Excess membrane binding of monomeric alpha-, beta- and gamma-synuclein is invariably associated with inclusion formation and toxicity.

α-Synuclein (αS) has been well-documented to play a role in human synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). First, the lesions found in PD/DLB brains-Lewy bodies and Lewy neurites-are rich in aggregated αS. Second, genetic evidence links missense mutations and increased αS expression to familial forms of PD/DLB. Third, toxicity and cellular stress can be caused by αS under certain experimental conditions. In contrast, the homologs ß-synuclein (ßS) and γ-synuclein (γS) are not typically found in Lewy bodies/neurites, have not been clearly linked to brain diseases and have been largely non-toxic in experimental settings. In αS, the so-called non-amyloid-ß component of plaques (NAC) domain, constituting amino acids 61-95, has been identified to be critical for aggregation in vitro. This domain is partially absent in ßS and only incompletely conserved in γS, which could explain why both homologs do not cause disease. However, αS in vitro aggregation and cellular toxicity have not been firmly linked experimentally, and it has been proposed that excess αS membrane binding is sufficient to induce neurotoxicity. Indeed, recent characterizations of Lewy bodies have highlighted the accumulation of lipids and membranous organelles, raising the possibility that ßS and γS could also become neurotoxic if they were more prone to membrane/lipid binding. Here, we increased ßS and γS membrane affinity by strategic point mutations and demonstrate that these proteins behave like membrane-associated monomers, are cytotoxic and form round cytoplasmic inclusions that can be prevented by inhibiting stearoyl-CoA desaturase.

Structure of a spliceosome remodelled for exon ligation.

The spliceosome excises introns from pre-mRNAs in two sequential transesterifications-branching and exon ligation-catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA). Recently reported structures of the spliceosomal C complex with the cleaved 5' exon and lariat-3'-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5' splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site. Here we present, at 3.8 Å resolution, the cryo-electron microscopy structure of a Saccharomyces cerevisiae spliceosome stalled after Prp16-mediated remodelling but before exon ligation. While the U6 snRNA catalytic core remains firmly held in the active site cavity of Prp8 by proteins common to both steps, the branch helix has rotated by 75° compared to the C complex and is stabilized in a new position by Prp17, Cef1 and the reoriented Prp8 RNase H-like domain. This rotation of the branch helix removes the branch adenosine from the catalytic core, creates a space for 3' exon docking, and restructures the pairing of the 5' splice site with the U6 snRNA ACAGAGA region. Slu7 and Prp18, which promote exon ligation, bind together to the Prp8 RNase H-like domain. The ATPase Prp22, bound to Prp8 in place of Prp16, could interact with the 3' exon, suggesting a possible basis for mRNA release after exon ligation. Together with the structure of the C complex, our structure of the C* complex reveals the two major conformations of the spliceosome during the catalytic stages of splicing.

Cryo-EM structure of the spliceosome immediately after branching.

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat-intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5'-splice site is cleaved but remains close to the catalytic Mg2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5'-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2'OH. The 5'-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson-Crick interactions between the branch helix and 5'-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation.

Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution.

U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Šresolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.

Acute association between heatwaves and stillbirth in six US states.

BACKGROUND: Heatwaves are becoming more frequent and may acutely increase the risk of stillbirth, a rare and severe pregnancy outcome. OBJECTIVES: Examine the association between multiple heatwave metrics and stillbirth in six U.S. states. METHODS: Data were collected from fetal death and birth records in California (1996-2017), Florida (1991-2017), Georgia (1994-2017), Kansas (1991-2017), New Jersey (1991-2015), and Oregon (1991-2017). Cases were matched to controls 1:4 based on maternal race/ethnicity, maternal education, and county, and exposure windows were aligned (gestational week prior to stillbirth). County-level temperature data were obtained from Daymet and linked to cases and controls by residential county and the exposure window. Five heatwave metrics (1 categorical, 3 dichotomous, 1 continuous) were created using different combinations of the duration and intensity of hot days (mean daily temperature exceeding the county-specific 97.5th percentile) during the exposure window, as well as a continuous measure of mean temperature during the exposure window modeled using natural splines to allow for nonlinear associations. State-specific odds ratios (ORs) and 95% confidence intervals (CI) were estimated using conditional logistic regression models. State-specific results were pooled using a fixed-effects meta-analysis. RESULTS: In our data set of 140,428 stillbirths (553,928 live birth controls), three of the five heatwave metrics examined were not associated with stillbirth. However, four consecutive hot days during the previous week was associated with a 3% increase in stillbirth risk (CI: 1.01, 1.06), and a 1 °C average increase over the threshold was associated with a 10% increase in stillbirth risk (CI: 1.04, 1.17). In continuous temperature analyses, there was a slight increased risk of stillbirth associated with extremely hot temperatures (≥ 35 °C). DISCUSSION: Most heat wave definitions examined were not associated with acute changes in stillbirth risk; however, the most extreme heatwave durations and temperatures were associated with a modest increase in stillbirth risk.

Using logic regression to characterize extreme heat exposures and their health associations: a time-series study of emergency department visits in Atlanta.

BACKGROUND: Short-term associations between extreme heat events and adverse health outcomes are well-established in epidemiologic studies. However, the use of different exposure definitions across studies has limited our understanding of extreme heat characteristics that are most important for specific health outcomes or subpopulations. METHODS: Logic regression is a statistical learning method for constructing decision trees based on Boolean combinations of binary predictors. We describe how logic regression can be utilized as a data-driven approach to identify extreme heat exposure definitions using health outcome data. We evaluated the performance of the proposed algorithm in a simulation study, as well as in a 20-year time-series analysis of extreme heat and emergency department visits for 12 outcomes in the Atlanta metropolitan area. RESULTS: For the Atlanta case study, our novel application of logic regression identified extreme heat exposure definitions that were associated with several heat-sensitive disease outcomes (e.g., fluid and electrolyte imbalance, renal diseases, ischemic stroke, and hypertension). Exposures were often characterized by extreme apparent minimum temperature or maximum temperature over multiple days. The simulation study also demonstrated that logic regression can successfully identify exposures of different lags and duration structures when statistical power is sufficient. CONCLUSION: Logic regression is a useful tool for identifying important characteristics of extreme heat exposures for adverse health outcomes, which may help improve future heat warning systems and response plans.

The architecture of the spliceosomal U4/U6.U5 tri-snRNP.

U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a precursor messenger RNA substrate bound to U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), and transforms into a catalytically active spliceosome after extensive compositional and conformational changes triggered by unwinding of the U4 and U6 (U4/U6) snRNAs. Here we use cryo-electron microscopy single-particle reconstruction of Saccharomyces cerevisiae tri-snRNP at 5.9 Å resolution to reveal the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3' stem-loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the amino-terminal domain of Prp8 position U5 snRNA to insert its loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome.

Time-series analysis of daily ambient temperature and emergency department visits in five US cities with a comparison of exposure metrics derived from 1-km meteorology products.

BACKGROUND: Ambient temperature observations from single monitoring stations (usually located at the major international airport serving a city) are routinely used to estimate heat exposures in epidemiologic studies. This method of exposure assessment does not account for potential spatial variability in ambient temperature. In environmental health research, there is increasing interest in utilizing spatially-resolved exposure estimates to minimize exposure measurement error. METHODS: We conducted time-series analyses to investigate short-term associations between daily temperature metrics and emergency department (ED) visits for well-established heat-related morbidities in five US cities that represent different climatic regions: Atlanta, Los Angeles, Phoenix, Salt Lake City, and San Francisco. In addition to airport monitoring stations, we derived several exposure estimates for each city using a national meteorology data product (Daymet) available at 1 km spatial resolution. RESULTS: Across cities, we found positive associations between same-day temperature (maximum or minimum) and ED visits for heat-sensitive outcomes, including acute renal injury and fluid and electrolyte imbalance. We also found that exposure assessment methods accounting for spatial variability in temperature and at-risk population size often resulted in stronger relative risk estimates compared to the use of observations at airports. This pattern was most apparent when examining daily minimum temperature and in cities where the major airport is located further away from the urban center. CONCLUSION: Epidemiologic studies based on single monitoring stations may underestimate the effect of temperature on morbidity when the station is less representative of the exposure of the at-risk population.

Acute associations between heatwaves and preterm and early-term birth in 50 US metropolitan areas: a matched case-control study.

BACKGROUND: The effect of heatwaves on adverse birth outcomes is not well understood and may vary by how heatwaves are defined. The study aims to examine acute associations between various heatwave definitions and preterm and early-term birth. METHODS: Using national vital records from 50 metropolitan statistical areas (MSAs) between 1982 and 1988, singleton preterm (< 37 weeks) and early-term births (37-38 weeks) were matched (1:1) to controls who completed at least 37 weeks or 39 weeks of gestation, respectively. Matching variables were MSA, maternal race, and maternal education. Sixty heatwave definitions including binary indicators for exposure to sustained heat, number of high heat days, and measures of heat intensity (the average degrees over the threshold in the past 7 days) based on the 97.5th percentile of MSA-specific temperature metrics, or the 85th percentile of positive excessive heat factor (EHF) were created. Odds ratios (OR) for heatwave exposures in the week preceding birth (or corresponding gestational week for controls) were estimated using conditional logistic regression adjusting for maternal age, marital status, and seasonality. Effect modification by maternal education, age, race/ethnicity, child sex, and region was assessed. RESULTS: There were 615,329 preterm and 1,005,576 early-term case-control pairs in the analyses. For most definitions, exposure to heatwaves in the week before delivery was consistently associated with increased odds of early-term birth. Exposure to more high heat days and more degrees above the threshold yielded higher magnitude ORs. For exposure to 3 or more days over the 97.5th percentile of mean temperature in the past week compared to zero days, the OR was 1.027 for early-term birth (95%CI: 1.014, 1.039). Although we generally found null associations when assessing various heatwave definitions and preterm birth, ORs for both preterm and early-term birth were greater in magnitude among Hispanic and non-Hispanic black mothers. CONCLUSION: Although associations varied across metrics and heatwave definitions, heatwaves were more consistently associated with early-term birth than with preterm birth. This study's findings may have implications for prevention programs targeting vulnerable subgroups as climate change progresses.

Dynamic protein-RNA interactions in mediating splicing catalysis.

The spliceosome is assembled via sequential interactions of pre-mRNA with five small nuclear RNAs and many proteins. Recent determination of cryo-EM structures for several spliceosomal complexes has provided deep insights into interactions between spliceosomal components and structural changes of the spliceosome between steps, but information on how the proteins interact with pre-mRNA to mediate the reaction is scarce. By systematic analysis of proteins interacting with the splice sites (SSs), we have identified many previously unknown interactions of spliceosomal components with the pre-mRNA. Prp8 directly binds over the 5'SS and the branch site (BS) for the first catalytic step, and the 5'SS and 3'SS for the second step. Switching the Prp8 interaction from the BS to the 3'SS requires Slu7, which interacts dynamically with pre-mRNA first, and then interacts stably with the 3'-exon after Prp16-mediated spliceosome remodeling. Our results suggest that Prp8 plays a key role in positioning the 5'SS and 3'SS, facilitated by Slu7 through interactions with Prp8 and substrate RNA to advance exon ligation. We also provide evidence that Prp16 first docks on the intron 3' tail, then translocates in the 3' to 5' direction on remodeling the spliceosome.

Molecular Mechanism and Evolution of Nuclear Pre-mRNA and Group II Intron Splicing: Insights from Cryo-Electron Microscopy Structures.

Nuclear pre-mRNA splicing and group II intron self-splicing both proceed by two-step transesterification reactions via a lariat intron intermediate. Recently determined cryo-electron microscopy (cryo-EM) structures of catalytically active spliceosomes revealed the RNA-based catalytic core and showed how pre-mRNA substrates and reaction products are positioned in the active site. These findings highlight a strong structural similarity to the group II intron active site, strengthening the notion that group II introns and spliceosomes evolved from a common ancestor. Prp8, the largest and most conserved protein in the spliceosome, cradles the active site RNA. Prp8 and group II intron maturase have a similar domain architecture, suggesting that they also share a common evolutionary origin. The interactions between maturase and key group II intron RNA elements, such as the exon-binding loop and domains V and VI, are recapitulated in the interactions between Prp8 and key elements in the spliceosome's catalytic RNA core. Structural comparisons suggest that the extensive RNA scaffold of the group II intron was gradually replaced by proteins as the spliceosome evolved. A plausible model of spliceosome evolution is discussed.

Loss of native α-synuclein multimerization by strategically mutating its amphipathic helix causes abnormal vesicle interactions in neuronal cells.

α-Synuclein (αS) forms round cytoplasmic inclusions in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Evidence suggests a physiological function of αS in vesicle trafficking and release. In contrast to earlier tenets, recent work indicates that αS normally exists in cells in a dynamic equilibrium between monomers and tetramers/multimers. We engineered αS mutants incapable of multimerization, leading to excess monomers at vesicle membranes. By EM, such mutants induced prominent vesicle clustering, leading to round cytoplasmic inclusions. Immunogold labeling revealed abundant αS intimately associated with vesicles of varied size. Fluorescence microscopy with marker proteins showed that the αS-associated vesicles were of diverse endocytic and secretory origin. An αS '3K' mutant (E35K + E46K + E61K) that amplifies the PD/DLB-causing E46K mutation induced αS-rich vesicle clusters resembling the vesicle-rich areas of Lewy bodies, supporting pathogenic relevance. Mechanistically, E46K can increase αS vesicle binding via membrane-induced amphipathic helix formation, and '3K' further enhances this effect. Another engineered αS variant added hydrophobicity to the hydrophobic half of αS helices, thereby stabilizing αS-membrane interactions. Importantly, substituting charged for uncharged residues within the hydrophobic half of the stabilized helix not only reversed the strong membrane interaction of the multimer-abolishing αS variant but also restored multimerization and prevented the aberrant vesicle interactions. Thus, reversible αS amphipathic helix formation and dynamic multimerization regulate a normal function of αS at vesicles, and abrogating multimers has pathogenic consequences.

Crystal structure of U2 snRNP SF3b components: Hsh49p in complex with Cus1p-binding domain.

Spliceosomal proteins Hsh49p and Cus1p are components of SF3b, which together with SF3a, Msl1p/Lea1p, Sm proteins, and U2 snRNA, form U2 snRNP, which plays a crucial role in pre-mRNA splicing. Hsh49p, comprising two RRMs, forms a heterodimer with Cus1p. We determined the crystal structures of Saccharomyces cerevisiae full-length Hsh49p as well as its RRM1 in complex with a minimal binding region of Cus1p (residues 290-368). The structures show that the Cus1 fragment binds to the α-helical surface of Hsh49p RRM1, opposite the four-stranded ß-sheet, leaving the canonical RNA-binding surface available to bind RNA. Hsh49p binds the 5' end region of U2 snRNA via RRM1. Its affinity is increased in complex with Cus1(290-368)p, partly because an extended RNA-binding surface forms across the protein-protein interface. The Hsh49p RRM1-Cus1(290-368)p structure fits well into cryo-EM density of the Bact spliceosome, corroborating the biological relevance of our crystal structure.

Crystal structure of Prp8 reveals active site cavity of the spliceosome.

The active centre of the spliceosome consists of an intricate network formed by U5, U2 and U6 small nuclear RNAs, and a pre-messenger-RNA substrate. Prp8, a component of the U5 small nuclear ribonucleoprotein particle, crosslinks extensively with this RNA catalytic core. Here we present the crystal structure of yeast Prp8 (residues 885-2413) in complex with Aar2, a U5 small nuclear ribonucleoprotein particle assembly factor. The structure reveals tightly associated domains of Prp8 resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease. Suppressors of splice-site mutations, and an intron branch-point crosslink, map to a large cavity formed by the reverse transcriptase thumb, and the endonuclease-like and RNaseH-like domains. This cavity is large enough to accommodate the catalytic core of group II intron RNA. The structure provides crucial insights into the architecture of the spliceosome active site, and reinforces the notion that nuclear pre-mRNA splicing and group II intron splicing have a common origin.

KTKEGV repeat motifs are key mediators of normal α-synuclein tetramerization: Their mutation causes excess monomers and neurotoxicity.

α-Synuclein (αS) is a highly abundant neuronal protein that aggregates into ß-sheet-rich inclusions in Parkinson's disease (PD). αS was long thought to occur as a natively unfolded monomer, but recent work suggests it also occurs normally in α-helix-rich tetramers and related multimers. To elucidate the fundamental relationship between αS multimers and monomers in living neurons, we performed systematic mutagenesis to abolish self-interactions and learn which structural determinants underlie native multimerization. Unexpectedly, tetramers/multimers still formed in cells expressing each of 14 sequential 10-residue deletions across the 140-residue polypeptide. We postulated compensatory effects among the six highly conserved and one to three additional αS repeat motifs (consensus: KTKEGV), consistent with αS and its homologs ß- and γ-synuclein all forming tetramers while sharing only the repeats. Upon inserting in-register missense mutations into six or more αS repeats, certain mutations abolished tetramer formation, shown by intact-cell cross-linking and independently by fluorescent-protein complementation. For example, altered repeat motifs KLKEGV, KTKKGV, KTKEIV, or KTKEGW did not support tetramerization, indicating the importance of charged or small residues. When we expressed numerous different in-register repeat mutants in human neural cells, all multimer-abolishing but no multimer-neutral mutants caused frank neurotoxicity akin to the proapoptotic protein Bax. The multimer-abolishing variants became enriched in buffer-insoluble cell fractions and formed round cytoplasmic inclusions in primary cortical neurons. We conclude that the αS repeat motifs mediate physiological tetramerization, and perturbing them causes PD-like neurotoxicity. Moreover, the mutants we describe are valuable tools for studying normal and pathological properties of αS and screening for tetramer-stabilizing therapeutics.

In vivo cross-linking reveals principally oligomeric forms of α-synuclein and ß-synuclein in neurons and non-neural cells.

Aggregation of α-synuclein (αSyn) in neurons produces the hallmark cytopathology of Parkinson disease and related synucleinopathies. Since its discovery, αSyn has been thought to exist normally in cells as an unfolded monomer. We recently reported that αSyn can instead exist in cells as a helically folded tetramer that resists aggregation and binds lipid vesicles more avidly than unfolded recombinant monomers (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110). However, a subsequent study again concluded that cellular αSyn is an unfolded monomer (Fauvet, B., Mbefo, M. K., Fares, M. B., Desobry, C., Michael, S., Ardah, M. T., Tsika, E., Coune, P., Prudent, M., Lion, N., Eliezer, D., Moore, D. J., Schneider, B., Aebischer, P., El-Agnaf, O. M., Masliah, E., and Lashuel, H. A. (2012) J. Biol. Chem. 287, 15345-15364). Here we describe a simple in vivo cross-linking method that reveals a major ~60-kDa form of endogenous αSyn (monomer, 14.5 kDa) in intact cells and smaller amounts of ~80- and ~100-kDa forms with the same isoelectric point as the 60-kDa species. Controls indicate that the apparent 60-kDa tetramer exists normally and does not arise from pathological aggregation. The pattern of a major 60-kDa and minor 80- and 100-kDa species plus variable amounts of free monomers occurs endogenously in primary neurons and erythroid cells as well as neuroblastoma cells overexpressing αSyn. A similar pattern occurs for the homologue, ß-synuclein, which does not undergo pathogenic aggregation. Cell lysis destabilizes the apparent 60-kDa tetramer, leaving mostly free monomers and some 80-kDa oligomer. However, lysis at high protein concentrations allows partial recovery of the 60-kDa tetramer. Together with our prior findings, these data suggest that endogenous αSyn exists principally as a 60-kDa tetramer in living cells but is lysis-sensitive, making the study of natural αSyn challenging outside of intact cells.

Saline suppression testing-induced hypocalcemia and implications for clinical interpretations.

BACKGROUND: Extracellular calcium critically regulates physiologic aldosterone production. Moreover, abnormal calcium flux and signaling are involved in the pathogenesis of the majority of primary aldosteronism cases. METHODS: We investigated the influence of the saline suppression test (SST) on calcium homeostasis in prospectively recruited participants (n = 86). RESULTS: During SST, 100% of participants had decreases in serum calcium, with 48% developing frank hypocalcemia. Serum calcium declined from 2.30 ± 0.08 mmol/L to 2.13 ± 0.08 mmol/L (P < .001) with parallel increases in parathyroid hormone from 6.06 ± 2.39 pmol/L to 8.13 ± 2.42 pmol/L (P < .001). In contrast, serum potassium and bicarbonate did not change, whereas eGFR increased and serum glucose decreased (P < .001). Lower body surface area (translating to greater effective circulating volume expansion during SST) was associated with greater reductions in (ß = .33, P = .001), and absolutely lower, serum calcium levels (ß = .25, P = .001). When evaluating clinically-relevant diagnostic thresholds, participants with post-SST aldosterone levels <138 pmol/L had lower post-SST calcium and 25-hydroxyvitamin D levels (P < .05), and higher post-SST parathyroid hormone levels (P < .05) compared with those with post-SST aldosterone levels >277 pmol/L. CONCLUSION: SST uniformly decreases serum calcium, which is likely to be due to the combination of variable dilution, increased renal clearance, and vitamin D status. These acute reductions in bioavailable calcium are associated with lower post-SST aldosterone. Given the critical role of extracellular calcium in regulating aldosterone production, these findings warrant renewed inquiry into the validity of SST interpretations for excluding primary aldosteronism.

Preterm and Early-Term Delivery After Heat Waves in 50 US Metropolitan Areas.

Importance: Heat waves are increasing in frequency, intensity, and duration and may be acutely associated with pregnancy outcomes. Objective: To examine changes in daily rates of preterm and early-term birth after heat waves in a 25-year nationwide study. Design, Setting, and Participants: This cohort study of singleton births used birth records from 1993 to 2017 from the 50 most populous US metropolitan statistical areas (MSAs). The study included 53 million births, covering 52.8% of US births over the period. Data were analyzed between October 2022 and March 2023 at the National Center for Health Statistics. Exposures: Daily temperature data from Daymet at 1-km2 resolution were averaged over each MSA using population weighting. Heat waves were defined in the 4 days (lag, 0-3 days) or 7 days (lag, 0-6 days) preceding birth. Main Outcomes and Measures: Daily counts of preterm birth (28 to <37 weeks), early-term birth (37 to <39 weeks), and ongoing pregnancies in each gestational week on each day were enumerated in each MSA. Rate ratios for heat wave metrics were obtained from time-series models restricted to the warm season (May to September) adjusting for MSA, year, day of season, and day of week, and offset by pregnancies at risk. Results: There were 53 154 816 eligible births in the 50 MSAs from 1993 to 2017; 2 153 609 preterm births and 5 795 313 early-term births occurring in the warm season were analyzed. A total of 30.0% of mothers were younger than 25 years, 53.8% were 25 to 34 years, and 16.3% were 35 years or older. Heat waves were positively associated with daily rates of preterm and early-term births, showing a dose-response association with heat wave duration and temperatures and stronger associations in the more acute 4-day window. After 4 consecutive days of mean temperatures exceeding the local 97.5th percentile, the rate ratio for preterm birth was 1.02 (95% CI, 1.00-1.03), and the rate ratio for early-term birth was 1.01 (95% CI, 1.01-1.02). For the same exposure, among those who were 29 years of age or younger, had a high school education or less, and belonged to a racial or ethnic minority group, the rate ratios were 1.04 (95% CI, 1.02-1.06) for preterm birth and 1.03 (95% CI, 1.02-1.05) for early-term birth. Results were robust to alternative heat wave definitions, excluding medically induced deliveries, and alternative statistical model specifications. Conclusions and Relevance: In this cohort study, preterm and early-term birth rates increased after heat waves, particularly among socioeconomically disadvantaged subgroups. Extreme heat events have implications for perinatal health.

Progressive 11ß-Hydroxysteroid Dehydrogenase Type 2 Insufficiency as Kidney Function Declines.

BACKGROUND: It has been postulated that chronic kidney disease (CKD) is a state of relative 11ß-hydroxysteroid dehydrogenase type 2 (11ßHSD2) insufficiency, resulting in increased cortisol-mediated mineralocorticoid receptor (MR) activation. We hypothesized that relative 11ßHSD2 insufficiency manifests across a wide spectrum of progressively declining kidney function, including within the normal range. METHODS: Adult participants were recruited at two academic centers. A discovery cohort (n=500) enrolled individuals with estimated glomerular filtration rate (eGFR) ranging from normal to CKD stage 5, in whom serum cortisol-to-cortisone (F/E) was measured as a biomarker of 11ßHSD2 activity. A validation cohort (n=101) enrolled only individuals with normal kidney function (eGFR ≥ 60 mL/min/1.73 m2) in whom 11ßHSD2 activity was assessed via serum F/E and 11-hydroxy-to-11-keto androgen (11OH/K) ratios following multiple maneuvers: oral sodium suppression test (OSST), dexamethasone suppression test (DST), and ACTH-stimulation test (ACTHstim). RESULTS: In the discovery cohort, lower eGFR was associated with higher F/E (P-trend<0.001). Similarly, in the validation cohort, with normal eGFR, an inverse association between eGFR and both F/E and 11OH/K ratios was observed (P-trend<0.01), which persisted following DST (P-trend<0.001) and ACTHstim (P-trend< 0.05). The fractional excretion of potassium, a marker of renal MR activity, was higher with higher F/E (P-trend < 0.01) and with lower eGFR (P-trend<0.0001). CONCLUSIONS: A continuum of declining 11ßHSD2 activity was observed with progressively lower eGFR in individuals spanning a wide spectrum of kidney function, including those with apparently normal kidney function. These findings implicate cortisol-mediated MR activation in the pathophysiology of hypertension and cardiovascular disease in CKD.

Estimating Heat-Related Exposures and Urban Heat Island Impacts: A Case Study for the 2012 Chicago Heatwave.

Accelerated urbanization increases both the frequency and intensity of heatwaves (HW) and urban heat islands (UHIs). An extreme HW event occurred in 2012 summer that caused temperatures of more than 40°C in Chicago, Illinois, USA, which is a highly urbanized city impacted by UHIs. In this study, multiple numerical models, including the High Resolution Land Data Assimilation System (HRLDAS) and Weather Research and Forecasting (WRF) model, were used to simulate the HW and UHI, and their performance was evaluated. In addition, sensitivity testing of three different WRF configurations was done to determine the impact of increasing model complexity in simulating urban meteorology. Model performances were evaluated based on the statistical performance metrics, the application of a multi-layer urban canopy model (MLUCM) helps WRF to provide the best performance in this study. HW caused rural temperatures to increase by ∼4°C, whereas urban Chicago had lower magnitude increases from the HW (∼2-3°C increases). Nighttime UHI intensity (UHII) ranged from 1.44 to 2.83°C during the study period. Spatiotemporal temperature fields were used to estimate the potential heat-related exposure and to quantify the Excessive Heat Factor (EHF). The EHF during the HW episode provides a risk map indicating that while urban Chicago had higher heat-related stress during this event, the rural area also had high risk, especially during nighttime in central Illinois. This study provides a reliable method to estimate spatiotemporal exposures for future studies of heat-related health impacts.