems very suggestive to me. This comment is related to the comment above (Major comment #2). See our reply to Reviewer #1 (Other points, Figure 2E).

It should be noted that, in Figure 3 (unlike in Figure 2), we did not compare the different magnitudes of the effect observed between H2 depletion and double depletion. Thus, the reviewer’s comment that “Similar to Fig. 2, the authors argue about the different magnitude of the effect (H2 depletion vs double depletion) ” does not accurately reflected our description.

Moreover, while the distance between two specific loci (Fig. 2E) and CT circularity (Fig. 3G) are intuitively related, they represent distinct parameters. Thus, it is not unexpected that double depletion resulted in apparently different outcomes for the two measurements. Thus, the reviewer’s counter-argument is not strictly applicable here.

That said, we agree with the reviewer that our descriptions here need to be clarified.

The differences between H2 depletion and double depletion are two-fold: (1) centromere dispersion is suppressed upon H2 depletion, but not upon double depletion (Fig 1G); (2) the distance between Cen 12 and 12q15 increased upon H2 depletion, but not upon double depletion (Fig 2E).

We have decided to remove the “homologous pair overlap” panel (formerly Fig. 3E) from the revised manuscript. Accordingly, the corresponding sentence has been deleted from the main text. Instead, we have added a new panel of “aspect ratio”, defined as the ratio of the major to the minor axis (new Fig. 3F). While this intuitive parameter was altered upon condensin II depletion and double depletion, again, we acknowledge that it is not sufficient to convincingly distinguish between the elongated and cloud-like phenotypes proposed in the original manuscript. For these reasons, in the revised manuscript, we have toned down our statements regarding the differences in CT morphology between the two conditions. Nonetheless, together with the data from Figs. 1 and 2, it is clear that the Rabl configuration observed upon condensin II depletion is further exacerbated in the absence of cohesin. Accordingly, we have modified the main text and the cartoon (Fig 3H) to more accurately depict the observations summarized above.

• 5 and accompanying text. I agree with the authors that this is a significant and very interesting effect. However, I believe the sharp bends is in most cases an artifact caused by the maximum intensity projection. I tried to illustrate this effect in two photographs: Reviewer Fig. 1, side view, and Reviewer Fig. 2, same situation top view (https://cloud.bio.lmu.de/index.php/s/77npeEK84towzJZ). As I said, in my opinion, there is a significant and important effect; the authors should simply adjust the description. This comment is well taken. We appreciate the reviewer’s effort to help clarify our original observations. We have therefore added a new section entitled “Limitations of the study” to explicitly describe the constrains of our current approach. That said, as the reviewer also acknowledges, our observations remain valid because all experiments were performed with appropriate controls.

Minor concerns:

• I would like to suggest proactively discussing possible artifacts that may arise from the harsh conditions during FISH sample preparation. We fully agree with the reviewer’s concerns. For FISH sample preparation, we used relatively harsh conditions, including (1) fixation under a hypotonic condition (0.3x PBS), (2) HCl treatment, and (3) a denaturation step. We recognize that these procedures inevitably affect the preservation of the original structure; however, they are unavoidable in the standard FISH protocol. We also acknowledge that our analyses were limited to 2D structures based on projected images, rather than full 3D reconstructions. These technical limitations are now explicitly described in a new section entitled “Limitations of the study”, and the technical details are provided in Materials and Methods.

• It would be helpful if the authors could provide the original data (microscopic image stacks) for download. We thank the reviewer for this suggestion and understand that providing the original image stacks could be of interest to readers. We agree that if the nuclei were perfectly spherical, as is the case for example in lymphocytes, 3D image stacks would contain much more information than 2D projections. However, as is typical for adherent cultured cells, including the HCT116-derived cells used in this study, the nuclei are flattened due to cell adhesion to the culture dish, with a thickness of only about one-tenth of the nuclear diameter (10–20 μm). Considering also the inevitable loss of structural preservation during FISH sample preparation, we were concerned that presenting 3D images might confuse rather than clarify. We therefore believe that representing the data as 2D projections, while explicitly acknowledging the technical limitations, provides the clearest and most interpretable presentation of our results. These limitations are now described in a new section of the manuscript.

• The authors use a blind deconvolution algorithm to improve image quality. It might be helpful to test other methods for this purpose (optional). We thank the reviewer for this valuable suggestion and fully agree that it is a valid point. We recognize that alternative image enhancement methods can offer advantages, particularly for smaller structures or when multiple probes are analyzed simultaneously. In our study, however, the focus was on detecting whole chromosome territories (CTs) and specific chromosomal loci, which can be visualized clearly with our current FISH protocol combined with blind deconvolution. We therefore believe that the image quality we obtained is sufficient to support the conclusions of this manuscript.

Reviewer #3 (Significance (Required)):

Advance:

Ono et al. addresses the important question on how the complex pattern of chromatin is reestablished after mitosis and maintained during interphase. In addition to affinity interactions (1,2), it is known that cohesin plays an important role in the formation and maintenance of chromosome organization interphase (3). However, current knowledge does not explain all known phenomena. Even with complete loss of cohesin, TAD-like structures can be recognized at the single-cell level (4), and higher structures such as chromosome territories are also retained (5). The function of condensin II during mitosis is another important factor that affects chromosome architecture in the following G1 phase (6). Although condensin II is present in the cell nucleus throughout interphase, very little is known about the role of this protein in this phase of the cell cycle. This is where the present publication comes in, with a new double degron cell line in which essential subunits of cohesin AND condensin can be degraded in a targeted manner. I find the data from the experiments in the G2 phase most interesting, as they suggest a previously unknown involvement of condensin II in the maintenance of larger chromatin structures such as chromosome territories.

The experiments regarding the M-G1 transition are less interesting to me, as it is known that condensin II deficiency in mitosis leads to elongated chromosomes (Rabl configuration)(6), and therefore the double degradation of condensin II and cohesin describes the effects of cohesin on an artificially disturbed chromosome structure.

For further clarification, we provide below a table summarizing previous studies relevant to the present work. We wish to emphasize three novel aspects of the present study. First, newly established cell lines designed for double depletion enabled us to address questions that had remained inaccessible in earlier studies. Second, to our knowledge, no study has previously reported condensin II depletion, cohesin depletion and double depletion in G2-arrested cells. Third, the present study represents the first systematic comparison of two different stages of the cell cycle using multiscale FISH under distinct depletion conditions. Although the M-to-G1 part of the present study partially overlaps with previous work, it serves as an important prelude to the subsequent investigations. We are confident that the reviewer will also acknowledge this point.

cell cycle

cond II depletion

cohesin depletion

double depletion

M-to-G1

Hoencamp et al (2021); Abramo et al (2019); Brunner et al (2025);

this study

Schwarzer et al (2017);

Wutz et al (2017);

this study

this study

G2

this study

this study

this study

Hoencamp et al (2021): Hi-C and imaging (CENP-A distribution)

Abramo et al (2019): Hi-C and imaging

Brunner et al (2025): mostly imaging (chromatin tracing)

Schwarzer et al (2017); Wutz et al (2017): Hi-C

this study: imaging (multi-scale FISH)

General limitations:

(1) Single cell imaging of chromatin structure typically shows only minor effects which are often obscured by the high (biological) variability. This holds also true for the current manuscript (cf. major concern 2 and 3).

See our reply above.

(2) A common concern are artefacts introduced by the harsh conditions of conventional FISH protocols (7). The authors use a method in which the cells are completely dehydrated, which probably leads to shrinking artifacts. However, differences between samples stained using the same FISH protocol are most likely due to experimental variation and not an artefact (cf. minor concern 1).

See our reply above.

• The anisotropic optical resolution (x-, y- vs. z-) of widefield microscopy (and most other light microscopic techniques) might lead to misinterpretation of the imaged 3D structures. This seems to be the cases in the current study (cf. major concern 4). See our reply above.

• In the present study, the cell cycle was synchronized. This requires the use of inhibitors such as the CDK1 inhibitor RO-3306. However, CDK1 has many very different functions (8), so unexpected effects on the experiments cannot be ruled out. The current approaches involving FISH inevitably require cell cycle synchronization. We believe that the use of the CDK1 inhibitor RO-3306 to arrest the cell cycle at G2 is a reasonable choice, although we cannot rule out unexpected effects arising from the use of the drug. This issue has now been addressed in the new section entitled “Limitations of the study”.

Audience:

The spatial arrangement of genomic elements in the nucleus and their (temporal) dynamics are of high general relevance, as they are important for answering fundamental questions, for example, in epigenetics or tumor biology (9,10). The manuscript from Ono et al. addresses specific questions, so its intended readership is more likely to be specialists in the field.

We are confident that, given the increasing interest in the 3D genome and its role in regulating diverse biological functions, the current manuscript will attract the broad readership of leading journals in cell biology.

About the reviewer:

By training I'm a biologist with strong background in fluorescence microscopy and fluorescence in situ hybridization. In recent years, I have been involved in research on the 3D organization of the cell nucleus, chromatin organization, and promoter-enhancer interactions.

We greatly appreciate the reviewer’s constructive comments on both the technical strengths and limitations of our fluorescence imaging approaches, which have been very helpful in revising the manuscript. As mentioned above, we have decided to add a special paragraph entitled “Limitations of the study” at the end of the Discussion section to discuss these issues.

All questions regarding the statistics of angularly distributed data are beyond my expertise. The authors do not correct their statistical analyses for "multiple testing". Whether this is necessary, I cannot judge.

We thank the reviewer for raising this important point. In our study, the primary comparisons were made between -IAA and +IAA conditions within the same cell line. Accordingly, the figures report P-values for these pairwise comparisons.

For the distance measurements, statistical evaluations were performed in PRISM using ANOVA (Kruskal–Wallis test), and the P-values shown in the figures are based on these analyses (Fig. 1, G and H; Fig. 2 E; Fig. 3 F and G; Fig. 4 F; Fig. 6 F [right]–H; Fig. S2 B and G; Fig. S3 D and H; Fig. S5 A [right] and B [right]; Fig. S8 B). While the manuscript focuses on pairwise comparisons between -IAA and +IAA conditions within the same cell line, we also considered potential differences across cell lines as part of the same ANOVA framework, thereby ensuring that multiple testing was properly addressed. Because cell line differences are not the focus of the present study, the corresponding results are not shown.

For the angular distribution analyses, we compared -IAA and +IAA conditions within the same cell line using the Mardia–Watson–Wheeler test; these analyses do not involve multiple testing (circular scatter plots; Fig. 5 C–E and Fig. S6 B, C, and E–H). In addition, to determine whether angular distributions exhibited directional bias under each condition, we applied the Rayleigh test to each dataset individually (Fig. 5 F and Fig. S6 I). As these tests were performed on a single condition, they are also not subject to the problem of multiple testing. Collectively, we consider that the statistical analyses presented in our manuscript appropriately account for potential multiple testing issues, and we remain confident in the robustness of the results.

Literature

Falk, M., Feodorova, Y., Naumova, N., Imakaev, M., Lajoie, B.R., Leonhardt, H., Joffe, B., Dekker, J., Fudenberg, G., Solovei, I. et al. (2019) Heterochromatin drives compartmentalization of inverted and conventional nuclei. Nature, 570, 395-399. Mirny, L.A., Imakaev, M. and Abdennur, N. (2019) Two major mechanisms of chromosome organization. Curr Opin Cell Biol, 58, 142-152. Rao, S.S.P., Huang, S.C., Glenn St Hilaire, B., Engreitz, J.M., Perez, E.M., Kieffer-Kwon, K.R., Sanborn, A.L., Johnstone, S.E., Bascom, G.D., Bochkov, I.D. et al. (2017) Cohesin Loss Eliminates All Loop Domains. Cell, 171, 305-320 e324. Bintu, B., Mateo, L.J., Su, J.H., Sinnott-Armstrong, N.A., Parker, M., Kinrot, S., Yamaya, K., Boettiger, A.N. and Zhuang, X. (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science, 362. Cremer, M., Brandstetter, K., Maiser, A., Rao, S.S.P., Schmid, V.J., Guirao-Ortiz, M., Mitra, N., Mamberti, S., Klein, K.N., Gilbert, D.M. et al. (2020) Cohesin depleted cells rebuild functional nuclear compartments after endomitosis. Nat Commun, 11, 6146. Hoencamp, C., Dudchenko, O., Elbatsh, A.M.O., Brahmachari, S., Raaijmakers, J.A., van Schaik, T., Sedeno Cacciatore, A., Contessoto, V.G., van Heesbeen, R., van den Broek, B. et al. (2021) 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science, 372, 984-989. Beckwith, K.S., Ødegård-Fougner, Ø., Morero, N.R., Barton, C., Schueder, F., Tang, W., Alexander, S., Peters, J.-M., Jungmann, R., Birney, E. et al. (2023) Nanoscale 3D DNA tracing in single human cells visualizes loop extrusion directly in situ. BioRxiv 8 of 9https://doi.org/10.1101/2021.04.12.439407. Massacci, G., Perfetto, L. and Sacco, F. (2023) The Cyclin-dependent kinase 1: more than a cell cycle regulator. Br J Cancer, 129, 1707-1716. Bonev, B. and Cavalli, G. (2016) Organization and function of the 3D genome. Nat Rev Genet, 17, 661-678. Dekker, J., Belmont, A.S., Guttman, M., Leshyk, V.O., Lis, J.T., Lomvardas, S., Mirny, L.A., O'Shea, C.C., Park, P.J., Ren, B. et al. (2017) The 4D nucleome project. Nature, 549, 219-226.

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Referee #3

Evidence, reproducibility and clarity

Summary:

The manuscript „Condensin II collaborates with cohesin to establish and maintain interphase chromosome territories" investigates how condensin II and cohesin contribute to chromosome organization during the M-to-G1 transition and in G2 phase using published auxin-inducible degron (AID) cell lines which render the respective protein complexes nonfunctional after auxin addition. In this study, a novel degron cell line was established that enables the simultaneous depletion of both protein complexes, thereby facilitating the investigation of synergistic effects between the two SMC proteins. The chromosome architecture is studied using fluorescence in situ hybridization (FISH) and light microscopy. The authors reproduce a number of already published data and also show that double depletion causes during the M-to-G1 transition defects on chromosome territories, producing expanded, irregular shapes that obscure condensin II-specific phenotypes. Findings in G2 cells point to a new role of condensin II for chromosome conformation at a scale of ~20Mb. Although individual depletion has minimal effects on large-scale CT morphology in G2, combined loss of both complexes produces marked structural abnormalities, including irregular crescent-shaped CTs displaced toward the nucleolus and increased nucleolus-CT contact. The authors propose that condensin II and cohesin act sequentially and complementarily to ensure proper post-mitotic CT formation and maintain chromosome architecture across genomic scales.

Concerns about statistics:

(1) The authors provide the information on how many cells are analyzed but not the number of independent experiments. My concern is that there might variations in synchronization of the cell population and in the subsequent preparation (FISH) affecting the final result.

(2) Statistically the authors analyze the effect of cells with induced degron vs. vehicle control (non-induced). However, the biologically relevant question is whether the data differ between cell lines when the degron system is induced. This is not tested here (cf. major concern 2 and 3).

(3) Some Journal ask for blinded analysis of the data which might make sense here as manual steps are involved in the data analysis (e.g. line 626 / 627the convex hull of the signals was manually delineated, line 635 / 636 Chromosome segmentation in FISH images was performed using individual thresholding). However personally I have no doubts on the correctness of the work.

Major concerns:

(1) Degron induction appears to delay in Rad21-AID#1 an Double-AID#1 cells the transition from M to G1, as shown in Fig. S1. After auxin treatment, more cells exhibit a G2 phenotype than in an untreated population. What are the implications of this for the interpretation of the experiments?

(2) Line 178 "In contrast, cohesin depletion had a smaller effect on the distance between the two site-specific probes compared to condensin II depletion (Fig. 2, C and E)." The data in Fig. 2 E show both a significant effect of H2 and a significant effect of RAD21 depletion. Whether the absolute difference in effect size between the two conditions is truly relevant is difficult to determine, as the distribution of the respective control groups also appears to be different.

(3) In Figures 3, S3 and related text in the manuscript I cannot follow the authors' argumentation, as H2 depletion alone leads to a significant increase in the CT area (Chr. 18, Chr. 19, Chr. 15). Similar to Fig. 2, the authors argue about the different magnitude of the effect (H2 depletion vs double depletion). Here, too, appropriate statistical tests or more suitable parameters describing the effect should be used. I also cannot fully follow the argumentation regarding chromosome elongation, as double depletion in Chr. 18 and Chr. 19 also leads to a significantly reduced circularity. Therefore, the schematic drawing Fig. 3 H (double depletion) seems very suggestive to me.

(4) Fig. 5 and accompanying text. I agree with the authors that this is a significant and very interesting effect. However, I believe the sharp bends is in most cases an artifact caused by the maximum intensity projection. I tried to illustrate this effect in two photographs: Reviewer Fig. 1, side view, and Reviewer Fig. 2, same situation top view (https://cloud.bio.lmu.de/index.php/s/77npeEK84towzJZ). As I said, in my opinion, there is a significant and important effect; the authors should simply adjust the description.

Minor concerns:

(1) I would like to suggest proactively discussing possible artifacts that may arise from the harsh conditions during FISH sample preparation..

(2) It would be helpful if the authors could provide the original data (microscopic image stacks) for download

(3) The authors use a blind deconvolution algorithm to improve image quality. It might be helpful to test other methods for this purpose (optional).

Significance

Advance:

Ono et al. addresses the important question on how the complex pattern of chromatin is reestablished after mitosis and maintained during interphase. In addition to affinity interactions (1,2), it is known that cohesin plays an important role in the formation and maintenance of chromosome organization interphase (3). However, current knowledge does not explain all known phenomena. Even with complete loss of cohesin, TAD-like structures can be recognized at the single-cell level (4), and higher structures such as chromosome territories are also retained (5). The function of condensin II during mitosis is another important factor that affects chromosome architecture in the following G1 phase (6). Although condensin II is present in the cell nucleus throughout interphase, very little is known about the role of this protein in this phase of the cell cycle. This is where the present publication comes in, with a new double degron cell line in which essential subunits of cohesin AND condensin can be degraded in a targeted manner. I find the data from the experiments in the G2 phase most interesting, as they suggest a previously unknown involvement of condensin II in the maintenance of larger chromatin structures such as chromosome territories. The experiments regarding the M-G1 transition are less interesting to me, as it is known that condensin II deficiency in mitosis leads to elongated chromosomes (Rabl configuration)(6), and therefore the double degradation of condensin II and cohesin describes the effects of cohesin on an artificially disturbed chromosome structure.

General limitations:

(1) Single cell imaging of chromatin structure typically shows only minor effects which are often obscured by the high (biological) variability. This holds also true for the current manuscript (cf. major concern 2 and 3).

(2) A common concern are artefacts introduced by the harsh conditions of conventional FISH protocols (7). The authors use a method in which the cells are completely dehydrated, which probably leads to shrinking artifacts. However, differences between samples stained using the same FISH protocol are most likely due to experimental variation and not an artefact (cf. minor concern 1).

(3) The anisotropic optical resolution (x-, y- vs. z-) of widefield microscopy (and most other light microscopic techniques) might lead to misinterpretation of the imaged 3D structures. This seems to be the cases in the current study (cf. major concern 4).

(4) In the present study, the cell cycle was synchronized. This requires the use of inhibitors such as the CDK1 inhibitor RO-3306. However, CDK1 has many very different functions (8), so unexpected effects on the experiments cannot be ruled out.

Audience:

The spatial arrangement of genomic elements in the nucleus and their (temporal) dynamics are of high general relevance, as they are important for answering fundamental questions, for example, in epigenetics or tumor biology (9,10). The manuscript from Ono et al. addresses specific questions, so its intended readership is more likely to be specialists in the field.

About the reviewer: By training I'm a biologist with strong background in fluorescence microscopy and fluorescence in situ hybridization. In recent years, I have been involved in research on the 3D organization of the cell nucleus, chromatin organization, and promoter-enhancer interactions.

All questions regarding the statistics of angularly distributed data are beyond my expertise. The authors do not correct their statistical analyses for "multiple testing". Whether this is necessary, I cannot judge.

Literature

1. Falk, M., Feodorova, Y., Naumova, N., Imakaev, M., Lajoie, B.R., Leonhardt, H., Joffe, B., Dekker, J., Fudenberg, G., Solovei, I. et al. (2019) Heterochromatin drives compartmentalization of inverted and conventional nuclei. Nature, 570, 395-399.
2. Mirny, L.A., Imakaev, M. and Abdennur, N. (2019) Two major mechanisms of chromosome organization. Curr Opin Cell Biol, 58, 142-152.
3. Rao, S.S.P., Huang, S.C., Glenn St Hilaire, B., Engreitz, J.M., Perez, E.M., Kieffer-Kwon, K.R., Sanborn, A.L., Johnstone, S.E., Bascom, G.D., Bochkov, I.D. et al. (2017) Cohesin Loss Eliminates All Loop Domains. Cell, 171, 305-320 e324.
4. Bintu, B., Mateo, L.J., Su, J.H., Sinnott-Armstrong, N.A., Parker, M., Kinrot, S., Yamaya, K., Boettiger, A.N. and Zhuang, X. (2018) Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science, 362.
5. Cremer, M., Brandstetter, K., Maiser, A., Rao, S.S.P., Schmid, V.J., Guirao-Ortiz, M., Mitra, N., Mamberti, S., Klein, K.N., Gilbert, D.M. et al. (2020) Cohesin depleted cells rebuild functional nuclear compartments after endomitosis. Nat Commun, 11, 6146.
6. Hoencamp, C., Dudchenko, O., Elbatsh, A.M.O., Brahmachari, S., Raaijmakers, J.A., van Schaik, T., Sedeno Cacciatore, A., Contessoto, V.G., van Heesbeen, R., van den Broek, B. et al. (2021) 3D genomics across the tree of life reveals condensin II as a determinant of architecture type. Science, 372, 984-989.
7. Beckwith, K.S., Ødegård-Fougner, Ø., Morero, N.R., Barton, C., Schueder, F., Tang, W., Alexander, S., Peters, J.-M., Jungmann, R., Birney, E. et al. (2023) Nanoscale 3D DNA tracing in single human cells visualizes loop extrusion directly in situ. BioRxiv https://doi.org/10.1101/2021.04.12.439407.
8. Massacci, G., Perfetto, L. and Sacco, F. (2023) The Cyclin-dependent kinase 1: more than a cell cycle regulator. Br J Cancer, 129, 1707-1716.
9. Bonev, B. and Cavalli, G. (2016) Organization and function of the 3D genome. Nat Rev Genet, 17, 661-678.
10. Dekker, J., Belmont, A.S., Guttman, M., Leshyk, V.O., Lis, J.T., Lomvardas, S., Mirny, L.A., O'Shea, C.C., Park, P.J., Ren, B. et al. (2017) The 4D nucleome project. Nature, 549, 219-226.

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Referee #2

Evidence, reproducibility and clarity

Summary:

• Ono et al use a variety of imaging and genetic (AID) depletion approaches to examine the roles of condensin II and cohesin in the reformation of interphase genome architecture in human HCT16 cells. Consistent with previous literature, they find that condensin II is required for CENP-A dispersion in late mitosis/early G1. Using in situ FISH at the centromere/q arm of chromosome 12 they then establish that condensin II removal causes lengthwise elongation of chromosomes that, interestingly, can be suppressed by cohesin removal. To better understand changes in whole-chromosome morphology, they then use whole chromosome painting to examine chromosomes 18 and 19. In the absence of condensin II, cells effectively fail to reorganise their chromosomes from rod-like structures into spherical chromosome territories (which may explain why CENP-A dispersion is suppressed). Cohesin is not required for spherical CT formation, suggesting condensin II is the major initial driver of interphase genome structure. Double depletion results in complete disorganisation of chromatin, leading the authors to conclude that a typical cell cycle requires orderly 'handover' from the mitotic to interphase genome organising machinery.

• The authors then move on to G2 phase, where they use a variety of different FISH probes to assess alterations in chromosome structure at different scales. They thereby establish that perturbation of cohesin or condensin II influences local and longer range chromosome structure, respectively. The effects of condensin II depletion become apparent at a genomic distance of 20 Mb, but are negligible either below or above. The authors repeat the G1 depletion experiment in G2 and now find that condensin II and cohesin are individually dispensable for CT organisation, but that dual depletion causes CT collapse. This rather implies that there is cooperation rather than handover per se.

• Overall this study is a broadly informative multiscale investigation of the roles of SMC complexes in organising the genome of postmitotic cells, and solidifies a potential relationship between condensin II and cohesin in coordinating interphase genome structure. The deeper investigation of the roles of condensin II in establishing chromosome territories and intermediate range chromosome structure in particular is a valuable and important contribution, especially given our incomplete understanding of what functions this complex performs during interphase.

Major comments:

• In general the claims and conclusions of the manuscript are well supported by multiscale FISH labelling. An important absent control is western blotting to confirm protein depletion levels. Currently only fluorescence is used as a readout for the efficiency of the AID depletion, and we know from prior literature that even small residual quantities of SMC complexes are quite effective in organising chromatin. I would consider a western blot a fairly straightforward and important technical control.

• I find the point on handover as a mechanism for maintaining CT architecture somewhat ambiguous, because the authors find that the dependence simply switches from condensin II to both condensin II and cohesin, between G1 and G2. To me this implies augmented cooperation rather than handover.

• I have two further suggestions, both of which I would strongly recommend but would consider desirable but 'optional' according to review commons guidelines.

Firstly, the depletions are performed at different stages of the cell cycle but have different outcomes. The authors suggest this is because handover is already complete, but an alternative possibility is that the phenotype is masked by other changes in chromosome structure (e.g. duplication/catenation). I would be very curious to see, for example, how the outcome of this experiment would change if the authors were to repeat the depletions in the presence of a topoisomerase II inhibitor.

Secondly, if the author's claim of handover is correct then one (not exclusive) possibility is that there is a relationship between condensin II and cohesin loading onto chromatin. There does seem to be a modest co-dependence (e.g. fig S4 and S7), could the authors comment on this? Another control experiment here would be to treat fully WT cells with IAA and test whether non-AID labelled H2 or RAD21 dip in intensity. If they do not, then perhaps there's a causal relationship between condensin II and cohesin levels?

• I recognise this is something considered in Brunner et al 2025 (JCB), but in their case they depleted SMC4 (so all condensins are lost or at least dismantled). Might bear further investigation.

Methods:

Data and methods are described in reasonable detail, and a decent number of replicates/statistical analyses have been. Documentation of the cell lines used could be improved. The actual cell line is not mentioned once in the manuscript. Although it is referenced, I'd recommend including the identity of the cell line (HCT116) in the main text when the cells are introduced and also in the relevant supplementary tables. Will make it easier for readers to contextualise the findings.

Minor comments:

Overall the manuscript is well-written and well presented. In the introduction it is suggested that no experiment has established a causal relationship between human condensin II and chromosome territories, but this is not correct, Hoencamp et al 2021 (cell) observed loss of CTs after condensin II depletion. Although that manuscript did not investigate it in as much detail as the present study, the fundamental relationship was previously established, so I would encourage the authors to revise this statement.

Significance

General assessment: Strengths: the multiscale investigation of genome architecture at different stages of interphase allow the authors to present convincing and well-analysed data that provide meaningful insight into local and global chromosome organisation across different scales. Limitations: As suggested in major comments.

Advance: Although the role of condensin II in generating chromosome territories, and the roles of cohesin in interphase genome architecture are established, the interplay of the complexes and the stage specific roles of condensin II have not been investigated in human cells to the level presented here. This study provides meaningful new insight in particular into the role of condensin II in global genome organisation during interphase, which is much less well understood compared to its participation in mitosis.

Audience: Will contribute meaningfully and be of interest to the general community of researchers investigating genome organisation and function at all stages of the cell cycle. Primary audience will be cell biologists, geneticists and structural biochemists. Importance of genome organisation in cell/organismal biology is such that within this grouping it will probably be of general interest.

My expertise is in genome organization by SMCs and chromosome segregation.

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Referee #1

Evidence, reproducibility and clarity

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

Major point:

They propose a functional 'handover' from condensin II to cohesin, for the organization of CTs at the M-to-G1 transition. However, the 'handover', i.e. difference in timing of executing their functions, was not experimentally substantiated. Ideally, they can deplete condensin II and cohesin at different times to prove the 'handover'. However, this would require the use of two different degron tags and go beyond the revision of this manuscript. At least, based on the literature, the authors should discuss why they think condensin II and cohesin should work at different timings in the CT organization.

Other points:

• Figure 2E: It seems that the chromosome length without IAA is shorter in Rad21-aid cells than H2-aid cells or H2-aid Rad21-aid cells. How can this be interpreted?

• Figure 3: Regarding the CT morphology, could they explain further the difference between 'elongated' and 'cloud-like (expanded)'? Is it possible to quantify the frequency of these morphologies?

• Figure 5: How did they assign C, P and D3 for two chromosomes? The assignment seems obvious in some cases, but not in other cases (e.g. in the image of H2-AID#2 +IAA, two D3s can be connected to two Ps in the other way). They may have avoided line crossing between two C-P-D3 assignments, but can this be justified when the CT might be disorganized e.g. by condensin II depletion?

• Figure 6F: The mean is not indicated on the right-hand side graph, in contrast to other similar graphs. Is this an error?

• Figure S1A: The two FACS profiles for Double-AID #3 Release-2 may be mixed up between -IAA and +IAA.

• The method section explains that 'circularity' shows 'how closely the shape of an object approximates a perfect circle (with a value of 1 indicating a perfect circle), calculated from the segmented regions'. It would be helpful to provide further methodological details about it.

Significance

Ono et al addressed how condensin II and cohesin work to define chromosome territories (CT) in human cells. They used FISH to assess the status of CT. They found that condensin II depletion leads to lengthwise elongation of G1 chromosomes, while double depletion of condensin II and cohesin leads to CT overlap and morphological defects. Although the requirement of condensin II in shortening G1 chromosomes was already shown by Hoencamp et al 2021, the cooperation between condensin II and cohesin in CT regulation is a new finding. They also demonstrated that cohesin and condensin II are involved in G2 chromosome regulation on a smaller and larger scale, respectively. Though such roles in cohesin might be predictable from its roles in organizing TADs, it is a new finding that the two work on a different scale on G2 chromosomes. Overall, this is technically solid work, which reports new findings about how condensin II and cohesin cooperate in organizing G1 and G2 chromosomes.

I like this because I am using this helpful information in my writing and research class while writing my research essay. I am trying not to rely on quotes but only paraphrase what I am reading into my own worlds, while still give the author credit when needed.

This is an important step that I feel like as writers we forget to do, and we just get in the habit of just writing for ourselves. Doing this may confuse our readers if we don’t explain ourselves and the meaning behind it.

This is a key part in the chapter, as it reviews why we cite sources and gives a detailed bullet point list as to why we do.

digital censorship remains non-discrete because silencing means something about the values of the platform

Although this did actually happen, I think it is symbolic of the current state of our country as so many people, regardless of political affiliation, are either afraid to speak their minds and/or are unable to do so.

extremely well put -- censorship is not as literal as it once was before the digital age; it instead involves convincing the public that they have the liberty to control what they do/don't engage with on social media, despite corporate agenda-setting through discrete, targeted content.

very well put; it is human nature to be drawn to things that fill a void.

It sounds to me that even though, yes, he was arrested, which isn't fun, I believe he is grateful for the time he was there because he truly understood and found his identity as a socialist, and not only that, but he also received letters of support from other socialists.

So he was arrested by "lawful" authorities who basically don't have the same belief as him? And those "lawful" authorities were just under capital control?

This part talks about the actions socialism does (by supporting the class struggle people) and also the fact that Americans were indirectly following socialism. It's crazy to think that it's "okay" to be either Republican or Democrat in today's day, but being a socialist is shunned (sometimes by ignorant people who don't understand the meaning of it) in today's day.

Not many people can admit that they were wrong at first and then learned from their mistake or misunderstanding.

It seems that he was more focused on making the conditions better instead of fixing it. But wouldn't that be better than nothing? Isn't bettering something giving it a little fix?

His thinking kind of sounds wrong in a way because everyone thinks differently. That's why it's so hard to communicate with people because sometimes they just assume things, and one person could think one way on a situation while the other does or says the complete opposite.

If you think about it, with the type of job he had and the commitment that goes into it, you kind of see he would believe in socialism even though he didn't know there was a specific name for it.

provided with what is necessary for life, health, and growth, like food.

In this part of his answer, it shows how deeply he felt for the working class and how he also was part of it.

I wonder if this job position is still a thing in today's time.

• Users appreciate Grafana's rich features but criticize its complexity and brittleness.
• Tools like Prometheus and Grafana are seen as over-engineered, especially with components like Mimir requiring dedicated infrastructure.
• Concerns exist about the future of self-hosted Grafana due to frequent breaking changes.
• Some suggest simpler, stable alternatives for monitoring such as Prometheus alone, Zabbix, VictoriaMetrics, or newer tools like SigNoz, OpenObserve, and Perses.
• The community desires a stable, reliable "boring" monitoring stack rather than constant feature churn.

Overall, there is notable dissatisfaction with Grafana’s direction and operational demands, particularly among smaller teams or startups seeking sustainable solutions.

Odysseus feels guilty and weak. Because he knows he can’t fight against a goddess’s anger.

This is a very good point. Students who are "gifted" and not challenged in ways that are appropriate for their level will end up assuming that this is the way life will always be for them, and it will cause them to potentially become unmotivated and unenthusiastic.

I totally agree with this. Receiving parent and teacher input when determining if a student is "gifted" or not can be really useful because those are the people who see the student the most, and in multiple different settings. Teachers get to see students not only in an academic light, but also how they interact socially, how they problem-solve, etc. Parents get to observe their children when they are in a home setting and how they interact there.

I agree, IQ testing can often miss important pieces of people's intelligence. Including other measures is a great way to make sure every part of someone's intelligence is assessed properly.

This is a really great addition to the testing. Some students can be very anxious about speaking in front of others, especially adults in an assessment-like scenario. Using nonverbal testing can be a game changer for some students who are very intelligent but who struggle with communicating verbally.

This makes a lot of sense, and it is something that needs to be remedied in the process of discerning which students are "gifted."

This is so important. "Gifted" students need to not only be enriched in academic areas, but also in creative ones. If the creative parts of ones life are neglected, the other areas of their life will start to fall short. Creativity is so important for maintaining a healthy and strong mind.

I agree with this statement; students with these qualities are more than likely going to excel in their pursuits, whether those be academic or creative/artistic.

This is so true, and it is something that is often overlooked by many. Students who are exceeding their grade level's "normal" content can become neglected because they are so far ahead, the teacher does not know how to help them learn at a pace that makes more sense for them.

Diş fırçasının kılları, dişlerin taç yönüne doğru 45 derece açıyla yerleştirilir.

① Exert gentle vibratory pressure, using short, back-and-forth motions without dislodging the tips of the bristles. Kılların uçlarını yerinden çıkarmadan, kısa ileri-geri hareketlerle hafif titreşimli baskı uygulayın. ② This motion forces the bristle ends into the gingival sulcus area, as well as partly into the interproximal embrasures. Bu hareket, kıl uçlarını diş eti oluğu (sulkus) bölgesine ve kısmen interproksimal aralara zorlar. ③ The pressure should be firm enough to blanch the gingiva. Baskı, diş etini hafifçe beyazlatacak kadar sıkı olmalıdır.

Kılları diş eti hattına yerleştirin ve dişlerin uzun eksenine 45 derece açıyla yönlendirin.

Yumuşak bir fırça başını oklüzal düzleme paralel yerleştirin, fırça başı üç ila dört dişi kapsayacak şekilde konumlandırın ve çenede en distal dişten başlayın.

n sık önerilen yöntem Bass tekniğidir çünkü kılların bu en önemli bölgeye yerleştirilmesine önem verir.

① It is important for patients to understand that the plaque biofilm removal at the dento-gingival junction is necessary to prevent caries as well as periodontal disease. Hastaların, dento-gingival birleşim bölgesindeki plak biyofilminin temizlenmesinin hem çürük hem de periodontal hastalıkları önlemek için gerekli olduğunu anlaması önemlidir.

② This is referred to as «target hygiene» Buna «hedef hijyen» denir.

Dişleri fırçalama için birçok yöntem tanımlanmış ve etkin ve verimli olduğu öne sürülmüştür.

Hesaptaş kontrol diş macunundan en yüksek etkiyi elde etmek için, bu ürünleri günlük ev bakım rutinine eklerken hastanın dişleri temizlenmiş ve diş taşı birikiminden tamamen arınmış olmalıdır.

“Tartar kontrol diş macunları” olarak da adlandırılan “Hesaptaş (tartar) kontrol diş macunları”, pirofosfat içerir ve dişlerde yeni tartar birikimini azaltabileceği gösterilmiştir.

Florür iyonunun çürük azaltıcı etkiler sağlamak için 1000 ila 1100 milyon parça (ppm) miktarında bulunması gerekir.

Diş macunları, tedavi edici ajanları dişlere ve diş etlerine iletmek için çok faydalıdır.

① Dentifrices aid in cleaning and polishing tooth surfaces. Diş macunları, diş yüzeylerinin temizlenmesine ve parlatılmasına yardımcı olur. ② Dentifrices increase the effectiveness of brushing but should cause a minimum of abrasion to root surfaces. Diş macunları fırçalamanın etkinliğini artırır, ancak kök yüzeylerinde minimum aşındırma yapmalıdır.

① Powered toothbrushes with oscillating and rotating motions remove plaque biofilm and reduce gingival bleeding slightly better than manual toothbrushes. Salınımlı ve döner hareketlere sahip elektrikli diş fırçaları, plak biyofilmini temizler ve diş eti kanamasını manuel diş fırçalarına göre biraz daha iyi azaltır.

② Patients who want to use powered toothbrushes should be encouraged to do so. Elektrikli diş fırçası kullanmak isteyen hastalar teşvik edilmelidir.

③ Patients need to be instructed in the proper use of powered devices. Hastalara elektrikli cihazların doğru kullanımı öğretilmelidir.

④ Patients who are poor brushers, children, and caregivers may particularly benefit from using powered toothbrushes. Dişlerini iyi fırçalamayan hastalar, çocuklar ve bakıcılar, elektrikli diş fırçası kullanmaktan özellikle fayda görebilir.

Bu fırçalar tarafından oluşturulan hidrodinamik kesme kuvvetleri, kılların uçlarından kısa bir mesafedeki biyofilmleri bozar ve bu, ek interproksimal biyofilm temizliğini açıklar.

Ne sesli titreşim ne de elektrikli diş fırçalarının mekanik hareketi, bakteriyel hücrelerin canlılığını etkilediği gösterilmemiştir.

Titreşimlerin, bakterilerin ağız yüzeylerine tutunmasını engellediği de gösterilmiştir.

"Village worthy" is a term that refers to a person who is popular in their community for appearing virtuous from the outside, but who lacks true inner virtue.

teachers have flexibility in understanding all 4 need to be included but can be in any order

important to note the intentionality of the interactions! I feel like this is the key. We have to be intentional.

India by itself is larger than Chinese registrations already

Which paper or article from Romeo Dean and Dwarkesh patel?

Effectively dealing with change

1 GW? that seems small. Last year, between 30 and 40 GW of battery capacity was added by batteries alone

Wow, if you were only looking at the US and EU, you'd totally miss this

This is the instance first exemplar of the exploreimental creation of corss peergos account publishing via hyperpost pages created by an other allied named peergos account

This is an actual practical instantiation of a true collaborative technology allieance!

between founding members of the indranet

This is description is how i understand it too, but the link does not seem to say this - it refers to open loop being about cooling a room, and closed loop as a sort of targetted cooling instead.

This seems incredibly fast. Perhaps it's a longer term historical average? Note to self - check the reference.

Update: no, it is indeed correct. Once construction is approved, the average has been 6-8 historically, and it's not that much slower globally. In UK and Europe, and the USA, the most recent ones have been been closer to 10 years (i.e. Hinckley, Vogtle and Flammanville). Permitting is a separate process though.

This is a really useful stat. You need a specific definition of datacentre, but it's still handy.

Dossier d'Information : Les Dynamiques de la Négociation de Paix selon Alberto Fergusson

Synthèse

Ce document de synthèse analyse les réflexions et les expériences d'Alberto Fergusson, un acteur clé du processus de paix colombien, qui allie une expertise en médecine, psychiatrie et psychanalyse à une pratique intensive des négociations.

Ses observations, issues de plus d'une décennie d'implication, notamment dans les pourparlers avec l'ELN, révèlent les dynamiques psychologiques et sociales complexes qui sous-tendent les processus de paix.

Les points à retenir sont les suivants :

• Le Paradoxe de l'Accord (Individu vs. Groupe) :

L'observation la plus frappante de Fergusson est qu'un accord est quasi systématiquement possible lors de discussions individuelles et privées avec les membres de la partie adverse, y compris les dirigeants.

Cependant, cet accord devient impossible à atteindre une fois que les discussions retournent à la table de négociation formelle, avec ses dynamiques de groupe et ses impératifs de représentation.

• L'Importance Capitale des Canaux Parallèles ("Back Channels") : Contrairement à l'idée reçue, la majorité des décisions cruciales ne sont pas prises lors des sessions officielles, mais dans le cadre de discussions informelles et de réunions secrètes.

La maîtrise de ces canaux parallèles est un art qui requiert l'identification des bons interlocuteurs et la gestion précise du format et de la durée des échanges.

• L'Application de la Psychopathologie à la Négociation : Fergusson tire ses principaux outils d'analyse de son travail avec des sans-abri atteints de maladies mentales graves.

Il postule que les mécanismes de défense et les perturbations émotionnelles observées dans la "folie" éclairent les comportements, parfois irrationnels, des acteurs dans des situations de haute tension comme les négociations de paix.

• La Question Fondamentale sur l'Impact Réel des Négociations : Fergusson s'interroge de manière critique sur la capacité des négociations à modifier durablement les processus sociaux.

Il se demande si les accords de paix réussis sont le fruit d'une habileté de négociation ou s'ils ne font que formaliser une évolution déjà inéluctable des dynamiques sociales, soulevant le risque de parvenir à des accords "artificiels" et prématurés.

Contexte et Objectifs du Chercheur

Alberto Fergusson, fort d'une formation en médecine, psychiatrie et psychanalyse, a consacré une part importante de sa carrière à des activités psychosociales.

Son travail initial auprès de sans-abri atteints de schizophrénie en Colombie lui a permis de développer un modèle, l'"auto-analyse accompagnée", pour comprendre et accompagner les personnes souffrant de troubles émotionnels sévères.

Depuis près de vingt ans, il applique les connaissances acquises dans ce domaine au processus de paix colombien.

Il a été directement impliqué dans les pourparlers, notamment en tant que membre de la délégation gouvernementale du président Santos lors des discussions avec l'ELN en Équateur et à Cuba.

Il a également été membre de la Commission de la Vérité en Colombie.

Actuellement professeur à l'Université du Rosaire, il consacre un mois à l'IEA de Paris (en mode virtuel) pour organiser, synthétiser et repenser une décennie d'expériences.

Ce travail de réflexion est crucial car il s'apprête à réintégrer le processus de paix colombien avec une perspective académique, visant à analyser la situation d'un point de vue plus large et moins partisan.

Thèmes Centraux et Observations Clés

De la "Folie" à la "Normalité" : Une Approche Inversée

Fergusson qualifie son approche de "confession" : il reconnaît que l'essentiel de sa compréhension des processus de négociation provient de son expérience avec des personnes atteintes de maladies mentales graves.

Sa présentation est intitulée "La normalité à la lumière de la folie" (Normality in the light of Madness), signifiant que les mécanismes psychologiques extrêmes observés chez ses patients offrent une grille de lecture pertinente pour les dynamiques apparemment "normales" des négociations politiques.

Le Paradoxe de l'Accord : Individu contre Groupe

L'observation la plus puissante et la plus récurrente de Fergusson est la dichotomie radicale entre les interactions individuelles et les dynamiques de groupe.

• En tête-à-tête : Fergusson affirme que, sans exception, lors de conversations approfondies et individuelles avec n'importe quel membre de la partie adverse (y compris les plus hauts dirigeants de l'ELN), il a toujours été possible de parvenir à un consensus.

Il déclare : "nous aurions toujours pu signer l'accord individuellement, en tête-à-tête."

• À la table de négociation : Dès que la discussion est portée à la table formelle, où les dynamiques de groupe, les hiérarchies (nécessité d'obtenir l'approbation du leader suprême, comme "Gabino" pour l'ELN) et les pressions de représentation entrent en jeu, l'accord devient compliqué, voire impossible.

Ce paradoxe constitue le cœur de son questionnement actuel : pourquoi ce qui est mutuellement acceptable en privé devient-il inacceptable en public ?

L'Irrationalité Apparente : Agir Contre Ses Propres Intérêts

Une autre observation centrale est que, dans le cadre des négociations, les individus et les groupes adoptent fréquemment des positions qui vont manifestement à l'encontre de leurs propres intérêts, ou du moins partiellement.

Fergusson cherche à dépasser la simple explication des "facteurs émotionnels et psychologiques" pour analyser en détail les mécanismes qui conduisent à ces décisions contre-productives.

Le Rôle Crucial des Canaux de Négociation Parallèles ("Back Channels")

Fergusson affirme sans équivoque que la plupart des décisions importantes ne sont pas prises à la table officielle de négociation.

• Lieu de décision réel : Les véritables avancées se produisent lors de réunions informelles, en marge des sessions officielles.

• L'art du "Back Channel" : Le succès de ces canaux parallèles dépend d'une stratégie fine :

1. Identifier l'interlocuteur clé : Il faut savoir repérer la personne de l'autre camp avec qui un accord de principe peut être trouvé.   

2. Rassembler les décideurs : Dans un exemple réussi, Fergusson et son homologue de l'ELN, après s'être mis d'accord, ont organisé une réunion privée entre leurs deux dirigeants respectifs pour leur présenter leur solution commune.

Ce fut le moment où les négociations ont le plus progressé.  

3. Maîtriser la durée : La longueur d'une réunion est un facteur critique. Fergusson note que si des êtres humains continuent de parler après avoir trouvé un accord, ils finiront par trouver un désaccord.

Savoir quand s'arrêter est essentiel.

La Question Fondamentale : Négociation et Évolution Sociale

La principale question de recherche de Fergusson, qu'il explore durant sa résidence, est la suivante :

"Jusqu'à quel point peut-on changer les processus sociaux par le biais des négociations ?"

Il illustre ce dilemme avec une analogie : celle d'une personne qui, toute la nuit, pousse de toutes ses forces pour faire venir le soleil et qui, à 6 heures du matin, lorsque le soleil se lève, s'écrie : "J'ai réussi !".

• Négociateur : Agent du changement ou simple facilitateur ?

Les négociateurs sont-ils les artisans d'un accord, ou leur intervention se contente-t-elle de faciliter ou d'accélérer une trajectoire que les dynamiques sociales et les conflits auraient de toute façon suivie ?

• Le risque des "lois sociales naturelles" : Il se demande si les négociateurs, en tentant de forcer un accord, ne vont pas à l'encontre des "lois sociales naturelles", créant ainsi des arrangements artificiels et prématurés.

• Le critère du succès : Pour Fergusson, un accord réussi n'est pas celui qui tient six mois ou deux ans.

Sa question porte sur les accords de paix durables et leur véritable origine : l'habileté des négociateurs ou l'évolution inéluctable de la société.

Perspectives Issues de la Discussion

Les échanges avec les autres chercheurs ont enrichi et précisé plusieurs points :

• Légitimer le Changement de Position sans "Perdre la Face" :

◦ Un participant a suggéré que le rôle du négociateur est de créer un cadre où les parties peuvent légitimement changer de position sans "perdre la face".  

◦ Cette idée est illustrée par une expérience de dégustation de vin : des dégustateurs ont radicalement changé leur évaluation d'un vin après avoir vu l'étiquette, mais n'ont jamais admis avoir changé d'avis.

Ils ont prétendu que c'était le vin qui avait "changé" (il s'était "ouvert").  

◦ Leçon pour le négociateur : Il ne s'agit pas de convaincre l'autre partie de changer d'avis, mais de présenter la situation différemment (par exemple, en invoquant de "nouveaux événements" ou de "nouveaux aspects") afin que l'adoption d'une nouvelle position apparaisse comme une réponse logique à un contexte modifié, et non comme une capitulation.

• L'Équilibre entre Secret et Public :

◦ Même les processus de paix qui semblent secrets, comme celui avec les FARC, sont en réalité un mélange complexe d'échanges publics et de canaux parallèles.  

◦ Fergusson confirme que l'accord final avec les FARC a été le résultat d'une "chaîne de canaux parallèles", souvent au grand dam des dirigeants qui n'apprécient pas ces manœuvres.

vs avoidant

oh OOF

doing it for someone else to break out of this: interesting

Where are we replaying low capacity?

This is helpful because these steps make your introduction clear and organized, making it easier for readers to understand your topic right away.

It explains the important background of your answer or topic the key who, what, when, where, why, and how questions based on your research.

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one of the hypothesis accounts that the developer of the indyWeb has

@to - origo-meta-design-folder

The main putpose of creating this indyWebSpace

is to provide a human readable/writable name to be passed to personal connections

and to have a web accessible name for links for entry points to a range of private, cyptographicall secured communication channels

This is the origo meta-design folder for - https://indy0.peergos.me/1/gyuri

@from - https://hyp.is/hHfR1MLVEfCbJSOeqS01og/indy0.peergos.me/1/gyuri

Various examples of world model work: Nex to Yann LeCun. Fei-Fei Li World Labs w Marble model, Google has Genie models, Nvidia Omniverse and Cosmos.

Yann LeCun says world models more promising. What are world models?

live by

Not just stories but

Myth metaphor and misticism etc

for - adjacency - war - interbrain - self/other gestalt - dualism

everthing but the kitchen sink ありとあらゆる物、何でもかんでも

Comics definitely don't exist on their own. They're shaped by other industries like film, TV, online media, etc. Creators pull ideas from everywhere, and comics influence those other media, too. This makes sense to me, considering that media today overlaps constantly, and audiences move between platforms without even thinking about it.

This shows how much comics have changed in price and accessibility. What used to be a super cheap, everyday thing is now more expensive, which affects who buys them and how the fandom works. As far as I've seen, comics aren't as popular or purchased nowadays. But the medium has evolved from simple entertainment and moved towards a bigger, commercialized industry.

People have treated the term "comic book" like an insult for decades. Society looks down on comics and uses the word to say that something is childish or low-quality. Comics never got taken seriously, even though they became a huge part of culture.

How you know you have a good source

Show you're passionate about your thesis and you can later back it up with reasoning

This not only highlights the key prupose of this passage and the book from which it is from, but it also connects to much our class discussions on feminist epistimologies and shows how inter-disiciplines vary in their perceptions on what is deemed "important" and the need for feminist epistimology that challenges what has traditionally (under colonialism and even now in our post colonial societies) have deemed as "knowledge."

Exactly the same

For media convergence to work, everyone, including companies, creators, and technology, needs to cooperate. However, the truth is that it's really hard. Everyone has their own goals and timelines, so even though teamwork could fix a lot of problems, no one really knows how to pull it off. Convergence isn't about new tech, it's about people figuring out how to work together as well.

Convergence isn't about everything becoming stable or perfectly unified. It's always pushing media and culture together, but change is still happening at the same time. Things are always shifting, so convergence is more about an ongoing movement and tension than a final, fixed outcome.

We all take pieces of media from TV, the internet, and other sources to make sense of our own lives. It's like everyone builds their own understanding from the information around them. This shows that media isn't something that we just passively watch. It actually shapes how we understand the world and communicate with others.

where is this calculated?

なるほどねー

I think another part of it is that Silicon Valley has a crazy amount of money to experiment and invest in new technology. In comparison, Brazil companies are significantly poorer and don't have as many resources to stand out in its own or evolve their tech (hence Silicon seeing their stuff as outdated). Especially the rise of AI, I don't think gap will get any better in the future

This article reflects on Zuckerberg's character and his campaign to spread the internet across the world. Zuckerberg defends his capitalistic model, saying that in order to do the actions he is doing right now, he has to make money somehow. He says that a free ad model is much better than a pay model which builds many barriers. For something as basic as social media and internet, I cannot help to agree with him, but for the case of facebook, I think the lack of transparency and consent is the main problem than the model itself as you still get collect data and advertise while allowing users to have some limited levels of privacy

This debunks all arguments that students may use it to completely write essays, ChatGPT is not programmed for that, only to specific questions. Backed up by statements from Ying Xu, an assistant professor of artificial intelligence in learning and education at Harvard University. She states, "There's this specific language used when talking to AI: It's very matter of fact. It's almost like a quiz versus when you see a child interacting with a teacher," (Jimenez 11)

Parry, the teacher from South Dakota, also claims that ChatGPT should be encouraged for students to edit and revise their essays stating that contrary to the common concern "If it's about the writing of the actual paper: Then you cannot have ChatGPT do it," she said. "It should help you prewrite, revise and edit. The middle belongs to the individual showcasing his or her writing abilities ... That’s what kids still have to produce."

Much like the instructor interviewed by PBS, this shows real life examples where AI has been used as more of a teaching assistant or a tutor than a cheating device.

A survey was conducted by a nonprofit think tank called RAND Corp.; they found that about 40% of US English teachers have used AI in their classrooms. This shows that the acceptance and use of AI in a learning capacity is gaining traction and may soon be common to find in any classroom, nation-wide (Jimenez 6)

Kayla Jimenez interviewed a 12th grade teacher from South Dakota who found a new solution to old writing prompts. Lisa Parry was having issues with worn out and un-inspiring prompts for her students' essays, but she found that ChatGPT could give students fresh new ideas(Jimenez 1-5)

grading ignores process-based growth, recognizing code-meshing strategies supports multilingual students’ development

even when SAE is mandated, students translanguage anyway because it helps them make meaning, SAE rules push this practice underground, reducing opportunities for feedback and assessment fairness, should we legitimize code-meshing for equity?

definition challenges SAE-only norms, programs enforcing SAE restrict multilingual students’ rhetorical resources, limiting academic success.

Even if emperor penguins can adapt to climate change, without enough resources, they still won't be able to survive successfully.

Emperor penguins have been able to adapt by themselves for a long period of time, mostly by relocating.

Figure 3 shows the sea ice concentration anomalies for each climate scenario.

Figure 2 shows some of the scenarios caused by global warming, including the Paris Agreement.

Peter Greenes suggestion "If it (ChatGPT) can come up with an essay that you would consider a good piece of work, then that prompt should be refined, reworked, or simply scrapped.” I think this is very interesting, having no experience with ChatGPT, the little I do have with other forms of AI is that it often makes small but obvious mistakes like 6 fingers, or images appear from thin air. Does this also apply in ChatGPT? Where an essay might have all of the information correct factually, but maybe the grammar doesn't make sense, or is structured so well it reads like it was written by a college professor as opposed to a high school English student? If either or even both end results are true, then like Pondiscio I feel that AI generated essays shouldn't necessarily be a realistic concern.

In this article, Robert Pondiscio shares some opposing points of view or fears that AI will be used as a cheating aid and not a learning aid. He references how in the past, teachers who were familiar with their students writing could tell if a paper had been influenced or plagiarized, and that AI generated essays will be no more difficult to weed out. Pondiscio counters many academics worries with the advice that AI should be embraced and that we should not assume it will destroy academic integrity.

Pondiscio warns that AI should be utilized as a learning tool for students and teachers, not shunned as some cheat to easy learning. He closes his argument with a short, but impactful statement"____"(Pondiscio 8)

Pondiscio often references Daniel Herman, an English teacher and writer for The Atlantic. Here, Pondiscio argues against Hermans worry that AI will allow students to cheat through school, without doing any of the actual hard work"_____"(Pondiscio 7).

Pondiscio claims"___"(Pondiscio 6). Meaning any student who attempts to plagiarize work or cheat in some way would be quickly sniffed out if the teacher was familiar with their students writing and uses this to defend fears of students using AI generated essays.

This is utterly hilarious.

I believe this quote gives insight to the current situation between Jews and Arabs and gives insight on the future between the two. It explains that the high birthrate of the younger Arab leads to anxiety among the Israeli polity. They fear that this birthrate will lead to the Arab population to surpass that of the Jewish population. They believe that this change in population will eventually lead to Israeli control on the area to come to an end. There are many solutions being thought up for this problem but all of them will most likely lead to more conflict between the two groups, which shows how peace does not look likely for the foreseeable future.

A supportive classroom climate is the most essential and important thing that you need to have in order for students to feel confident enough to take those risks and truly engage in the learning.

This is very true that we all have our own unique styles but we are all also held to the same standards as educators.

for the ME LTS example of the processes

Being classified together as both Asian Americans will hide the real differences in their lives and experiences.

Hsu Hua’s quiet writing style makes the story feel sincere and let the reader easy to connect with.

Ken’s influence didn’t appear as direct teaching, but changed the way he sees the world.

It’s interesting to see that the pope promised land, wealth, and forgiveness of sins to men who joined the quest.

I wonder why so many people in 1066 lived as farmers in the feudal system across England, Wales, Scotland, and Ireland.

It’s interesting how their missionary work helped spread the Byzantine Church’s religion, alphabet, and culture int Bulgaria, Serbia, and later Russia.

for - comparison - climate crisis - need war time mobilization - like WWIi Canadian mobilization

Crowdsourcing is when a group of participants disperses the collective work it would take to complete something amongst themselves and is then able to complete the task more efficiently and effectively. Frequently, their task is some sort of product that requires many people to ponder over it at the same time. Crowdsourcing is also not limited to online examples. and has been around for much of human history.

I've played video games where everyone was participating in an objective that would complete some greater thing beyond the scale of what we could do on our own. Completing these objectives was a collective commuinity effort and was only accomplished because everyone did their part.

Inspired by this section, though not specifically suggested:

In Greek, Roman, and even Christian (saint) traditions, most gods (saints), were closely associated with one or potentially a few attributes which made it easier to give them short hand identifications and also to use them in mnemonic traditions. It would seem that in Celtic traditions, that the gods (or heroes) were better delineated people with broader and fuller characters which didn't play into this sort of mnemonic/oral piece in the same way.

Donn in Irish traditions seems to fit this mold. What other evidences could be brought to bear to back this up?

Is this a reference to continuum robotics, where links are very flexible so you need to use structural models which enables large non-rigid body motions (like cosserat rod theory), since these robots are usually "soft" so their behaviour can't be really captured by euler-bernoulli or timoshenko beams?

Effects of social influence

This concept of visualizing data as "streams visualizing the ebb and flow of coins over the centuries" is highly relevant to my research. I need to show change over time, specifically the sudden drop in coins or shift in material (like the debasement of currency) that occurs during and immediately after the plague years (1347-1351). This idea of "dynamic streams" helps me think past a static map and towards visualizing economic instability and recovery across Europe.

Whiile open education should be available to all it is still challenging due to having full access to the resources for use or links being changed.

A reading of childhood rearing conditions of british working class households post-WWII can give a fair idea of the adverse circumstances operating against any such "well-intentioned efforts" at that time & place. Furthermore, such efforts likely failed to account for early (educational) developmental windows, combined nutritional differences, & given wide-spread racism & classism at the time. The efforts would be unlikely to counteract disparity in opportunity. Thus it is likely Fisher's (1951) assertion would not stand scrutiny today & may not be the best statement to be cited for Lynn's argument.

For a somewhat entertaining account of 1950s working class conditions & social mores, see Jennifer Worth's book (or tv series?) Call the Midwife.

Use this link to view the page in Peergs

close the view and the enclosing folder is shown

where the page can be edited using indy0pad.next

First explorimental exemplar of relegating Page-in-a-Page into a named subfolder in the current working focal context using iPeergos ndy0pad app

what the fuck

what the hail

connected to the wrong word?

for - MAGA base - when the old economy dies, they will be looking for defenders of the working class - adjacency - corporation to cooperation - MAGA base

for - definition - civilian labor corp - future of work - there will have to be new labor pools in the economy that free market has ignored to date - caretakers for the elderly - climate adaptation - affordable housing

for - stats - elites - top 10% - responsible for 50% of consumer spending

for - definition - prime age rate - an important rate to follow for economics

for - stats - US economy - 25% is gig economy

This is a very common concern for new Tailwind users, and the answer is twofold:

1. No, you do not have to memorize all the classes. The developer workflow is built around powerful code editor tools that make this unnecessary.
2. Yes, interactive editors like you described absolutely exist. They are excellent for certain workflows, especially for building pages quickly.

Here is a detailed breakdown of the solutions available.

Solution 1: The Standard Developer Workflow (Code Editor Extensions)

This is how the vast majority of developers use Tailwind CSS. Instead of memorizing classes, you rely on an intelligent plugin in your code editor (like VS Code).

The most essential tool is the Tailwind CSS IntelliSense extension for Visual Studio Code.

This plugin solves the "memorization" problem in three specific ways:

1. Autocomplete: You rarely type the full class name. You start typing a prefix, and the editor shows you all possible options.
   • If you type bg-, it will pop up a list of all available colors (bg-blue-500, bg-red-700, etc.), complete with a color swatch.
   • If you type p-, it will list all padding options (p-1, p-2, p-4, p-6, etc.).
2. Hover-to-Preview: If you are unsure what a class does, you can hover your mouse over it. The plugin will show you the exact CSS it generates.
   • Hovering over p-4 will show a popup that says padding: 1rem;.
   • Hovering over rounded-lg will show border-radius: 0.5rem;.
   • This feature turns the editor into a powerful learning tool.
3. Linting (Error Checking): The plugin will underline conflicting classes, helping you avoid mistakes. For example, if you accidentally type p-2 and p-4 on the same element, it will flag this as an issue because you are applying two different padding values.

This workflow is not based on memory, but on a logical, discoverable system. The class names are consistent: * p is padding, m is margin. * t is top, b is bottom, l is left, r is right. * Therefore, pt-4 is padding-top of 1rem. This logic becomes second nature very quickly.

Solution 2: Visual (WYSIWYG) Editors

For the "PowerPoint-like" scenario you described, several tools provide a full graphical user interface (GUI) for styling with Tailwind. These are often called visual builders or page builders.

With these tools, you would click an element, and then use a properties panel on the side to adjust its padding, color, or margin. The tool then writes the correct Tailwind HTML for you.

Examples of these tools include:

• Windframe: A visual editor and AI tool designed specifically for Tailwind CSS. It features a drag-and-drop interface and a properties panel to adjust styles, then exports production-ready code.
• Pinegrow: A professional desktop web editor that has a dedicated Tailwind Visual Editor add-on. It allows you to visually edit your project and provides controls for all Tailwind properties.
• Shuffle (and Tailwind.build): An online editor with a large library of pre-built UI components. It allows you to drag components onto a canvas, customize their styles with visual controls, and export the final HTML.
• GrayGrids: Another online tool that functions as a "Tailwind CSS Website UI Builder" with drag-and-drop functionality.

These tools are excellent for rapidly building landing pages or prototyping. The primary trade-off is that for complex, dynamic applications, many developers find it faster and more precise to work directly in the code using the IntelliSense plugin (Solution 1).

Solution 3: Component Libraries (The Middle Ground)

There is a third option that also reduces the need to "memorize" individual classes: using pre-built component libraries.

The official Tailwind UI is the most popular example.

This is not a visual editor, but a paid library of over 500+ professionally designed components (navbars, forms, buttons, page sections, etc.).

• Your Workflow: Instead of building a complex form from 100 different utility classes, you find the form you need in the Tailwind UI library, copy its HTML, and paste it into your project.
• How it Helps: This solves the problem by giving you large, complete, and perfectly-styled blocks, so you only need to make minor adjustments (like changing bg-blue-500 to bg-indigo-500) rather than building everything from scratch.

Would you like me to elaborate on how to install and configure the Tailwind CSS IntelliSense plugin for VS Code?

I think a comment added on the Identity operator's use here, would be useful

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To begin with, the widespread adoption of global fast food undermines family cohesion. (解释事实：) This is due to the fact that the cookery and preparation of traditional meals offer an invaluable occasion for family members to gather around the table, engage in meaningful conversations, and strengthen emotional bonds. In contrast, fast food consumption tends to be individualistic and rushed/hurried, often taking place outside the home, resulting in fewer opportunities for family interactions and (回扣：) gradually eroding family cohesion in the long run.

Key Idea: The overall goal is culturally relevant and rigorous instruction for emergent bilinguals. Course Link: Ties into culturally sustaining pedagogy and asset-based teaching. Reflection: Summarizes the book’s contribution to equitable language education.

Key Idea: Reviewer notes a missed opportunity to discuss student identity and social belonging. Reflection: Important sociolinguistic dimension often overlooked in policy discourse.

Key Idea: Monolingual policies harm multilingual students. Course Link: Mirrors class discussions about inequity in language policy and Standard English ideology. Reflection: Reinforces systemic barriers to linguistic diversity.

Key Idea: Teachers need specialized PD to support emergent bilinguals effectively. Course Link: Aligns with research showing gaps in teacher preparation for multilingual learners.

Key Idea: Literacy is cross-disciplinary; inquiry and reading share similar cognitive processes. Reflection: Literacy instruction belongs in every subject area. Course Link: Status of academic language across discourse communities.

Key Idea: CSR integrates language learning with comprehension strategy instruction. Course Link: Example of codemeshing—using all linguistic resources to build understanding. Reflection: Shows value of integrated models rather than separate ESL tracks.

Key Idea: Supporting home language development accelerates English learning. Course Link: Connects to translanguaging and codeswitching research we studied. Reflection: Counters the myth that bilingualism slows academic progress.

Key Idea: Asset-based approaches view bilingualism as a strength rather than a deficit. Course Link: Reflects Ruiz’s (1984) language-as-resource framework. Question: Why do deficit views still dominate educational policy?

Key Idea: The book challenges the monolingual and monocultural assumptions built into the Common Core. Course Connection: Links to class themes on language policies and critiques of Standard English expectations. Why Important: Shows how policies can marginalize emergent bilingual students.

Watching a movie always requires interpretation. Audiences automatically fill in gaps and connect details. It's a part of understanding the film. If we see a character get into a car and then later the car arrives at another location, we get that they drove there even though the movie doesn't show the whole trip.

Films don't have a single message. Viewers interpret them based on their own experiences. meaning comes from both the filmmaker's choices and how the audience sees it.

Every part of a film is planned. Costumes, props, lighting, and shots are chosen deliberately. Even things that look spontaneous are controlled. This shows how precise filmmaking is and how much thought goes into every detail.

Filmmaking has always depended on teamwork. Even under strict studio control, movies required many departments working together. The system created a structure where each role contributed to the final product.

This highlights how the studio system's focus on efficiency caused films to become repetitive. With producers pushing for quick, standardized output, creativity suffered. The industry had to recognize that prioritizing speed and control made the movies themselves weaker.