Widespread decline in winds delayed autumn foliar senescence over high latitudes

The high northern latitudes (>50°) experienced a pronounced surface stilling (i.e., decline in winds) with climate change. As a drying factor, the influences of changes in winds on the date of autumn foliar senescence (DFS) remain largely unknown and are potentially important as a mechanism explaining the interannual variability of autumn phenology. Using 183,448 phenological observations at 2,405 sites, long-term site-scale water vapor and carbon dioxide flux measurements, and 34 y of satellite greenness data, here we show that the decline in winds is significantly associated with extended DFS and could have a relative importance comparable with temperature and precipitation effects in contributing to the DFS trends. We further demonstrate that decline in winds reduces evapotranspiration, which results in less soil water losses and consequently more favorable growth conditions in late autumn. In addition, declining winds also lead to less leaf abscission damage which could delay leaf senescence and to a decreased cooling effect and therefore less frost damage. Our results are potentially useful for carbon flux modeling because an improved algorithm based on these findings projected overall widespread earlier DFS than currently expected by the end of this century, contributing potentially to a positive feedback to climate.

Understanding the responses of the date of autumn foliar senescence (DFS) to climate change has recently received increased focus for a better interpretation of carbon uptake, but accurately predicting DFS globally using models remains challenging (1). Nonurbanized lands in the high northern latitudes (>50°) are currently a large carbon sink but have experienced the greatest increase in air temperature (2–6). In those ecosystems, the annual net ecosystem productivity (NEP) has increased for years with an earlier start of spring leaf unfolding (7–11). A delay of DFS has been reported for middle to high latitudes from both eddy covariance (FLUXNET) measurements and remotely sensed observations of vegetation reflectance (8, 12–15). Such a trend of DFS was found to contribute to an overall increase of annual NEP for temperate forests (16, 17) but may potentially offset carbon uptake due to extended ecosystem respiration for higher latitude ecosystems (3).Climate change over the last few decades has had substantial effects on vegetation phenology (18, 19). Global increases in autumn temperature could delay DFS (14, 20), yet there are also studies showing either earlier or relatively stable DFS, with a possible explanation from opposite changes in DFS in response to daytime and nighttime warming (21). Decreases in precipitation and associated drought may have more complicated influences on DFS, depending on the severity of drought and on regional characteristics of plant functional types (22). In addition to changes in temperature and precipitation, wind speed over the last three decades shows widespread decreasing trends in the northern hemisphere (23, 24) with possible impacts on plant growth, chemical composition, structure, and morphology (25–28). The drying effect of wind affects foliar gas and heat exchange and could increase water stress by reducing the thickness of foliar boundary layers (27), so the responses of DFS to wind may depend on water stress. We therefore ask 1) what are the physical and physiological impacts of declined winds on plant growth and 2) how these changes affect DFS accordingly at high northern latitudes where a pronounced decline in winds was observed. To this end, we used gridded meteorological data (temperature, precipitation, cloud cover, and wind speed) together with DFS data from three independent data sets: 1) 183,448 phenological observations at 2,405 ground sites since the 1980s, 2) 267 site-years of data from the FLUXNET eddy covariance network, across 18 long-term sites over 1994 to 2014 (SI Appendix, Fig. S1), and 3) latest Normalized Difference Vegetation Index data (NDVI, GIMMS3g.v1) for 1982 to 2015.     

Structural basis of Chikungunya virus inhibition by monoclonal antibodies

Chikungunya virus (CHIKV) is an emerging viral pathogen that causes both acute and chronic debilitating arthritis. Here, we describe the functional and structural basis as to how two anti-CHIKV monoclonal antibodies, CHK-124 and CHK-263, potently inhibit CHIKV infection in vitro and in vivo. Our in vitro studies show that CHK-124 and CHK-263 block CHIKV at multiple stages of viral infection. CHK-124 aggregates virus particles and blocks attachment. Also, due to antibody-induced virus aggregation, fusion with endosomes and egress are inhibited. CHK-263 neutralizes CHIKV infection mainly by blocking virus attachment and fusion. To determine the structural basis of neutralization, we generated cryogenic electron microscopy reconstructions of Fab:CHIKV complexes at 4- to 5-Å resolution. CHK-124 binds to the E2 domain B and overlaps with the Mxra8 receptor-binding site. CHK-263 blocks fusion by binding an epitope that spans across E1 and E2 and locks the heterodimer together, likely preventing structural rearrangements required for fusion. These results provide structural insight as to how neutralizing antibody engagement of CHIKV inhibits different stages of the viral life cycle, which could inform vaccine and therapeutic design.

Chikungunya virus (CHIKV), a single-stranded positive-sense RNA envelope virus, is an emerging alphavirus transmitted to humans by Aedes species mosquitoes (1, 2). CHIKV consists of three related genotypes: Asian, East/Central/South African (ECSA), and West African (3). According to the Centers for Disease Control and Prevention, there have been millions of cases reported in approximately 100 countries. CHIKV infection causes an acute febrile illness accompanied by musculoskeletal disease (4, 5). A subset of cases (∼30%) showed that chronic arthritis can develop and persist for months to years (6, 7).The 12-kb positive-sense RNA genome is packaged within an icosahedral nuclear capsid core composed of 240 copies of capsid proteins, which is surrounded by a host-derived lipid bilayer. The surface of the mature CHIKV particle (diameter ∼700 Å) has 80 trimeric envelope E1-E2 heterodimer protein spikes anchored on the lipid bilayer membrane (SI Appendix, Fig. S1A) arranged in T = 4 icosahedral symmetry. E1 and E2 protein ectodomains each consist of three domains: E1-DI; E1-DII and E1-DIII; and E2-DA, E2-DB, and E2-DC (SI Appendix, Fig. S1B). The fusion loop on the distal end of E1-DII mediates endosomal membrane fusion. The groove formed by E2-DA and E2-DB shields the fusion loop of E1 protein from premature membrane fusion at neutral pH (8). Multiple attachment factors have been implicated in CHIKV entry of cells (9), and E2-DB reportedly contains receptor-binding sites (10, 11). Mxra8, a recently identified alphavirus receptor (12), recognizes an epitope spanning both the E1 and E2 proteins (13, 14).The virus infection cycle starts with the E1-E2 proteins binding to the cell-surface receptors (12). The virion is then internalized into the endosome (15, 16). The acidic condition of the endosome causes E1-E2 heterodimers to undergo conformational changes, exposure of the E1 fusion loop for insertion into the endosomal membrane, and subsequent reorganization of the E1 protein into E1 trimers to allow endosomal membrane fusion (17, 18). After fusion, the capsid and RNA genome are released into the cytoplasm (19) to allow translation and replication of the viral genome. The newly synthesized virus buds at the plasma membrane (20).Currently, there exist no licensed CHIKV vaccine or therapeutics. Neutralizing antibodies have been shown to confer both prophylactic and therapeutic protection in animal models (21–28). Here we show the potencies of two CHIKV antibodies, CHK-124 and CHK-263, in vivo and demonstrate that they inhibit multiple steps in the virus infection cycle in vitro. We also determined the cryogenic electron microscopy (cryo-EM) structures of their Fab fragments complexed with CHIKV to 4- to 5-Å resolution. For CHK-124, the predominant neutralization mechanisms are aggregation of virus particles and inhibition of receptor binding. For CHK-263, the mechanism is the inhibition of fusion by locking E1 and E2 proteins together. Altogether, our study provides a structural understanding as to how potent antibodies block CHIKV infection.     

Anthropogenic depletion of Iran’s aquifers

Global groundwater assessments rank Iran among countries with the highest groundwater depletion rate using coarse spatial scales that hinder detection of regional imbalances between renewable groundwater supply and human withdrawals. Herein, we use in situ data from 12,230 piezometers, 14,856 observation wells, and groundwater extraction points to provide ground-based evidence about Iran’s widespread groundwater depletion and salinity problems. While the number of groundwater extraction points increased by 84.9% from 546,000 in 2002 to over a million in 2015, the annual groundwater withdrawal decreased by 18% (from 74.6 to 61.3 km³/y) primarily due to physical limits to fresh groundwater resources (i.e., depletion and/or salinization). On average, withdrawing 5.4 km³/y of nonrenewable water caused groundwater tables to decline 10 to 100 cm/y in different regions, averaging 49 cm/y across the country. This caused elevated annual average electrical conductivity (EC) of groundwater in vast arid/semiarid areas of central and eastern Iran (16 out of 30 subbasins), indicating “very high salinity hazard” for irrigation water. The annual average EC values were generally lower in the wetter northern and western regions, where groundwater EC improvements were detected in rare cases. Our results based on high-resolution groundwater measurements reveal alarming water security threats associated with declining fresh groundwater quantity and quality due to many years of unsustainable use. Our analysis offers insights into the environmental implications and limitations of water-intensive development plans that other water-scarce countries might adopt.

Groundwater is the backbone of water and food security in arid/semiarid areas, including Iran, with spatial and temporal changes due to natural surface water variability and scarcity. Groundwater provides about 60% of the total water supply in Iran (1), where agriculture is responsible for more than 90% of water withdrawal (2). Systematic groundwater extraction in Iran dates back at least two and a half millennia, when underground aqueducts known as “qanats” were excavated to transfer groundwater to the surface under the force of gravity (3). The Persian qanats that had facilitated development and agricultural production in Iran for thousands of years mostly dried up with technological advances and modernization of agriculture in the 20th century (4). Deep well drilling made groundwater overexploitation possible, while increased surface water damming and diversion reduced groundwater recharge, together drawing down groundwater tables (SI Appendix, Fig. S1) and making groundwater harvesting through historical qanats less feasible. Aggressive water resources development (1, 2, 5) to support the livelihood of over 80 million people and irrigate about 5.9 million ha of agricultural land heightened the pressure on groundwater. Iran’s water scarcity in the 21st century has been exacerbated by frequent droughts and climatic changes (2, 6). On average, more than half of the design capacity of Iran’s reservoirs was empty from 2003 to 2017 (7), increasing the reliance on groundwater. Consequently, Iran was ranked among the countries with the highest groundwater depletion rate in the 21st century, along with India, the United States, Saudi Arabia, and China (8).Iran is grappling with acute water management problems and tensions (9, 10). Groundwater overdraft has contributed to a host of contemporary socioecological problems, including the drying up of wetlands, desertification, sand and dust storms, deteriorating water quality, and population displacement (10, 11). Land subsidence due to groundwater depletion is now a manmade hazard to vital infrastructure and residents in vulnerable plains (12). Further, declining groundwater tables have degraded groundwater quality due to natural processes such as saltwater intrusion (13–15). The increasing strain on rural livelihoods and mounting tensions among groundwater users exacerbate food and water security risks (16), and create sociopolitical issues related to the migration of rural populations to urban areas (17).Groundwater assessments based on global models and remote sensing approaches have offered high-level characterizations of Iran’s groundwater resources (8, 18, 19). However, these investigations are limited by coarse spatial scales and large uncertainties due to lack of ground-truth data, hindering the detection of regional imbalances between renewable groundwater supply and human withdrawals. This study provides a statistical analysis of the major groundwater characteristics using a rich ground-based dataset (2002 to 2015) to determine the groundwater depletion and salinization in all 30 subbasins of Iran (SI Appendix, Fig. S2). The investigation of the temporal trend and spatial distribution of groundwater depletion and salinity provides valuable information for effective management of aquifers across Iran, and offers insights to other countries facing similar water security issues.     

Sea spray aerosol concentration modulated by sea surface temperature

Natural aerosols in pristine regions form the baseline used to evaluate the impact of anthropogenic aerosols on climate. Sea spray aerosol (SSA) is a major component of natural aerosols. Despite its importance, the abundance of SSA is poorly constrained. It is generally accepted that wind-driven wave breaking is the principle governing SSA production. This mechanism alone, however, is insufficient to explain the variability of SSA concentration at given wind speed. The role of other parameters, such as sea surface temperature (SST), remains controversial. Here, we show that higher SST promotes SSA mass generation at a wide range of wind speed levels over the remote Pacific and Atlantic Oceans, in addition to demonstrating the wind-driven SSA production mechanism. The results are from a global scale dataset of airborne SSA measurements at 150 to 200 m above the ocean surface during the NASA Atmospheric Tomography Mission. Statistical analysis suggests that accounting for SST greatly enhances the predictability of the observed SSA concentration compared to using wind speed alone. Our results support implementing SST into SSA source functions in global models to better understand the atmospheric burdens of SSA.

Over two-thirds of the Earth is covered by the ocean. The material exchange between the ocean and the atmosphere affects the balance of the Earth’s energy on a global scale (1). Sea spray aerosol (SSA) is the major particulate material directly emitted from the ocean. Studies have shown that SSA dominates the aerosol mass in the marine boundary layer (MBL). Such dominance renders SSA an important player in climate change (2). However, the exact processes by which the SSA is introduced to the atmosphere still remains to be learned, making the SSA budget highly uncertain (3).It is generally established that SSA is produced by mechanical processes (4–6). Wind stress induces breaking waves that entrain bubbles into the surface ocean (7). Film and jet drops formed during bubble bursting are the main sources of SSA particles (8). The wind-driven mechanism is supported by the positive correlation between wind speed and SSA concentration from field observations (9, 10). Therefore, wind speed is used as the sole parameter to characterize SSA in many models (1, 4, 11).In addition to wind speed, sea surface temperature (SST) may play a large role in SSA production (12–15). SST affects the drop formation process by modifying the physical properties of the surface ocean water. An increase of SST reduces the kinematic viscosity and surface tension of the ocean, thereby enhancing the entrainment efficiency and rising speed of bubbles (12, 16). As a result, the number size distribution of the bubbles may change, leading to varying SSA properties (14, 15, 17).Limited laboratory and field studies regarding the effects of SST on SSA production have shown disparate results. Some argue that SSA production is independent of SST (18) or suppressed by increasing SST (14, 15, 19, 20) from 0 to 10 °C, while other laboratory (12, 21–23) and field measurements (3, 5) suggest that SSA production increases monotonically with water temperature. Furthermore, recent observations in the remote Atlantic Ocean shows that increasing SST enhances the modal mean diameter of SSA (24). On the other hand, model simulations have demonstrated that incorporating SST into SSA source functions generally improves the SSA prediction (3, 25, 26). The inconsistency in the previous work suggests that the impacts of SST on SSA formation remain unclear.In this study, we conducted unprecedented aircraft measurements of SSA concentration on a global scale during the Atmospheric Tomography Mission (ATom). These measurements consist of a series of flights spanning three seasons (summer, fall, and winter) over remote oceans (Fig. 1 and SI Appendix, Fig. S1). Our observations again confirm that wind speed is the dominant factor controlling the concentration of SSA. Further, we show that increasing SST enhances the mass concentration of SSA.Open in a separate windowFig. 1.Flight tracks during ATom2. The color indicates the flight altitude. The size of the markers represents the sea salt number fraction. The inset in the bottom right shows the vertical profile of sea salt number fraction. The flight tracks during ATom3 and ATom4 are similar to ATom2.     

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

Stage-specific overcompensation,the hydra effect,and the failure to eradicate an invasive predator

As biological invasions continue to increase globally, eradication programs have been undertaken at significant cost, often without consideration of relevant ecological theory. Theoretical fisheries models have shown that harvest can actually increase the equilibrium size of a population, and uncontrolled studies and anecdotal reports have documented population increases in response to invasive species removal (akin to fisheries harvest). Both findings may be driven by high levels of juvenile survival associated with low adult abundance, often referred to as overcompensation. Here we show that in a coastal marine ecosystem, an eradication program resulted in stage-specific overcompensation and a 30-fold, single-year increase in the population of an introduced predator. Data collected concurrently from four adjacent regional bays without eradication efforts showed no similar population increase, indicating a local and not a regional increase. Specifically, the eradication program had inadvertently reduced the control of recruitment by adults via cannibalism, thereby facilitating the population explosion. Mesocosm experiments confirmed that adult cannibalism of recruits was size-dependent and could control recruitment. Genomic data show substantial isolation of this population and implicate internal population dynamics for the increase, rather than recruitment from other locations. More broadly, this controlled experimental demonstration of stage-specific overcompensation in an aquatic system provides an important cautionary message for eradication efforts of species with limited connectivity and similar life histories.

Theoretical population models can produce counterintuitive predictions regarding the consequences of harvest or removal of predatory species. These models show that for simple predator-prey systems, there can be positive population responses to predator mortality resulting from harvest for fisheries or population management, which can create an increased equilibrium level of that predator species (1–5). Among these mortality processes is the “hydra effect,” named after the mythical multi-headed serpent that grew two new heads for each one that was removed (6, 7). This counterintuitive outcome can be driven by a density-dependent process known as overcompensation. The hydra effect typically refers to higher equilibrium or time-averaged densities in response to increased mortality, typically involving consumer populations undergoing population cycles. Population increases in response to mortality can be the result of stage-specific overcompensation, which involves an increase in a specific life history stage or a size class following increased mortality. The first analysis of overcompensatory responses to mortality did not depend on stage specificity and was applied initially to fisheries harvests (1). Subsequent models have included stage specificity and have been applied to a broad range of systems in which species have been harvested for consumption or removed for population control of non-native species (4, 5, 8–15).Theory suggests that overcompensation in response to harvest or removal can occur for a variety of reasons, including 1) reduced competition for resources and increased adult reproduction rates, 2) faster rates of juvenile maturation or greater success in reaching the adult stage, and 3) increased juvenile or adult survival rates (1–7). An increase in reproductive output in response to reduced adult density can be the result of a reduction in resource competition (SI Appendix, Fig. S1).While there is substantial evidence that conditions that could produce density-dependent overcompensation occur frequently, evidence for overcompensation in natural populations is rare. For only a few populations do we have the long-term demographic data collected over a sufficiently long duration and for population densities over a wide enough range to detect this effect. Unfortunately, recent reviews of population increases in response to increased mortality do not include field studies with explicit controls for removals (13–17).There are examples of density-dependent overcompensation from field populations (4, 13–15), as well as a larger number of studies from the laboratory and greenhouse typically involving plant and insect populations (18–22). Among the field examples is a population control program for smallmouth bass in a lake in upstate New York, which paradoxically resulted in greater bass abundance, primarily of juveniles, after 7 y of removal efforts (23, 24). Another field study in the United Kingdom showed that perch populations responded similarly when an unidentified pathogen decimated adults (25). Other programs that attempted to remove invasive fishes, including pikeperch in England (26), brook trout in Idaho (27), and Tilapia in Australia (28), showed similar results. However, although many of these examples involved well-executed studies with substantial field data, none had explicit controls for removal, such as comparable populations without harvest (or disease). Thus, despite the support of current theory in these studies, the contribution of external factors to observed population responses to harvest remains uncertain. To date, we are unaware of any experimental studies with comparable controls in a field population that demonstrates overcompensation in a single species (13–15).     

Dynamic competition between SARS-CoV-2 NSP1 and mRNA on the human ribosome inhibits translation initiation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a beta-CoV that recently emerged as a human pathogen and is the causative agent of the COVID-19 pandemic. A molecular framework of how the virus manipulates host cellular machinery to facilitate infection remains unclear. Here, we focus on SARS-CoV-2 NSP1, which is proposed to be a virulence factor that inhibits protein synthesis by directly binding the human ribosome. We demonstrate biochemically that NSP1 inhibits translation of model human and SARS-CoV-2 messenger RNAs (mRNAs). NSP1 specifically binds to the small (40S) ribosomal subunit, which is required for translation inhibition. Using single-molecule fluorescence assays to monitor NSP1–40S subunit binding in real time, we determine that eukaryotic translation initiation factors (eIFs) allosterically modulate the interaction of NSP1 with ribosomal preinitiation complexes in the absence of mRNA. We further elucidate that NSP1 competes with RNA segments downstream of the start codon to bind the 40S subunit and that the protein is unable to associate rapidly with 80S ribosomes assembled on an mRNA. Collectively, our findings support a model where NSP1 proteins from viruses in at least two subgenera of beta-CoVs associate with the open head conformation of the 40S subunit to inhibit an early step of translation, by preventing accommodation of mRNA within the entry channel.

Beta-coronaviruses (CoVs) are a family of RNA viruses that include human pathogens (1). In the last two decades, two beta-CoVs have emerged from animal hosts to cause epidemic diseases of the human respiratory tract: severe acute respiratory syndrome (SARS-CoV, in 2002) (2, 3) and Middle East respiratory syndrome (MERS-CoV, in 2012) (4). A third beta-CoV emerged in late 2019—SARS-CoV-2—that is responsible for the ongoing COVID-19 pandemic (5). Given the lack of effective therapies against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus manipulates the machineries present in human cells.SARS-CoV-2 and the closely related SARS-CoV have single-stranded, positive-sense RNA genomes nearly 30 kb in length (6, 7). Upon entry of a virion into human cells, the genomic RNA is released into the cytoplasm where it must hijack human translation machinery to synthesize viral proteins (8). As the genomic RNA has a 7-methylguanosine (m⁷G) cap on the 5′ terminus, viral protein synthesis likely proceeds via a process reminiscent of that which occurs on typical human messenger RNAs (mRNAs) (9). However, as viral proteins accumulate, human translation is inhibited and host mRNAs are destabilized, which facilitates suppression of the host immune response (10–13).Studies on SARS-CoV have implicated nonstructural protein 1 (NSP1), the first encoded viral protein, as a virulence factor with a key role in the shutdown of host translation (10, 11, 14). In infected cells or upon its ectopic expression, NSP1 inhibits human translation, which is dependent on its association with the small (40S) subunit of the human ribosome (12–17). In a linked but separable activity, NSP1 destabilizes at least a subset of human mRNAs, likely via recruitment of an unidentified human endonuclease (12, 13, 15, 16, 18). NSP1 from SARS-CoV-2 is expected to employ similar mechanisms, given its ∼85% sequence identity with the SARS-CoV protein. Indeed, SARS-CoV-2 NSP1 inhibits translation by binding to the 40S subunit (19–21). Thus, NSP1 has a near-singular ability to disrupt host gene expression dramatically; yet, the mechanism by which this inhibition occurs is not clear.The 40S subunit is the nexus of translation initiation, recruiting an m⁷G-capped mRNA through a multistep, eukaryotic initiation factor (eIF)-mediated process. Prior to recruitment of an mRNA, the 40S subunit is bound by numerous eIFs, which include eIF1, eIF1A, eIF3, eIF5, and the ternary complex (TC) of eIF2–GTP–methionine initiator transfer RNA (tRNA_i^Met) (22). The eIFs make extensive contacts with the 40S subunit, including the ribosomal A and P sites (23, 24). They also manipulate the dynamics of the 40S head region to facilitate mRNA recruitment, which has structural consequences at both the mRNA entry (3′ side of mRNA) and exit (5′ end of mRNA) channels. Following mRNA recruitment and directional scanning of the 5′ untranslated region (UTR) to a start codon, a series of compositional and conformational changes occur (25, 26). This ultimately repositions the 40S subunit head into the closed conformation and accommodates the anticodon stem loop of the initiator tRNA at the start codon (23–26), enabling recruitment of the 60S subunit and entry into the elongation phase.Recently, structures of NSP1 bound to human ribosomes have been reported (19–21), including 40S preinitiation and 80S ribosomal complexes. In all instances, the N-terminal globular domain of NSP1 is flexibly localized to the solvent-exposed surface of the 40S subunit, near the entrance to the mRNA entry channel (SI Appendix, Fig. S1A). This domain is anchored by the two most C-terminal α-helices of NSP1, which were dynamic and unstructured in the free SARS-CoV NSP1 structure solved by NMR (27); in the NSP1–40S subunit complex, these helices were well resolved and docked within the mRNA entry channel, where they contact ribosomal proteins uS3 and uS5, and helix 18 of the 18S ribosomal RNA (rRNA). As noted above, this location on the ribosome is structurally flexible, adopting open and closed states upon swiveling of the 40S subunit head (23–25). The position of NSP1 in the mRNA channel may also conflict with the position of fully accommodated mRNA. Thus, the intrinsic dynamics of translation initiation present many opportunities and obstacles for NSP1 association with the ribosome, and its subsequent inhibition of translation.Here, we merge biochemical and single-molecule approaches to probe the molecular function of SARS-CoV-2 NSP1 and its interaction with the human ribosome. We showed that NSP1 potently inhibited translation of human and SARS-CoV-2 model mRNAs, and determined how the NSP1–40S subunit interaction was modulated by eIFs and mRNA. Our results reveal allosteric control of NSP1 association by key eIFs and identify a conformation of the ribosomal subunit compatible with rapid NSP1 association. They also define the dynamic competition between NSP1 and mRNA to bind the ribosome. When synthesized with recent structures, our study suggests a mechanism for how NSP1 inhibits translation initiation.     

Neural stem cells secreting bispecific T cell engager to induce selective antiglioma activity

Glioblastoma (GBM) is the most lethal primary brain tumor in adults. No treatment provides durable relief for the vast majority of GBM patients. In this study, we''ve tested a bispecific antibody comprised of single-chain variable fragments (scFvs) against T cell CD3ε and GBM cell interleukin 13 receptor alpha 2 (IL13Rα2). We demonstrate that this bispecific T cell engager (BiTE) (BiTE^LLON) engages peripheral and tumor-infiltrating lymphocytes harvested from patients'' tumors and, in so doing, exerts anti-GBM activity ex vivo. The interaction of BiTE^LLON with T cells and IL13Rα2-expressing GBM cells stimulates T cell proliferation and the production of proinflammatory cytokines interferon γ (IFNγ) and tumor necrosis factor α (TNFα). We have modified neural stem cells (NSCs) to produce and secrete the BiTE^LLON (NSC^LLON). When injected intracranially in mice with a brain tumor, NSC^LLON show tropism for tumor, secrete BiTE^LLON, and remain viable for over 7 d. When injected directly into the tumor, NSC^LLON provide a significant survival benefit to mice bearing various IL13Rα2⁺ GBMs. Our results support further investigation and development of this therapeutic for clinical translation.

Routine treatment of newly diagnosed glioblastoma (GBM) consists of surgical resection, chemotherapy, and radiation, which results in a median GBM patient survival of less than 2 y, with just 5% of patients surviving beyond 5 y (1). The blood–brain barrier (BBB) limits therapeutic access to the tumor (2). An immunosuppressive microenvironment and molecular heterogeneity of GBM present a unique set of challenges for developing effective therapies for this type of brain tumor (3–10).The development of treatments for lessening the immunosuppressive effects of GBM represents an active area of preclinical and clinical neuro-oncology research. Many, if not all, approaches being tested involve increasing T cell cytotoxic antitumor activity (11–17). Large numbers of functional cytotoxic tumor-infiltrating lymphocytes (TILs) correlate with improved progression-free survival for GBM patients (18–21). However, the immunosuppressive milieu of GBM impairs T cell cytolytic function, altering the effectiveness of T cell-based therapies for treating GBM (22–26). Numerous lymphocyte-directed treatments are being investigated, including the use of bispecific T cell engagers (BiTEs) (17, 27, 28). BiTEs can be produced and used without patient-specific BiTE individualization and can, therefore, be considered “off-the-shelf” therapeutics (29–32). The use of BiTEs targeting tumor-associated antigens (TAAs) has been approved by the Food and Drug Administration (FDA) in treating liquid malignancies, and BiTE-associated treatments are currently being evaluated in multiple clinical studies for solid tumors (e.g., {"type":"clinical-trial","attrs":{"text":"NCT03792841","term_id":"NCT03792841"}}NCT03792841, {"type":"clinical-trial","attrs":{"text":"NCT04117958","term_id":"NCT04117958"}}NCT04117958, and {"type":"clinical-trial","attrs":{"text":"NCT03319940","term_id":"NCT03319940"}}NCT03319940) (33–36).BiTEs consist of two single-chain variable fragments (scFvs) connected by a flexible linker (37). One of the scFvs is directed to a TAA and the other to CD3 epsilon (ε) that is expressed on T cells (38). BiTEs engage TILs and cancer cells in a major histocompatibility complex (MHC)-independent manner and are, therefore, unaffected by MHC down-regulation that occurs in GBM cells (37–40). The specificity of BiTE''s tumor antigen-directed scFv is imperative to harness the full therapeutic potential of the recombinant molecule (41). BiTE anticancer activity requires BiTE binding with malignant and immune cells simultaneously; single-arm binding to a tumor antigen or CD3ε is therapeutically ineffective (42, 43).The short half-life of BiTEs in plasma necessitates a frequent or continuous infusion into patient circulation to maintain therapeutic levels of BiTE (43–45). Several approaches have been proposed and tested to compensate for the rate of BiTE biologic life in treating peripheral cancers (45–47). These include the recombinant protein''s sustained production by subcutaneous injection of mesenchymal stem cells (MSCs) seeded into a synthetic extracellular matrix scaffold, liver translation of BiTE messenger RNA (mRNA), and peritumoral delivery of MSCs secreting BiTEs. A recent study also explored the use of modified immune cell delivery of BiTE to GBM (48), and the reduction of tumor burden in treated animals has been observed. It remains to be determined if these bicistronic antiglioma treatments share chimeric antigen receptor (CAR) T cells'' fate, which includes a low penetrance and short survival of CARs within the brain (27, 49, 50); both are limiting factors for sustained and efficient delivery of BiTEs by CAR T cells.Neural stem cells (NSCs) have inherent advantages as a cellular carrier of antineoplastic agents to the site of GBM since they are native to the brain. NSCs have demonstrated tropism to brain tumors in several preclinical models. These cells can withstand a harsh oxygen-deprived environment of GBM. Here, we investigated NSCs as producers of BiTEs targeting the tumor-associated antigen interleukin 13 receptor alpha 2 (IL13Rα2) and their antitumor activity using in vitro and in vivo models of GBM. In vitro, BiTEs show significant antitumor activity when used in cocultures that include T cells harvested from patients’ blood and tumor tissue. In vivo, NSCs modified for BiTE synthesis migrate to a tumor in animal subjects’ brains while functioning as intra- and peritumoral BiTE producers. The following are details of the results from our experiments in characterizing NSCs that produce IL13Rα2-directed BiTEs. We interpret these findings as support for additional safety and efficacy analysis for their potential clinical translation in treating GBM patients.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

From the Cover: Radiation-induced neoantigens broaden the immunotherapeutic window of cancers with low mutational loads

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use Kras^G12D x p53^−/− sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8⁺ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.

Immune checkpoint therapy (ICT) can lead to durable responses in subsets of cancer patients (1–8). On the basis of computational analyses, the patients who most benefit from ICT are those with cancers that have high mutational burden (9–18). For example, patients bearing tumors with high mutational burden caused by environmental exposure (such as ultraviolet-induced melanoma) or deficiencies in DNA repair (such as microsatellite instability-high colorectal cancers) tend to respond well to immunotherapy (18–26). Presumably the sensitivity of such cancers reflects the increased likelihood of formation of immunogenic, tumor-specific mutant neoantigens (27). We and others previously showed that certain tumor-specific neoantigens are major targets of natural and therapeutically induced antitumor responses in both mice and humans (28–41). Therefore, the presence of immunogenic tumor neoantigens is currently thought to contribute to tumor sensitivity to immunotherapy.However, many cancer patients do not respond to ICT, suggesting that their neoantigen burden is either of insufficient magnitude or immunogenicity to function as targets for T cell-dependent antitumor mechanisms. Indeed, there are many tumor types, such as acute myeloid leukemia, estrogen receptor-positive breast, and prostate cancers, that have limited mutational burdens and display low response rates to ICT (9, 13, 42, 43). Additionally, tumor cell clones expressing immunogenic neoantigens that develop during tumor evolution may be eliminated from tumors with high mutational burden by the process of cancer immunoediting, resulting in outgrowth of tumor cell clones with reduced immunogenicity that can then grow progressively in the presence of the unmanipulated immune system (33, 44, 45). Therefore, a process by which tumors with low neoantigen burden can acquire immunogenic mutations has the potential to expand the number of patients able to benefit from ICT.Ionizing radiation has been shown to elicit DNA damage in tumor cells, leading to an increase in overall mutational load (46–52). This damage is thought to occur primarily through generation of reactive oxygen species which induce base pair substitutions by mechanisms involving transitions, transversions, and/or faulty DNA repair (53). Multiple preclinical studies have demonstrated antitumor responses when focal radiation is combined with ICT in tumors that do not respond to ICT alone (54–60) and several clinical studies have demonstrated that human tumor patients have improved responsiveness to ICT following focal radiation (e.g., {"type":"clinical-trial","attrs":{"text":"NCT02303990","term_id":"NCT02303990"}}NCT02303990, {"type":"clinical-trial","attrs":{"text":"NCT02298946","term_id":"NCT02298946"}}NCT02298946, {"type":"clinical-trial","attrs":{"text":"NCT02383212","term_id":"NCT02383212"}}NCT02383212) (61–67). Radiation has been demonstrated to function as an in vivo tumor vaccine by inducing damage-associated molecular patterns (DAMP)-dependent immunogenic cell death (68), inducing DNA damage sensed by pattern recognition receptors (69, 70), enhancing access of immune effector cells to their cognate targets through tumor cell debulking and vasculature changes (71, 72), up-regulating major histocompatibility complex class I (MHC-I) receptors (73), up-regulating cell-surface molecules such as Fas (74), and augmenting tumor antigen cross-presentation by specific subsets of dendritic cells through up-regulation of type I interferon (IFN), which results in increased numbers and action of tumor-specific CD8⁺ T cells (75–77). However, none of these explanations take into account that following irradiation, tumor cells acquire novel mutations that may function as effective tumor neoantigens. In fact, two groups have demonstrated broadening of the T cell repertoire following radiation treatment of mouse 4T1 mammary tumors and B16F10 melanoma tumors (56, 78). Radiation-induced neoantigens may partially explain the broadening of the T cell repertoire reported during noncurative doses of irradiation.Given the above observations, we specifically explored whether one dose of in vitro irradiation could increase the immunogenicity of poorly immunogenic tumor cell lines through mechanisms involving the de novo generation of tumor-specific mutant neoantigens. For this purpose, we used a mouse Kras^G12D x p53^−/− sarcoma cell line as a model system since the R.D.S. and T.J. laboratories have previously shown that these tumor cells express a very limited number of somatic mutations, are essentially devoid of mutational neoantigens, and are nonimmunogenic and grow progressively in syngeneic wild-type (WT) mice either following treatment with control antibody or the combination of anti–PD-1/anti–CTLA-4 (34, 41). We find that treating these cell lines with noncurative doses of irradiation induces expression of somatic mutations, some of which function as neoantigens and render the sarcoma cells susceptible to ICT in vivo. These data support the concept that an additional mechanism underlying the synergy between radiation therapy and immunotherapy is that the former induces immunogenic mutations in tumors that now function as targets for the latter.     

Anthropogenic climate change is worsening North American pollen seasons

Airborne pollen has major respiratory health impacts and anthropogenic climate change may increase pollen concentrations and extend pollen seasons. While greenhouse and field studies indicate that pollen concentrations are correlated with temperature, a formal detection and attribution of the role of anthropogenic climate change in continental pollen seasons is urgently needed. Here, we use long-term pollen data from 60 North American stations from 1990 to 2018, spanning 821 site-years of data, and Earth system model simulations to quantify the role of human-caused climate change in continental patterns in pollen concentrations. We find widespread advances and lengthening of pollen seasons (+20 d) and increases in pollen concentrations (+21%) across North America, which are strongly coupled to observed warming. Human forcing of the climate system contributed ∼50% (interquartile range: 19–84%) of the trend in pollen seasons and ∼8% (4–14%) of the trend in pollen concentrations. Our results reveal that anthropogenic climate change has already exacerbated pollen seasons in the past three decades with attendant deleterious effects on respiratory health.

Human-caused climate change is expected to have widespread negative impacts on public health through a range of pathways (1–3). Climate change could trigger spatial and temporal shifts in plant airborne pollen loads, which have major respiratory health consequences for allergies and asthma (4–7), viral infections (8), school performance and downstream economic impacts (9), and emergency room visits (5, 10). Because pollen concentrations are often highly temperature-sensitive (11, 12), anthropogenic climate change could substantially harm respiratory health by increasing pollen concentrations and/or lengthening pollen seasons and exposure times (13–15). Thus, understanding the spatial and temporal variation in pollen loads and whether anthropogenic climate change is a major contributor to such changes at large geographical (e.g., continental) scales is urgently needed to estimate potential changes in respiratory health.Climate change detection and attribution analysis is a powerful tool for linking long-term climate change and observed impacts (16, 17). However, detection and attribution techniques have not been widely applied to public health impacts, despite major implications for policy and public health interventions (18). Detection and attribution approaches provide a substantial advance by connecting societal impacts to ongoing climate change and rigorously quantifying the role of human forcing of the climate in trends of impacts (18). Detection and attribution approaches aim to statistically detect whether a variable/impact is changing and attribute how much of the observed change was contributed by anthropogenic climate change (19–21).Among climate-related health impacts, pollen trends may be particularly suited to detection and attribution because both elevated temperature and CO₂ concentrations have been found to increase pollen production in greenhouse or growth chamber experimental studies (22–25). A few long-term observational studies on selected plant taxa or at a small number of sites have found increases in pollen concentrations and pollen season length over time, often correlated with temperature (11, 12, 15, 26), although temperature–pollen season relationships may depend on chilling requirements in some taxa (27, 28). Yet a continental-scale detection of long-term pollen trends with a formal attribution to anthropogenic climate change is lacking.Here, we leverage a continental-scale dataset of long-term pollen records from 60 North American cities spanning 1990–2018 (821 site-years of data; SI Appendix, Table S1), observational climate datasets, and a suite of simulations from 22 Earth system models to conduct a detection and attribution analysis on spatial and temporal characteristics of aero-allergenic pollen trends. We ask: 1) What are the long-term trends in common pollen metrics; i.e., can trends be detected in various estimates of pollen season severity? 2) Do climate—temperature and precipitation variables—and/or increasing atmospheric CO₂ concentrations play a prominent role in driving interannual variation and trends in pollen metrics? 3) How much of the observed temporal trends in pollen metrics can be attributed to recent human-caused changes in climate?     

CAP1 binds and activates adenylyl cyclase in mammalian cells

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

CAP/srv2 was originally identified in yeast biochemically as an adenylyl cyclase–associated protein (1) and genetically as a suppressor of the hyperactive Ras2-V19 allele (2). CAP/srv2-deficient yeast cells are unresponsive to active Ras2, and adenylyl cyclase activity is no longer regulated by Ras2 in these cells (1, 2), indicating the involvement of CAP/srv2 in the Ras/cyclase pathway. However, some mutant CAP/srv2 alleles presented phenotypes not observed in strains with impaired Ras/cyclase pathway (1–3), indicating the existence of Ras/cyclase-independent functions downstream of CAP/srv2. These two phenotype groups, that is, Ras/cyclase-linked and Ras/cyclase-independent, could be suppressed by expression of an N-terminal half and a C-terminal half of CAP/srv2, respectively (4). Subsequent studies showed that the C-terminal half of CAP/srv2 was able to bind monomeric G-actin (5–8) and other actin regulators establishing a role in F-actin dynamics (9–16). Thus, CAP/srv2 is a bifunctional protein with an N-terminal domain involved in Ras/cyclase regulation and a C-terminal domain involved with F-actin dynamics regulation (16–18).CAP1 is structurally conserved in all eukaryotes (18–22); however, their functions are not. Expression of the closely related Schizosaccharomyces pombe cap or mammalian CAP1 in yeast can only suppress the phenotypes associated with deletion of CAP/srv2’s C-terminal but not its N-terminal domain (19, 20, 22), suggesting that only the F-actin dynamics function was conserved while the Ras/cyclase regulation diverged early on in evolution (16–18). CAP/srv2’s N-terminal 1 to 36 domain was sufficient for cyclase binding in yeast involving a conserved RLE motif with predicted coiled-coil folding (23). Interestingly, this domain is also involved in CAP1 oligomerization both in yeast and mammalian cells (24–26), where it purifies as a high-molecular complex of ∼600 kDa consistent with a 1:1 stoichiometric CAP1-actin hexameric organization (12, 25, 27, 28). Importantly, removal of this domain disrupted CAP1 oligomerization, reduced F-actin turnover in vitro and caused defects in cell growth, cell morphology, and F-actin organization in vivo (24, 29). However, whether the conserved RLE motif in mammalian CAP1 interacts with other coiled-coil–containing proteins is for the moment unknown.Ras2-mediated cyclase regulation in yeast requires its farnesylation (30–32). However, the lipid target involved was not identified in the original studies. We have recently shown that mammalian CAP1 interacts with the small GTPase Rap1. The interaction involves Rap1’s C-terminal hypervariable region (HVR) and its lipid moiety in a geranylgeranyl-specific manner; that is, neither the closely related Ras1 nor engineered farnesylated Rap1 interacted with CAP1 (33). Thus, we raised the question whether CAP1-Rap1, similar to CAP/srv2-Ras2 in yeast, plays a role in cAMP dynamics in mammalian cells.In this study, we report that CAP1 binds to and activates mammalian adenylyl cyclase in vitro. The interaction involves CAP1’s conserved RLE motifs and cyclase’s conserved catalytic subdomains (e.g., C1a and/or C2a). Most importantly, we show that both CAP1 and Rap1 modulate cAMP dynamics in live cells and are critical players in cAMP-dependent proliferation.     

Cryo-EM structure of Mycobacterium smegmatis DyP-loaded encapsulin

Encapsulins containing dye-decolorizing peroxidase (DyP)-type peroxidases are ubiquitous among prokaryotes, protecting cells against oxidative stress. However, little is known about how they interact and function. Here, we have isolated a native cargo-packaging encapsulin from Mycobacterium smegmatis and determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). This encapsulin comprises an icosahedral shell and a dodecameric DyP cargo. The dodecameric DyP consists of two hexamers with a twofold axis of symmetry and stretches across the interior of the encapsulin. Our results reveal that the encapsulin shell plays a role in stabilizing the dodecameric DyP. Furthermore, we have proposed a potential mechanism for removing the hydrogen peroxide based on the structural features. Our study also suggests that the DyP is the primary cargo protein of mycobacterial encapsulins and is a potential target for antituberculosis drug discovery.

Compartmentalization is used by cells to overcome many difficult metabolic and physiological challenges (1). Eukaryotes employ membrane-bound organelles such as the mitochondrion (2); however, most prokaryotes rely on alternative proteinaceous compartments to achieve spatial control (3), one of which is the encapsulin nanocompartment.Encapsulins are newly identified nanocompartments but have already been applied in various scientific fields due to the unique structures (4, 5). It has been reported that more than 900 putative encapsulin systems in bacteria and archaea exist and are distributed across 15 bacterial and two archaeal phyla (6, 7), suggesting they are functionally diverse. Encapsulins are made of one type of shell protein, as opposed to several as is observed in many bacterial microcompartments (8, 9). The key feature of encapsulin systems is that cargo proteins can be specifically encapsulated and targeted to the encapsulin capsid interior, using a selective C-terminal sequence referred to as targeting peptides (TPs) (10). The functions of the nanocompartment are associated with the functions of its protein cargo. Many functionally diverse cargo proteins are associated with encapsulins, including dye-decolorizing peroxidases (DyPs) (11), ferritin-like proteins (FLP) (12), hydroxylamine oxidoreductase (HAO) (13), and cysteine desulfurases (14). Moreover, it has been shown that some encapsulin systems may possess multiple cargo proteins, which are made up of one core cargo protein and up to three secondary cargo proteins according to the TPs (6). Notably, a large proportion of native cargo proteins are DyP-type peroxidases, conferring the resistance of the cell to oxidative stress (6, 7, 11, 15–18). However, to date, the structural information on the cargo-encapsulated encapsulins is not yet available (SI Appendix, Table S1), and thus, little is known about the structural arrangement and mechanistic features of the cargo proteins loaded in the encapsulins.Actinobacteria harbors the largest number of encapsulin or encapsulin-like systems (6). DyP-containing encapsulins have already been reported from mycobacteria, including Mycobacterium smegmatis (15) and Mycobacterium tuberculosis (19). These have been considered as potential biomarkers to detect active tuberculosis (TB) (20). In the present study, we have isolated and characterized a DyP-loaded encapsulin system from M. smegmatis, which is commonly used as a model organism in studying the biology of the M. tuberculosis (21). We have determined its complete high-resolution structure by cryogenic electron microscopy (cryo-EM). Our results have revealed the interactions between the CFP-29 (a 29 kDa culture filtrate protein) shell and DyP cargo and a potential antioxidation mechanism. Our study also lays the foundation for the discovery of new diagnosis protocols and treatments of TB.     

An engineered 4-1BBL fusion protein with “activity on demand”

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues.

Cytokines are immunomodulatory proteins that have been considered for pharmaceutical applications in the treatment of cancer patients (1–3) and other types of disease (2). There is a growing interest in the use of engineered cytokine products as anticancer drugs, capable of boosting the action of T cells and natural killer (NK) cells against tumors (3, 4), alone or in combination with immune checkpoint inhibitors (3, 5–7).Recombinant cytokine products on the market include interleukin-2 (IL-2) (Proleukin) (8, 9), IL-11 (Neumega) (10, 11), tumor necrosis factor (TNF; Beromun) (12), interferon (IFN)-α (Roferon A, Intron A) (13, 14), IFN-β (Avonex, Rebif, Betaseron) (15, 16), IFN-γ (Actimmune) (17), granulocyte colony-stimulating factor (Neupogen) (18), and granulocyte macrophage colony-stimulating factor (Leukine) (19, 20). The recommended dose is typically very low (often <1 mg/d) (21–23), as cytokines may exert biological activity in the subnanomolar concentration range (24). Various strategies have been proposed to develop cytokine products with improved therapeutic index. Protein PEGylation or Fc fusions may lead to prolonged circulation time in the bloodstream, allowing the administration of low doses of active payload (25, 26). In some implementations, cleavable polyethylene glycol polymers may be considered, yielding prodrugs that regain activity at later time points (27). Alternatively, tumor-homing antibody fusions have been developed, since the preferential concentration of cytokine payloads at the tumor site has been shown in preclinical models to potentiate therapeutic activity, helping spare normal tissues (28–34). Various antibody-cytokine fusions are currently being investigated in clinical trials for the treatment of cancer and of chronic inflammatory conditions (reviewed in refs. 2, 33, 35–37).Antibody-cytokine fusions display biological activity immediately after injection into patients, which may lead to unwanted toxicity and prevent escalation to therapeutically active dosage regimens (9, 22, 38). In the case of proinflammatory payloads (e.g., IL-2, IL-12, TNF-α), common side effects include hypotension, nausea, and vomiting, as well as flu-like symptoms (24, 39–42). These side effects typically disappear when the cytokine concentration drops below a critical threshold, thus providing a rationale for slow-infusion administration procedures (43). It would be highly desirable to generate antibody-cytokine fusion proteins with excellent tumor-targeting properties and with “activity on demand”— biological activity that is conditionally gained on antigen binding at the site of disease, helping spare normal tissues.Here we describe a fusion protein consisting of the F8 antibody specific to the alternatively spliced extra domain A (EDA) of fibronectin (44, 45) and of murine 4-1BBL, which did not exhibit cytokine activity in solution but could regain potent biological activity on antigen binding. The antigen (EDA+ fibronectin) is conserved from mouse to man (46), is virtually undetectable in normal adult tissues (with the exception of the placenta, endometrium, and some vessels in the ovaries), but is expressed in the majority of human malignancies (44, 45, 47, 48). 4-1BBL, a member of the TNF superfamily (49), is expressed on antigen-presenting cells (50, 51) and binds to its receptor, 4-1BB, which is up-regulated on activated cytotoxic T cells (52), activated dendritic cells (52), activated NK and NKT cells (53), and regulatory T cells (54). Signaling through 4-1BB on cytotoxic T cells protects them from activation-induced cell death and skews the cells toward a more memory-like phenotype (55, 56).We engineered nine formats of the F8-4-1BBL fusion protein, one of which exhibited superior performance in quantitative biodistribution studies and conditional gain of cytokine activity on antigen binding. The antigen-dependent reconstitution of the biological activity of the immunostimulatory payload represents an example of an antibody fusion protein with “activity on demand.” The fusion protein was potently active against different types of cancer without apparent toxicity at the doses used. The EDA of fibronectin is a particularly attractive antigen for cancer therapy in view of its high selectivity, stability, and abundant expression in most tumor types (44, 45, 47, 48).     

A dark quencher genetically encodable voltage indicator (dqGEVI) exhibits high fidelity and speed

Voltage sensing with genetically expressed optical probes is highly desirable for large-scale recordings of neuronal activity and detection of localized voltage signals in single neurons. Most genetically encodable voltage indicators (GEVI) have drawbacks including slow response, low fluorescence, or excessive bleaching. Here we present a dark quencher GEVI approach (dqGEVI) using a Förster resonance energy transfer pair between a fluorophore glycosylphosphatidylinositol–enhanced green fluorescent protein (GPI-eGFP) on the outer surface of the neuronal membrane and an azo-benzene dye quencher (D3) that rapidly moves in the membrane driven by voltage. In contrast to previous probes, the sensor has a single photon bleaching time constant of ∼40 min, has a high temporal resolution and fidelity for detecting action potential firing at 100 Hz, resolves membrane de- and hyperpolarizations of a few millivolts, and has negligible effects on passive membrane properties or synaptic events. The dqGEVI approach should be a valuable tool for optical recordings of subcellular or population membrane potential changes in nerve cells.

The most widely used genetically encodable indicators of neuronal activity are Ca²⁺-binding proteins that change fluorescence upon binding Ca²⁺ after it enters through voltage-gated Ca²⁺ channels. However, these genetically encodable Ca²⁺ indicators are not ideally suited for accurate detection of single action potentials (APs) and are unable to record membrane hyperpolarizations or depolarizations below AP threshold (1, 2). In contrast, direct optical voltage sensing using genetically expressed probes is highly promising for large-scale recordings of neuronal activity. Many of the various genetically encodable voltage indicators (GEVIs) currently in use were subject to several recent reviews and comparative studies (3–10) observing the rapid development of these valuable probes together with highly sensitive fluorescent probes for membrane voltage monitoring that are not genetically encoded (11).One of the most promising starting approaches has been to fuse the voltage sensor of the Acetabularia chemigenetiacetabulum rhodopsin (Ace2N) and the fluorescent protein mNeonGreen to enable voltage-sensitive Förster resonance energy transfer (FRET) (12). This sensor has been shown to work in expression systems, neurons in culture, slices, in the intact brains of awake mice, and in dendrites of olfactory neurons in intact flies. Since then, several other approaches have been developed (13–15) that work well for determining cell-specific behavioral correlates in mice. The development of the Optopatch3 mouse line (13) was a revolutionary milestone in advancing research using GEVIs. The most recent advances in the field combine a voltage-sensitive microbial rhodopsin with a self-labeling protein domain that covalently binds the synthetic Janelia Fluor fluorophore that has to be administered separately. The degree of FRET is modulated by the voltage-sensitive absorption spectrum of the rhodopsin. This type of GEVI has been termed chemigenetic as it has a chemical and a genetic component. Voltron (16) and its latest derivative, Positron (17), have been used in various expression systems including intact mouse brains. Another chemigenetic fluorescent voltage sensing approach, also called hybrid GEVI, is based on FRET, or rather quenching (18) between two components, but the voltage sensor is not genetically encoded. A fluorescent particle is anchored to one side of the membrane and a small lipophilic anion (dark quencher) is used as the voltage sensor that is rapidly moved inside the membrane by the electric field. The approach was pioneered over 20 y ago (19) and has been refined by using the FRET reaction between a stationary fluorescent lipid and a mobile dye, which gave an astonishing >50% fluorescence change per 100 mV with a time constant of <0.4 ms (20). The principle was turned into a genuine GEVI approach by using a genetically encodable membrane-targeted fluorescent protein as the membrane-anchored fluorophore (usually myristoylated and palmtoylated at Gly and Cys residues), and dipicrylamine (DPA) as its FRET quencher pair (21). DPA was known from early charge-pulse relaxation experiments to electrophorese through lipid membranes with a submillisecond translocation rate (22) that was as fast as gating currents in the squid axon (23). When used in combination with the lipophilic fluorescent dye DiO, that had to be fastidiously loaded into individual neurons, DPA gave very fast and large optical voltage signals (24–26). As the DPA absorption and enhanced green fluorescent protein (eGFP) emission spectra do not greatly overlap (SI Appendix, Fig. S1A), improvements in the method have been attained by using the blue-shifted enhanced cyan fluorescent protein (18), by developing a membrane localized fluorophore (hVOS 1.5, a cerulean fluorescent protein tagged at its C terminus with a truncated farnesylation motif) (27), and by proposing various optimizations of the approach (28).In general, this two-component approach provides good signal-to-noise ratio (SNR) for the detection of APs and for recording subthreshold synaptic events in various preparations (21, 29–31), but in all of these studies the voltage-dependent dark quencher remained the same: DPA. While this molecule satisfies many of the original requirements (19) for voltage sensing, it also has several drawbacks. These include its accumulation on the outer surface of the membrane below approximately −50 mV, thus making it difficult to report small membrane hyperpolarizatios (29), the considerable capacitive membrane load near the required concentrations for voltage sensing (21, 28, 32) that causes time-dependent deterioration of APs (26), and its interactions with various neurotransmitter systems (33–35). In addition, as it is made up of two trinitrotoluene molecules joined together, DPA (also known as hexanitrodiphenylamine, or HND) is highly explosive; indeed, it was actively used as an explosive in World War II.In order to replace DPA in the two-component GEVI approach we have set out to carry out a systematic search for different dark quenching molecules. After testing several dozens of compounds, we identified an organic nitroazobenzene dye (Disperse Orange 3 or 4-amino-4′-nitroazobenzene, CAS number 730-40-5, herein referred to as D3) with an absorption spectrum better suited than DPA to quench eGFP fluorescence (SI Appendix, Fig. S1A). Importantly, as a dark quencher D3 does not have any autofluorescence, and therefore, despite being a voltage indicator, it does not report a fluorescence signal by itself. Here we report on the superior properties of the dark quencher D3 in combination with eGFP for a dark quencher GEVI (dqGEVI) approach with the fluorophore anchored to the outside of the membrane by the glycosylphosphatidylinositol (GPI) motif (36) (GPI-eGFP; SI Appendix, Fig. S1B) in neuronal cultures and demonstrate its innocuous effects on passive membrane properties and synaptic events. Some GEVIs are quite adequate for 2P imaging (37–39), and our approach should also perform well in this mode, as 2P fluorescent voltage measurements have been very successful with another quencher (DPA) paired with a nongenetically encoded membrane fluorophore (25, 26). We have compared the speed of our voltage sensor to one of the GEVIs used for 2P imaging, ASAP2s (37), and find a considerable faster fluorescence response during APs.     

From the Cover: Childhood self-control forecasts the pace of midlife aging and preparedness for old age

The ability to control one’s own emotions, thoughts, and behaviors in early life predicts a range of positive outcomes in later life, including longevity. Does it also predict how well people age? We studied the association between self-control and midlife aging in a population-representative cohort of children followed from birth to age 45 y, the Dunedin Study. We measured children’s self-control across their first decade of life using a multi-occasion/multi-informant strategy. We measured their pace of aging and aging preparedness in midlife using measures derived from biological and physiological assessments, structural brain-imaging scans, observer ratings, self-reports, informant reports, and administrative records. As adults, children with better self-control aged more slowly in their bodies and showed fewer signs of aging in their brains. By midlife, these children were also better equipped to manage a range of later-life health, financial, and social demands. Associations with children’s self-control could be separated from their social class origins and intelligence, indicating that self-control might be an active ingredient in healthy aging. Children also shifted naturally in their level of self-control across adult life, suggesting the possibility that self-control may be a malleable target for intervention. Furthermore, individuals’ self-control in adulthood was associated with their aging outcomes after accounting for their self-control in childhood, indicating that midlife might offer another window of opportunity to promote healthy aging.

The ability to control one’s own emotions, thoughts, and behaviors in early life sets the stage for many positive outcomes in later life. These include educational attainment, career success, healthy lifestyles (1–4), and, in particular, longevity (5–8). Prospective studies of children, adolescents, and adults have shown that individuals with better self-control—often measured as higher conscientiousness or lower impulsivity—live longer lives (5–8). But, do they also exhibit better midlife aging? Answering this question could reveal opportunities to extend not only life span (how long we live) but also health span [how long we live free of disease and disability (9)]. Here, we used data collected across five decades to connect children’s self-control to their pace of aging in midlife. We also linked children’s self-control with their midlife aging preparedness: the health, financial, and social reserves that may help prepare individuals for longer life span and better health span.Midlife represents a useful window during which to measure individual differences in aging and their relation to childhood self-control. Meaningful variation between individuals in the speed of both physiological and cognitive aging can be detected already at this life stage (10, 11), and prior work has established that individual differences in midlife health are linked to early-life factors (12–14). Furthermore, midlife is a critical period for preparing for the demands of older age (15). Now past their healthy young adult years, individuals must devote greater attention to preventing age-related diseases, increasing their financial reserves for retirement, and building the social networks that will provide practical and emotional supports in old age. Signs of one’s own aging emerge at this life stage, reminding us that multiple health, financial, and social demands are approaching: menopause and presbyopia set in, we start paying attention to our savings accounts, and we see our own futures in our parents’ decline.If outcomes of self-control extend as far as midlife, then it could be a key intervention target. It would also suggest the hypothesis that there may be opportunities to build aging preparedness while individuals are still in their robust forties (15, 16). Much emphasis has been placed on the importance of intervening early in development, and there is vigorous debate over the optimal timing for implementing early-years programs (17–19). Midlife, however, remains a largely unexplored potential window of opportunity for self-control intervention.We tested associations between childhood self-control and midlife aging using data from the Dunedin Longitudinal Study, a prospective study of a complete birth cohort of 1,037 individuals followed from birth to age 45 with 94% retention. As previously reported in this journal, we measured study members’ self-control across their first decade of life using a multi-occasion/multi-informant strategy (2, 20). We measured their pace of aging as well as their aging preparedness in midlife using a range of prespecified measures known to be associated with life span and/or health span (Fig. 1 and SI Appendix, Table S1), which were derived from biological and physiological assessments, structural brain-imaging scans, observer ratings, self-reports, informant reports, and administrative records. We used these data to test two hypotheses. First, we tested the hypothesis that individuals with better self-control in childhood exhibit slower aging of the body and fewer signs of brain aging in midlife. Second, we tested the hypothesis that individuals with better self-control in childhood exhibit better preparedness for the health, financial, and social demands that emerge in later life. Research has shown that self-control predicts health behaviors such as diet, smoking, alcohol consumption, and exercise (4, 21–24). Here, we extend the reach of this research by testing whether self-control predicts outcomes beyond health behaviors, including individuals’ practical health knowledge, their attitudes toward and expectancies about aging, their practical financial knowledge and financial behavior, their social integration, and their satisfaction with life.Open in a separate windowFig. 1.Aging domains and measures assessed in the current study. Column three indicates reference numbers for prior studies documenting associations between the given measure and life span and/or health span. The complete reference list is included in SI Appendix, Table S1.Children’s self-control is correlated with their socioeconomic circumstances (25, 26) and their intelligence (27, 28). Both social class and intelligence have been implicated in life span and health span (29, 30); in fact, both social class and intelligence have been called “fundamental causes” of later-life health (31–33). Social class has been proposed as a fundamental cause because it influences multiple disease outcomes through multiple mechanisms, it embodies access to important resources, and its associations with health outcomes are maintained even when intervening mechanisms change (31). Intelligence has been conceptualized as a fundamental cause for similar reasons (32). For childhood self-control to be implicated as an active ingredient in healthy aging, it is important to show that its effects are independent of these two fundamental influences on children’s futures. We therefore tested whether associations between self-control and aging survived after accounting for children’s social class and intelligence quotient (IQ) [assessing each, like self-control, using repeated measurements across childhood (Methods)].