The x2-versus-t relationship was linear for at least an hour (fig. S4C), consistent with random walk diffusion. Thus, over this time scale and this distance scale, we found no evidence for a trigger wave
In the absence of the mitochondria, the authors found that the spread of apoptosis signals were not consistent with propagation by trigger waves.
Every egg that showed a wave had increased caspase activity (Fig. 4, E and F, red symbols). We also collected eggs during the same time period that had not displayed a wave. None of these eggs had increased caspase activity
The authors detected caspase activity in eggs that displayed wave propagation under apoptotic conditions and did not detect any caspase activity in eggs that did not have any wave propagation.
This evidence indicated that caspase activation is involved in the propagation of apoptotic waves in intact eggs.
The TMRE wave propagated at the same speed as the caspase trigger wave reported by Z-DEVD-R110 in the same tube (38 μm/min)
Here, the authors confirmed that TMRE (which measures mitochondrial membrane potential) can be used to monitor and assess apoptotic trigger waves.
This video shows the propagation of apoptosis through a frog egg extract as visualized using the fluorescent dye TMRE: https://www.youtube.com/watch?v=3rIS7Y4LB6U
As shown in movie S1 and Fig. 1, B and C, apoptosis progressed up the thin tube at a constant speed of 27 μm/min over a distance of several millimeters. In five independent experiments, apoptosis always propagated linearly, without showing any signs of slowing down or diminishing, and the average speed was 29 ± 2 μm/min (mean ± SD). In contrast, the 10-kDa dye spread only a few hundred micrometers (Fig. 1B), implying that neither simple diffusion nor any unintended mixing could account for the spread of apoptosis.
The goal here was to find out if diffusion was responsible for the spread of apoptotic signals. Because diffusive spread is known to slow down over time, its was used by the author's as a mark to determine if the spread of apoptosis was by diffusion.
Results showed that apoptosis spread at a constant speed and did not slow down indicating that diffusion was not responsible.
These experiments implicate caspase-3 and/or -7 as well as Bcl-2 in the regulation of the apoptotic trigger waves. The experiments also show that the trigger waves are relatively robust; they are still present, though with reduced speeds, in extracts depleted of mitochondria or treated with maximal doses of GST–Bcl-2 or Ac-DEVD-CHO
Together, the results presented in Figure 3 indicate that caspase (-3, -7) and pro-apoptotic proteins (Bax and Bak) activity are essential in dictating the speed of apoptotic trigger waves in Xenopus egg extracts
We found that dRECS1KO cells exhibited increased formation of autophagosomes and autolysosomes together with higher overall autophagy flux (fig. S8, D to F). Together, these results suggest that CG9722 has a proapoptotic activity in vivo and sensitizes flies to lysosomal- and ER stress–induced cell death.
Flies without RECS1 exhibit increased autophagy under stress
In addition, dRECS1KO female animals were more resistant to death induced by nutrient starvation, an effect that was still observed but not as prominent in male flies or larvae (Fig. 9, D and E).
Female flies without RECS1 also exhibit increased lifespan even when starved by withholding food.
Moreover, dRECS1 deficiency conferred resistance to Tm in male and female flies, increasing life span under ER stress (Fig. 9, B and C).
Male flies without RECS1 have longer lifespan, even when they were stressed with Tm
Mutation of the di-aspartyl sensor results in a marked decrease in the channel’s permeability to Ca2+ and Na+ but not to Ba2+ or Cs+ (Fig. 7G). Together, these results suggest that RECS1 is a pH-dependent cationic channel, with a main preference for calcium.
Reduced calcium entry is observed when RECS1 is non functional
ltage clamp technique with potassium as the reference ion. Analyses of permeability ratios indicated that RECS1 allows the flux of Ca2+ and, to a lesser extent, Na+ (Fig. 7F).
RECS1 allows in more Calcium than Sodium
Notably, overexpression of RECS1 triggered cell death and structural abnormalities in the oocytes in a dose-dependent manner, consistent with its role as a cell death regulator (Fig. 7E)
Increasing the dose of RECS1 amplified cell death in oocytes.
ependent on BAX and BAK expression (Fig. 6D)
RECS1 requires BAX and BAK to induce lysosomal pH and calcium level changes.
RECS1 overexpression resulted in lysosomal acidification in HeLa cells, with lysosomal pH (pHL) decreasing from 4.77 to 4.38 (Fig. 6A). The acidification of lysosomes correlated with an increase in lysosomal intraluminal calcium concentration ([Ca2+]lys) from an average of 149 to 286 μM (1.9-fold increase) (Fig. 6B),
Overexpression of RECS1 in cells is associated with reduced lysosomal pH and increased calcium in the lysosome.
had no effects in basal cell viability (fig. S5, O and P), but it conferred protection against ER stress induced by Tm and Tg (Fig. 5, B and C).
Reduced RECS1 expression blocks cell death even when ER stress is induced by Tm or Tg, suggesting that RECS1 induces cell death under ER stress
(Fig. 4A). Since ER stress affects protein trafficking through the secretory pathway and the expression of RECS1 alone does not trigger ER stress, we allowed RECS1 to accumulate for 16 hours before challenging cells with Tm or other ER stressors (Fig. 4B). Using this protocol, we found that RECS1 overexpression hastened cell death to ER stress, as evidenced in cell death kinetic experiments (Fig. 4C). Moreover, we confirmed these observations in HeLa cells in dose-response experiments (fig. S5, M and N). We further validated these results in cells stimulated with Tg, followed by cell death measurements using both FACS and live microscopy (Fig. 4, D and E). Long-term survival assessment by clonogenic assays indicated that RECS1 expression reduced the growth of cells under ER stress (Fig. 4F). Last, we treated MEFs expressing Flag-RECS1 with QvD together with Tm and determined cell death by live microscopy. As expected, QvD treatment reduced cell death under ER stress (Fig. 4G). Similarly, BAX and BAK double deficiency fully inhibited cell death triggered by RECS1 overexpression under ER stress (Fig. 4H).
Increased cell death is observed in cells overexpressing RECS1 and treated with ER stress inducers, Tm and Tg, suggesting that RECS1 induces ER-stressed cells to die.
These results indicate that RECS1 overexpression alone does not cause ER stress. We also determined whether CQ treatment could induce ER stress. Treatment of Flag-RECS1–overexpressing MEFs with CQ failed to induce Xbp1 mRNA splicing (fig. S5, K and L), Bip/Grp78, or Chop/Gadd153 (fig. S5L). These results suggest that cell death induced by RECS1 under lysosomal stress conditions is not associated with the activation of the UPR.
Neither overexpression of RECS1 a lone nor treatment with CQ induce UPR. Therefore, ER stress caused by Tm and other ER-stress inducing chemicals is not connected to UPR (does not cause UPR)
Several proteins of the TMBIM family, such as BI-1 and GRINA, inhibit apoptosis triggered by ER stress, probably through the modulation of ER calcium (10, 13, 15, 17, 38). To gain further insights into the role of RECS1 in the control of cell death under cellular stress, we measured its expression in cells undergoing ER stress induced by the treatment with the N-glycosylation inhibitor tunicamycin (Tm). We found that Recs1 mRNA levels were up-regulated under ER stress, whereas Bi-1 mRNA levels remained unaltered (fig. S5A). This expression profile parallels that of certain BCL-2 proteins known to be induced by ER stress (i.e., Puma), while antiapoptotic components were unchanged (i.e., Bcl-2) (fig. S5B). Recs1 mRNA was also up-regulated in cells undergoing ER stress induced by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) calcium pump inhibitor thapsigargin (Tg) (fig. S5C). The kinetics of the up-regulation of Recs1 mRNA under ER stress paralleled the up-regulation of the ER chaperone Bip/Grp78 and the proapoptotic factor Chop/Gadd153 (fig. S5D)
Recs1 expression (mRNA) is increased in cells experiencing Endoplasmic Reticulum (ER) stress. Compounds that cause ER stress include tunicamycin (Tm), SERCA and thapsigargin (Tg)
As expected, BAX and BAK DKO cells were almost completely resistant to cell death induced by treatment with 25 μM CQ (Fig. 3D). However, we observed 30% of cell death in BAX and BAK DKO Flag-RECS1 cells treated with the higher CQ dose (50 μM), an effect that could not be reversed by inhibition of mitochondrial transition permeability pore (mPTP) (cyclosporin A) or necroptosis (necrostatin-1s) (Fig. 3D and fig. S4G).
Without BAX and BAK in cells, RECS1 cannot induce cell death even under stress conditions.
In addition, we measured the enzymatic activities of three lysosomal enzymes—cathepsin D, acid phosphatase, and hexosaminidase—in cytosolic lysosomal-free fractions. In line with previous results, we found that the activity of these three enzymes is increased in the cytosol of MEFs expressing Flag-RECS1 after CQ treatment but not in control cells (Fig. 3C)
Cells stressed with CQ and with high dose of RECS1 have more lysosomal protein leakage to the cytoplasm, suggesting that RECS1 induces LMP under lysosomal stress
, treatment with CQ resulted in a dose-dependent increase in the number of LGALS1/galectin-1 puncta (Fig. 3, A and B
Cells overexpressing RECS1 and stressed with CQ resulted in increased leak of proteins, suggesting that LMP was initiated
We then explored the effects of lysosomal stress on RECS1 localization. Analysis of the distribution of endogenous mouse RECS1 indicated that, under basal conditions, RECS1 colocalizes mainly in lysosomes and late endosomes (around ~20%) (fig. S3, C to E). However, in cells undergoing starvation or starvation and treated with CQ, RECS1 was further recruited to lysosomes but not to late endosomes, suggesting that RECS1 distribution is highly dynamic (fig. S3, C to E). Notably, the levels of Recs1 mRNA were up-regulated in cells undergoing nutrient starvation (fig. S3F). In addition, RECS1 overexpression induced autophagosome formation, an effect that was increased by CQ treatment (fig. S3G). Overall, our results indicate that RECS1 is located at lysosomes and regulates the susceptibility of cells to lysosomal stress.
RECS1 is localized at the lysosomal membrane where it regulates cellular responses to stress conditions. It increases chances of cell death in these conditions.
colocalization analysis
A technique to detect the location of one protein by use of the location of another protein. If two proteins exist in the same location together, then they are colocalized. Here, FLAG-RECS1 colocalizes with lysosomal proteins LAMP1 and LAMP2, suggesting that RECS1 is associated with the lysosome
These results indicate that RECS1 potentiates cell death triggered by lysosomal stress.
Altering lysosomal pH leads to stress. RECS1 causes rapid cell death under these conditions.
nexpectedly, RECS1 overexpression sensitized cells to the death triggered by ~14 compounds, whereas it repressed the activity of 12 compounds (fig. S2A). The top sensitizing drugs were chloroquine (CQ) and hydroxychloroquine (HCQ), which are known to induce lysosomal stress by inhibiting their acidification (Fig. 2, A and B).
Up to 14 chemicals induced cell death in cells with high doses of RECS1 compared to cells with low RECS1. Chloroquine (CQ) and Hydroxychloroquine(HCQ), which are known to cause lysosome stress had more significant effect, suggesting that the observed death was related to lysosome function/stress.
o determine the requirement of caspases, we treated RECS1-overexpressing cells with the pan-caspase inhibitor QvD-OPh (QvD), followed by cell death kinetics and fluorescence-activated cell sorting (FACS) analysis. Caspase inhibition completely abrogated cell death induced by RECS1
Without BAX and BAK, cell death was inhibited even when RECS1 was overexpressed, suggesting that to promote cell death, RECS1requires BAX and BAK function.
These observations suggest that RECS1 overexpression is sufficient to induce cell death.
RECS1 alone without any other treatment to cells induces cell death
(D295Q)
Aspartic acid (D) at position 295 in RECS1 is substituted for Glutamine (Q). This amino acid is necessary for regulation of lysosome membrane properties.
ECS1 expression highly sensitizes cells to lysosomotropic agents and microtubule-destabilizing drugs.
Expression of RECS1 causes an acute response of cells to lysosomal and microtubule stress, leading to cell death
TMBIM family, RECS1 expression results in cell death via the mitochondrial pathway of apoptosis.
RECS1 is pro cell death, although other members of TMBIM family were reported as inhibitors of cell death
to ER stress. RECS1 overexpression resulted in reduced survival when zebrafish were exposed to Tg in the water, while overexpression of the mouse GRINA conferred protection (Fig. 10, H and I) as reported (17). W
Zebrafish expressing high dose of RECS1 exhibit reduced survival under ER stress
Next, we determined the importance of RECS1 channel activity to cell death control during zebrafish development. We injected single-cell embryos with RECS1 WT or the D295Q mutant and assessed animal viability 24 hpf. In line with our in vitro results, overexpression of the RECS1 D295Q mutant failed to induce cell death in these embryos (Fig. 10E). Moreover, while the injection of RECS1 WT resulted in a high proportion of morphological-altered embryos, expression of the mutant protein did not have any adverse effect (Fig. 10, F and G).
The ion channel activity of RECS1 (through D295) is essential in regulating cell death in Zebrafish.
As expected, RECS1 overexpression induced a high number of AO spots, distributed along the head and, to a lesser extent, the body of the embryo (Fig. 10, C and D). Deletion of the C-terminal domain of RECS1 completely abrogated its cytotoxic effects (Fig. 10, C and D). T
Relative to whole body, head cells were more affected by a high dose of RECS1. Cell death was also dependent on the presence of C-terminal segment of RECS1.
RECS1 overexpression significantly decreased animal survival, a phenotype that was independent of its C-terminal domain (Fig. 10A). As a control, we overexpressed the TMBIM protein GRINA, which, under the same conditions, did not result in any developmental defects (Fig. 10A).
High dose of RECS1 is associated with reduced survival in Zebrafish. The effect of RECS1 here seems to not require its C-terminal domain unlike in mammalian cells in which the authors reported that the RECS1 C-terminal is necessary for RECS1-mediated cell death (Supplementary figure 1).
verexpression of dRECS1 strongly sensitizes to lysosomal perturbations and ER stress, resulting in exacerbated wing abnormalities (Fig. 9A).
More flies with high dose of RECS1 develop severely abnormal wings when exposed to Tm and CQ, compared to flies that have normal RECS1 dose, suggesting that RECS1 leads to abnormal development under stress conditions.
Adding GST–Bcl-2 decreased the wave speed (Fig. 3, A and B, and movie S5). The maximum effect [at 400 nM added GST–Bcl-2, which compares to the estimated endogenous Bcl-2 concentration of approximately 140 nM (14)] was a reduction of the speed to about 13 μm/min, the speed seen in purified cytosol. Added GST–Bcl-2 had no effect on the trigger wave speed in cytosolic extracts (Fig. 3, C and D), which emphasizes that the waves seen in purified cytosol are probably not caused by contaminating mitochondria. GST–Bcl-2 decreased the trigger wave speed in reconstituted (cytosol plus mitochondria) extracts (Fig. 3, E and F), just as it did in cytoplasmic extracts
The authors found that blocking the activity of Bax and Bak (via Bcl-2) reduces the speed of apoptotic trigger waves in cytoplasmic and reconstituted cytosolic extracts (both of which have mitochondria) but does not have any effect on the relatively slower speed of apoptotic trigger waves in purified cytosolic extracts (extracts without mitochondria).
This evidence suggested that Bax and Bak positively contribute to the generation and speed of apoptotic trigger waves in extracts containing the mitochondria.
During this same time period, a surface wave often appeared, originating near the vegetal pole and propagating toward the animal pole, with a typical apparent speed of ~30 μm/min
After treating eggs under apoptotic-inducing conditions, the authors observed the movement of a wave (with a speed characteristic of the speed of apoptotic trigger waves seen in the extracts) on the surface of the egg from one end of the egg (called vegetal pole) to the other end of the egg (called animal pole)
The vegetal pole is the portion of an ovum (mature female reproductive cell) containing most of the yolk and little cytoplasm while the animal pole is the portion of an ovum that contains the nucleus and less yolk.
The vegetal pole and the animal pole are opposite each other on the ovum.
These findings support the idea that spontaneous apoptosis typically initiates near the vegetal pole of the egg and propagates outward and upward from there as a ~30-μm/min trigger wave
In summary, the evidence presented in Figure 4 demonstrated that apoptotic trigger waves were also propagated in intact oocytes and eggs.
The surface waves propagated at an apparent speed of ~30 μm/min, similar to the wave speeds seen in apoptotic extracts. Control oocytes injected with Texas Red–dextran in water did not exhibit these surface waves. These findings indicate that apoptotic trigger waves can be produced in oocytes
The authors visualized apoptotic waves in intact oocytes (immature eggs) of Xenopus laevis suggesting that apoptotic trigger waves occur in oocytes.
Both reporters showed that the wave speed decreased as the inhibitor concentration increased
The authors observed that increasing the concentration of the caspase inhibitor in the extract resulted in decreasing speed of the apoptotic trigger waves.
As a result, the higher the concentration of the caspase inhibitor, the lower the speed of the apoptotic trigger waves.
This evidence indicated that caspase-3 and caspase-7 contribute positively to the speed of apoptotic trigger waves.
However, in experiments with either cytoplasmic extracts or reconstituted extracts, more than half of the time (in 12 of 22 or 21 of 37 tubes, respectively) a second spontaneous apoptotic wave emerged elsewhere in the tube
Here, the authors found that mitochondria-containing apoptotic (that is cytoplasmic or reconstituted) extracts sometimes generated a second apoptotic wave somewhere in the tube in addition to the first wave which originates from the bottom of the tube.
we determined the trigger wave speed to be half maximal at a mitochondrial concentration of 1.3 ± 0.6% (mean ± SE), which is estimated to be ~40% of the physiological mitochondrial concentration in Xenopus eggs. Thus, an average concentration of mitochondria is sufficient to generate apoptotic trigger waves of near-maximal speed, and the wave speed would be expected to drop in mitochondrion-poor regions of the cytoplasm
The authors found that only an average concentration of mitochondria is required to produce apoptotic trigger waves at their maximum speed.
As was the case in Fig. 2B, the speed of the wave front fell during the first ~120 min, consistent with diffusive propagation, but once the speed reached ~14 μm/min it remained constant for many hours (Fig. 2E). This suggests that purified cytosol is capable of generating apoptotic trigger waves, albeit with a substantially lower speed than that seen in cytoplasm or in cytosol supplemented with mitochondria
Here, the authors found that the purified cytosol (without mitochondria) produces slower apoptotic trigger waves when compared to that of the reconstituted cytosol (with mitochondria).
This evidence indicates the mitochondria positively contributes to the speed of apoptotic trigger waves.
Moreover, the reconstitution restored the trigger waves (movie S3 and Fig. 2D). The propagation distance increased linearly with time (Fig. 2D), as it did in crude cytoplasmic extracts (Fig. 1), and propagation occurred at a constant speed of 39 μm/min, somewhat faster than that observed in cytoplasmic extracts. The signal propagated over a long distance (6000 μm) with little loss of amplitude and no loss of speed (Fig. 2D)
The authors found that the addition of the mitochondria caused apoptosis to spread through the extracts by trigger waves.
This evidence showed that the mitochondria is required for the propagation of apoptotic signals via trigger waves.
In agreement with previous reports (3, 13), the caspases were briskly activated (Fig. 2A). Thus, mitochondria are not essential for cytochrome c–induced activation of executioner caspases in Xenopus extracts
The authors found that caspase activity was not blocked in the absence of the mitochondria indicating that the mitochondria was not necessary for the activation of caspases by cytochrome c.
Fluorescence spread up the tube at a constant speed (in this experiment, 33 μm/min). In eight experiments, the average speed was 30 ± 3 μm/min (mean ± SD)
Using a second method, the authors confirmed that apoptosis spread through the extract at a constant speed exhibiting the properties of trigger waves
The apoptotic activity propagated from the induction terminus to the distal terminus at a constant speed of 32 μm/min, consistent with a self-sustaining process
Apoptosis is spread by trigger waves as indicated by the spread of the apoptosis waves/signals at a steady speed.
trends noted here as being independent of the observational and analysis techniques used
The trends noted here appear to be real and not due to an error in the analysis, instruments, or observations.
Airplane weather measurements have been extensively compared against satellite data, especially for the North Atlantic, and has been shown to well estimate hurricane categories through satellite imagery.
As all the satellite images were analyzed in a similar fashion (the Dvorak scheme of cloud pattern comparison), the trends of increasing category 4 and 5 hurricanes are not caused by some problem with the analysis and thus appear to be driven by an environmental trend.
large increase was seen in the number and proportion of hurricanes reaching categories 4 and 5
After examining decades worth of satellite data on storms, it is now more likely a category 4 or 5 hurricane will form than how many used to occur in the 1970's. Thus the number of intense, life-threatening storms appears to have increased over the 35 year record of satellite data analyzed in this article.
global data indicate a 30-year trend toward more frequent and intense hurricanes
There appear to be more category 4 and 5 hurricanes forming in the 2000's than there were thirty years ago according to analyzed satellite data.
This trend towards an increased frequency of some of the strongest, most intense storms has also been a result of other studies and model simulations.
These changes occur in all of the ocean basins.
There has been an increase in the number of storms that reach a category 4 or 5. This increase has occurred in every ocean basin: the Pacific, Atlantic, and Indian Oceans.
has decreased monotonically as a percentage of the total number of hurricanes throughout the 35-year period
There are fewer category 1 hurricanes in the 2000's relative to the other category storms than there were in the 1970's.
A monotonic decrease means that for the whole of the record, there has been a decline in the percentage of category 1 storms. There were few if any periods where percentage values were constant or increasing.
a simple attribution of the increase in numbers of storms to a warming SST environment is not supported
Based on the satellite data from the 1970's to 2004, it is not possible to link warming ocean temperatures with a change in the number of hurricanes per year.
While the North Atlantic may be showing an increase in hurricane days with warming temperatures, the same is not happening in other ocean basins.
decreasing by 40% from 1995 to 2003
The number of hurricanes and storm days in the west Pacific Ocean increased from the mid 1970's to the early 1990's before declining to 2004.
Sea surface temperatures have been increasing in the west Pacific from the 1970's to 2000's. So there is not a clear relationship between storm frequency and sea surface temperatures for this ocean region.
Decadal variability is particularly evident in the eastern Pacific
There have been changes to the number of hurricanes and storm days per year that occur on the order of ten years or less in the eastern Pacific Ocean.
Examining Fig. 3, the eastern Pacific (EPAC) has a maximum number of storms occurring around 1984 and again near 1993. Between 1993 and 2004, the number of hurricanes and storm days has been declining.
Check out Fig. 1 again. Temperatures in the eastern Pacific have increased from the 1970's to 1992 before beginning to decline around 1993 to 2001.
North Atlantic Ocean, which possesses an increasing trend in frequency and duration
Of all the ocean basins examined, only the North Atlantic had a trend of increasing hurricane and storm days from 1970 to 2004.
a substantial decadal-scale oscillation
Between 1990 and 2000, there was a decade-long period of more frequent cyclonic storm days.
None of these time series shows a trend that is statistically different from zero over the period
Both the number of hurricanes and the number of storms, there was no statistical trends in the data. Thus it is not possible to state that the number of hurricane and storm events has increased over the period 1970 to 2004.
trends in each of the ocean basins are significantly different from zero at the 95% confidence level or higher, except for the southwest Pacific Ocean
From the 1970's to the 2000's, sea surface temperatures have been increasing through time for every ocean basin except the southwest Pacific Ocean. The statistics performed (Kendall Test) suggest this trend is real and significant.
Significance
Climate change will result in a shifting of where plants are usually located on the planet. Southern plants will move into higher latitudes, for example.
(H2) Flower and leaf bud break of high Arctic plants should be more sensitive to temperature increases than those of low Arctic and alpine plants.
The trends found indicated that high-elevation Arctic plants grew, flowered and aged at lower heat thresholds than low-elevation Arctic and alpine plants.
Flower and leaf bud break are occurring earlier as sites become warmer.
There was a strong negative relationship between the temporal trend of phenological events and the summer temperature trend for each species and site.
temporal trends of heat sums (βTDD_x_YEAR) showed significantly increased heat sums over time for greening but a tendency for lower heat sums over time for flowering and senescence.
The accumulated daily temperature required for plant growth has increased over time.
The accumulated daily temperature required for flowering and aging has decreased over time.
Trends in the timing of events as represented by βDOY_x_YEAR showed significantly later greening (positive slope) but tendencies for earlier flowering and earlier senescence (negative slopes) over the study.
With warmer temperatures, plants are growing at a later time in the year but flowering and aging at an earlier time in the year.
Plant phenological responses to these increases in season length and temperature are uncertain, but understanding changes as they occur is essential for predicting future changes in tundra vegetation processes
Constantly observing and following the seasonal changes that occur with these plants is very important in understanding the future of the plants and their habitat.
First, anti-ISIS agencies can thwart development of large aggregates that are potentially far more potent (21) by breaking up smaller ones.
This result may seem counter-intuitive, but it makes sense when we consider the costs of shutting down larger aggregates versus the costs of shutting down smaller ones. It is easier to shut down smaller aggregates.