A U.S. Lead Exposure Hotspots Analysis.

To identify U.S. lead exposure risk hotspots, we expanded upon geospatial statistical methods from a published Michigan case study. The evaluation of identified hotspots using five lead indices, based on housing age and sociodemographic data, showed moderate-to-substantial agreement with state-identified higher-risk locations from nine public health department reports (45-78%) and with hotspots of children's blood lead data from Michigan and Ohio (e.g., Cohen's kappa scores of 0.49-0.63). Applying geospatial cluster analysis and 80th-100th percentile methods to the lead indices, the number of U.S. census tracts ranged from ∼8% (intersection of indices) to ∼41% (combination of indices). Analyses of the number of children <6 years old living in those census tracts revealed the states (e.g., Illinois, Michigan, New Jersey, New York, Ohio, Pennsylvania, Massachusetts, California, Texas) and counties with highest potential lead exposure risk. Results support use of available lead indices as surrogates to identify locations in the absence of consistent, complete blood lead level (BLL) data across the United States. Ground-truthing with local knowledge, additional BLL data, and environmental data is needed to improve identification and analysis of lead exposure and BLL hotspots for interventions. While the science evolves, these screening results can inform "deeper dive" analyses for targeting lead actions.

A Systematic Review and Meta-analysis of Chemical Exposures and Attention-Deficit/Hyperactivity Disorder in Children.

Exposure to certain chemicals prenatally and in childhood can impact development and may increase risk for attention-deficit/hyperactivity disorder (ADHD). Leveraging a larger set of literature searches conducted to synthesize results from longitudinal studies of potentially modifiable risk factors for childhood ADHD, we present meta-analytic results from 66 studies that examined the associations between early chemical exposures and later ADHD diagnosis or symptoms. Studies were eligible for inclusion if the chemical exposure occurred at least 6 months prior to measurement of ADHD diagnosis or symptomatology. Included papers were published between 1975 and 2019 on exposure to anesthetics (n = 5), cadmium (n = 3), hexachlorobenzene (n = 4), lead (n = 22), mercury (n = 12), organophosphates (n = 7), and polychlorinated biphenyls (n = 13). Analyses are presented for each chemical exposure by type of ADHD outcome reported (categorical vs. continuous), type of ADHD measurement (overall measures of ADHD, ADHD symptoms only, ADHD diagnosis only, inattention only, hyperactivity/impulsivity only), and timing of exposure (prenatal vs. childhood vs. cumulative), whenever at least 3 relevant effect sizes were available. Childhood lead exposure was positively associated with ADHD diagnosis and symptoms in all analyses except for the prenatal analyses (odds ratios (ORs) ranging from 1.60 to 2.62, correlation coefficients (CCs) ranging from 0.14 to 0.16). Other statistically significant associations were limited to organophosphates (CC = 0.11, 95% confidence interval (CI): 0.03-0.19 for continuous measures of ADHD outcomes overall), polychlorinated biphenyls (CC = 0.08, 95% CI: 0.02-0.14 for continuous measures of inattention as the outcome), and both prenatal and childhood mercury exposure (CC = 0.02, 95% CI: 0.00-0.04 for continuous measures of ADHD outcomes overall for either exposure window). Our findings provide further support for negative impacts of prenatal and/or childhood exposure to certain chemicals and raise the possibility that primary prevention and targeted screening could prevent or mitigate ADHD symptomatology. Furthermore, these findings support the need for regular review of regulations as our scientific understanding of the risks posed by these chemicals evolves.

Segmental Dynamics of Membranous Cholesterol are Coupled.

Cholesterol promotes the structural integrity of the fluid cell membrane and interacts dynamically with many membrane proteins to regulate function. Understanding site-resolved cholesterol structural dynamics is thus important. This long-standing challenge has thus far been addressed, in part, by selective isotopic labeling approaches. Here we present a new 3D solid-state NMR (SSNMR) experiment utilizing scalar 13C-13C polarization transfer and recoupling of the 1H-13C interactions in order to determine average dipolar couplings for all 1H-13C vectors in uniformly 13C-enriched cholesterol. The experimentally determined order parameters (OP) agree exceptionally well with molecular dynamics (MD) trajectories and reveal coupling among several conformational degrees of freedom in cholesterol molecules. Quantum chemistry shielding calculations further support this conclusion and specifically demonstrate that ring tilt and rotation are coupled to changes in tail conformation and that these coupled segmental dynamics dictate the orientation of cholesterol. These findings advance our understanding of physiologically relevant dynamics of cholesterol, and the methods that revealed them have broader potential to characterize how structural dynamics of other small molecules impact their biological functions.

Lead Data Mapping to Prioritize US Locations for Whole-of-Government Exposure Prevention Efforts: State of the Science, Federal Collaborations, and Remaining Challenges.

For this state-of-science overview of geospatial approaches for identifying US communities with high lead-exposure risk, we compiled and summarized public data and national maps of lead indices and models, environmental lead indicators, and children's blood lead surveillance data. Currently available indices and models are primarily constructed from housing-age and sociodemographic data; differing methods, variables, data, weighting schemes, and geographic scales yield maps with different exposure risk profiles. Environmental lead indicators are available (e.g., air, drinking water, dust, soil) at different spatial scales, but key gaps remain. Blood lead level data have limitations as testing, reporting, and completeness vary across states. Mapping tools and approaches developed by federal agencies and other groups for different purposes present an opportunity for greater collaboration. Maps, data visualization tools, and analyses that synthesize available geospatial efforts can be evaluated and improved with local knowledge and blood lead data to refine identification of high-risk locations for prioritizing prevention efforts and targeting risk-reduction strategies. Remaining challenges are discussed along with a work-in-progress systematic approach for cross-agency data integration, toward advancing "whole-of-government" public health protection from lead exposures. (Am J Public Health. 2022;112(S7):S658-S669. https://doi.org/10.2105/AJPH.2022.307051).

Hybrid measurement of respiratory aerosol reveals a dominant coarse fraction resulting from speech that remains airborne for minutes.

Accurate measurements of the size and quantity of aerosols generated by various human activities in different environments are required for efficacious mitigation strategies and accurate modeling of respiratory disease transmission. Previous studies of speech droplets, using standard aerosol instrumentation, reported very few particles larger than 5 µm. This starkly contrasts with the abundance of such particles seen in both historical slide deposition measurements and more recent light scattering observations. We have reconciled this discrepancy by developing an alternative experimental approach that addresses complications arising from nucleated condensation. Measurements reveal that a large volume fraction of speech-generated aerosol has diameters in the 5- to 20-µm range, making them sufficiently small to remain airborne for minutes, not hours. This coarse aerosol is too large to penetrate the lower respiratory tract directly, and its relevance to disease transmission is consistent with the vast majority of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections initiating in the upper respiratory tract. Our measurements suggest that in the absence of symptoms such as coughing or sneezing, the importance of speech-generated aerosol in the transmission of respiratory diseases is far greater than generally recognized.

Concordance of X-ray and AlphaFold2 Models of SARS-CoV-2 Main Protease with Residual Dipolar Couplings Measured in Solution.

The 68-kDa homodimeric 3C-like protease of SARS-CoV-2, Mpro (3CLpro/Nsp5), is a promising antiviral drug target. We evaluate the concordance of models generated by the newly introduced AlphaFold2 structure prediction program with residual dipolar couplings (RDCs) measured in solution for 15N-1HN and 13C'-1HN atom pairs. The latter were measured using a new, highly precise TROSY-AntiTROSY Encoded RDC (TATER) experiment. Three sets of AlphaFold2 models were evaluated: (1) MproAF, generated using the standard AlphaFold2 input structural database; (2) MproAFD, where the AlphaFold2 implementation was modified to exclude all candidate template X-ray structures deposited after Jan 1, 2020; and (3) MproAFS, which excluded all structures homologous to coronaviral Mpro. Close agreement between all three sets of AlphaFold models and experimental RDC data is found for most of the protein. For residues in well-defined secondary structure, the agreement decreases somewhat upon Amber relaxation. For these regions, MproAF agreement exceeds that of most high-resolution X-ray structures. Residues from domain 2 that comprise elements of both the active site and the homo-dimerization interface fit less well across all structures. These results indicate novel opportunities for combining experimentation with molecular dynamics simulations, where solution RDCs provide highly precise input for QM/MM simulations of substrate binding/reaction trajectories.

Update of the Blood Lead Reference Value - United States, 2021.

The negative impact of lead exposure on young children and those who become pregnant is well documented but is not well known by those at highest risk from this hazard. Scientific evidence suggests that there is no known safe blood lead level (BLL), because even small amounts of lead can be harmful to a child's developing brain (1). In 2012, CDC introduced the population-based blood lead reference value (BLRV) to identify children exposed to more lead than most other children in the United States. The BLRV should be used as a guide to 1) help determine whether medical or environmental follow-up actions should be initiated for an individual child and 2) prioritize communities with the most need for primary prevention of exposure and evaluate the effectiveness of prevention efforts. The BLRV is based on the 97.5th percentile of the blood lead distribution in U.S. children aged 1-5 years from National Health and Nutrition Examination Survey (NHANES) data. NHANES is a complex, multistage survey designed to provide a nationally representative assessment of health and nutritional status of the noninstitutionalized civilian adult and child populations in the United States (2). The initial BLRV of 5 µg/dL, established in 2012, was based on data from the 2007-2008 and 2009-2010 NHANES cycles. Consistent with recommendations from a former advisory committee, this report updates CDC's BLRV in children to 3.5 µg/dL using NHANES data derived from the 2015-2016 and 2017-2018 cycles and provides helpful information to support adoption by state and local health departments, health care providers (HCPs), clinical laboratories, and others and serves as an opportunity to advance health equity and environmental justice related to preventable lead exposure. CDC recommends that public health and clinical professionals focus screening efforts on populations at high risk based on age of housing and sociodemographic risk factors. Public health and clinical professionals should collaborate to develop screening plans responsive to local conditions using local data. In the absence of such plans, universal BLL testing is recommended. In addition, jurisdictions should follow the Centers for Medicare & Medicaid Services requirement that all Medicaid-enrolled children be tested at ages 12 and 24 months or at age 24-72 months if they have not previously been screened (3).

Blood Lead Levels in U.S. Children Ages 1-11 Years, 1976-2016.

BACKGROUND: Lead can adversely affect child health across a wide range of exposure levels. We describe the distribution of blood lead levels (BLLs) in U.S. children ages 1-11 y by selected sociodemographic and housing characteristics over a 40-y period. METHODS: Data from the National Health and Nutrition Examination Survey (NHANES) II (1976-1980), NHANES III (Phase 1: 1988-1991 and Phase II: 1991-1994), and Continuous NHANES (1999-2016) were used to describe the distribution of BLLs (in micrograms per deciliter; 1µg/dL=0.0483µmol/L) in U.S. children ages 1-11 y from 1976 to 2016. For all children with valid BLLs (n=27,122), geometric mean (GM) BLLs [95% confidence intervals (CI)] and estimated prevalence ≥5µg/dL (95% CI) were calculated overall and by selected characteristics, stratified by age group (1-5 y and 6-11 y). RESULTS: The GM BLL in U.S. children ages 1-5 y declined from 15.2µg/dL (95% CI: 14.3, 16.1) in 1976-1980 to 0.83µg/dL (95% CI: 0.78, 0.88) in 2011-2016, representing a 94.5% decrease over time. For children ages 6-11 y, GM BLL declined from 12.7µg/dL (95% CI: 11.9, 13.4) in 1976-1980 to 0.60µg/dL (95% CI: 0.58, 0.63) in 2011-2016, representing a 95.3% decrease over time. Even so, for the most recent period (2011-2016), estimates indicate that approximately 385,775 children ages 1-11 y had BLLs greater than or equal to the CDC blood lead reference value of 5µg/dL. Higher GM BLLs were associated with non-Hispanic Black race/ethnicity, lower family income-to-poverty-ratio, and older housing age. DISCUSSION: Overall, BLLs in U.S. children ages 1-11 y have decreased substantially over the past 40 y. Despite these notable declines in population exposures to lead over time, higher GM BLLs are consistently associated with risk factors such as race/ethnicity, poverty, and housing age that can be used to target blood lead screening efforts. https://doi.org/10.1289/EHP7932.

Risk of Clinical Severity by Age and Race/Ethnicity Among Adults Hospitalized for COVID-19-United States, March-September 2020.

BACKGROUND: Older adults and people from certain racial and ethnic groups are disproportionately represented in coronavirus disease 2019 (COVID-19) hospitalizations and deaths. METHODS: Using data from the Premier Healthcare Database on 181 813 hospitalized adults diagnosed with COVID-19 during March-September 2020, we applied multivariable log-binomial regression to assess the associations between age and race/ethnicity and COVID-19 clinical severity (intensive care unit [ICU] admission, invasive mechanical ventilation [IMV], and death) and to determine whether the impact of age on clinical severity differs by race/ethnicity. RESULTS: Overall, 84 497 (47%) patients were admitted to the ICU, 29 078 (16%) received IMV, and 27 864 (15%) died in the hospital. Increased age was strongly associated with clinical severity when controlling for underlying medical conditions and other covariates; the strength of this association differed by race/ethnicity. Compared with non-Hispanic White patients, risk of death was lower among non-Hispanic Black patients (adjusted risk ratio, 0.96; 95% CI, 0.92-0.99) and higher among Hispanic/Latino patients (risk ratio [RR], 1.15; 95% CI, 1.09-1.20), non-Hispanic Asian patients (RR, 1.16; 95% CI, 1.09-1.23), and patients of other racial and ethnic groups (RR, 1.13; 95% CI, 1.06-1.21). Risk of ICU admission and risk of IMV were elevated among some racial and ethnic groups. CONCLUSIONS: These results indicate that age is a driver of poor outcomes among hospitalized persons with COVID-19. Additionally, clinical severity may be elevated among patients of some racial and ethnic minority groups. Public health strategies to reduce severe acute respiratory syndrome coronavirus 2 infection rates among older adults and racial and ethnic minorities are essential to reduce poor outcomes.

Hydrating the respiratory tract: An alternative explanation why masks lower severity of COVID-19.

The seasonality of respiratory diseases has been linked, among other factors, to low outdoor absolute humidity and low indoor relative humidity, which increase evaporation of water in the mucosal lining of the respiratory tract. We demonstrate that normal breathing results in an absorption-desorption cycle inside facemasks, in which supersaturated air is absorbed by the mask fibers during expiration, followed by evaporation during inspiration of dry environmental air. For double-layered cotton masks, which have considerable heat capacity, the temperature of inspired air rises above room temperature, and the effective increase in relative humidity can exceed 100%. We propose that the recently reported, disease-attenuating effect of generic facemasks is dominated by the strong humidity increase of inspired air. This elevated humidity promotes mucociliary clearance of pathogens from the lungs, both before and after an infection of the upper respiratory tract has occurred. Effective mucociliary clearance can delay and reduce infection of the lower respiratory tract, thus mitigating disease severity. This mode of action suggests that masks can benefit the wearer even after an infection in the upper respiratory tract has occurred, complementing the traditional function of masks to limit person-to-person disease transmission. This potential therapeutical use should be studied further.

Decreases in Young Children Who Received Blood Lead Level Testing During COVID-19 - 34 Jurisdictions, January-May 2020.

Exposure to lead, a toxic metal, can result in severe effects in children, including decreased ability to learn, permanent neurologic damage, organ failure, and death. CDC and other health care organizations recommend routine blood lead level (BLL) testing among children as part of well-child examinations to facilitate prompt identification of elevated BLL, eliminate source exposure, and provide medical and other services (1). To describe BLL testing trends among young children during the coronavirus disease 2019 (COVID-19) pandemic, CDC analyzed data reported from 34 state and local health departments about BLL testing among children aged <6 years conducted during January-May 2019 and January-May 2020. Compared with testing in 2019, testing during January-May 2020 decreased by 34%, with 480,172 fewer children tested. An estimated 9,603 children with elevated BLL were missed because of decreased BLL testing. Despite geographic variability, all health departments reported fewer children tested for BLL after the national COVID-19 emergency declaration (March-May 2020). In addition, health departments reported difficulty conducting medical follow-up and environmental investigations for children with elevated BLLs because of staffing shortages and constraints on home visits associated with the pandemic. Providers and public health agencies need to take action to ensure that children who missed their scheduled blood lead screening test, or who required follow-up on an earlier high BLL, be tested as soon as possible and receive appropriate care.

Hydroxychloroquine and Chloroquine Prescribing Patterns by Provider Specialty Following Initial Reports of Potential Benefit for COVID-19 Treatment - United States, January-June 2020.

Hydroxychloroquine and chloroquine, primarily used to treat autoimmune diseases and to prevent and treat malaria, received national attention in early March 2020, as potential treatment and prophylaxis for coronavirus disease 2019 (COVID-19) (1). On March 20, the Food and Drug Administration (FDA) issued an emergency use authorization (EUA) for chloroquine phosphate and hydroxychloroquine sulfate in the Strategic National Stockpile to be used by licensed health care providers to treat patients hospitalized with COVID-19 when the providers determine the potential benefit outweighs the potential risk to the patient.* Following reports of cardiac and other adverse events in patients receiving hydroxychloroquine for COVID-19 (2), on April 24, 2020, FDA issued a caution against its use† and on June 15, rescinded its EUA for hydroxychloroquine from the Strategic National Stockpile.§ Following the FDA's issuance of caution and EUA rescindment, on May 12 and June 16, the federal COVID-19 Treatment Guidelines Panel issued recommendations against the use of hydroxychloroquine or chloroquine to treat COVID-19; the panel also noted that at that time no medication could be recommended for COVID-19 pre- or postexposure prophylaxis outside the setting of a clinical trial (3). However, public discussion concerning the effectiveness of these drugs on outcomes of COVID-19 (4,5), and clinical trials of hydroxychloroquine for prophylaxis of COVID-19 continue.¶ In response to recent reports of notable increases in prescriptions for hydroxychloroquine or chloroquine (6), CDC analyzed outpatient retail pharmacy transaction data to identify potential differences in prescriptions dispensed by provider type during January-June 2020 compared with the same period in 2019. Before 2020, primary care providers and specialists who routinely prescribed hydroxychloroquine, such as rheumatologists and dermatologists, accounted for approximately 97% of new prescriptions. New prescriptions by specialists who did not typically prescribe these medications (defined as specialties accounting for ≤2% of new prescriptions before 2020) increased from 1,143 prescriptions in February 2020 to 75,569 in March 2020, an 80-fold increase from March 2019. Although dispensing trends are returning to prepandemic levels, continued adherence to current clinical guidelines for the indicated use of these medications will ensure their availability and benefit to patients for whom their use is indicated (3,4), because current data on treatment and pre- or postexposure prophylaxis for COVID-19 indicate that the potential benefits of these drugs do not appear to outweigh their risks.

Hydrating the Respiratory Tract: An Alternative Explanation Why Masks Lower Severity of COVID-19 Disease.

Seasonality of respiratory diseases has been linked, among other factors, to low outdoor absolute humidity and low relative humidity in indoor environments, which increase evaporation of water in the mucosal layer lining the respiratory tract. We demonstrate that normal breathing results in an absorption-desorption cycle inside facemasks, where super-saturated air is absorbed by the mask fibers during expiration, followed by evaporation during inspiration of dry environmental air. For double-layered cotton masks, which have considerable heat capacity, the temperature of inspired air rises above room temperature, and the effective increase in relative humidity can exceed 100%. We propose that the recently reported, disease-attenuating effect of generic facemasks is dominated by the strong humidity increase of inspired air. SIGNIFICANCE STATEMENT: Facemasks are the most widely used tool for mitigating the spread of the COVID-19 pandemic. Decreased disease severity by the wearer has also been linked to the use of cloth facemasks. This well-documented finding is surprising considering that such masks are poor at filtering the smallest aerosol particles, which can reach the lower respiratory tract and have been associated with severe disease. We show that facemasks strongly increase the effective humidity of inhaled air, thereby promoting hydration of the respiratory epithelium which is known to be beneficial to the immune system. Increased humidity of inspired air could be an alternate explanation for the now well-established link between mask wearing and lower disease severity.

Interrupted Pressure-Jump NMR Experiments Reveal Resonances of On-Pathway Protein Folding Intermediate.

Previous pressure-jump NMR experiments on a pressure-sensitized double mutant of ubiquitin showed evidence that its folding occurs via two parallel, comparably efficient pathways: a single barrier and a two-barrier pathway. An interrupted folding NMR experiment is introduced, where for a brief period the pressure is dropped to atmospheric conditions (1 bar), followed by a jump back to high pressure for signal detection. Conventional, forward sampling of the indirect dimension during the low-pressure period correlates the 15N or 13C' chemical shifts of the unfolded protein at 1 bar to the 1H frequencies of both the unfolded and folded proteins at high pressure. Remarkably, sampling the data of the same experiment in the reverse direction yields the frequencies of proteins present at the end of the low-pressure interval, which include unfolded, intermediate, and folded species. Although the folding intermediate 15N shifts differ strongly from natively folded protein, its 13C' chemical shifts, which are more sensitive probes for secondary structure, closely match those of the folded protein and indicate that the folding intermediate must have a structure that is quite similar to the native state.

Monitoring 15N Chemical Shifts During Protein Folding by Pressure-Jump NMR.

Pressure-jump hardware permits direct observation of protein NMR spectra during a cyclically repeated protein folding process. For a two-state folding protein, the change in resonance frequency will occur nearly instantaneously when the protein clears the transition state barrier, resulting in a monoexponential change of the ensemble-averaged chemical shift. However, protein folding pathways can be more complex and contain metastable intermediates. With a pseudo-3D NMR experiment that utilizes stroboscopic observation, we measure the ensemble-averaged chemical shifts, including those of exchange-broadened intermediates, during the folding process. Such measurements for a pressure-sensitized mutant of ubiquitin show an on-pathway kinetic intermediate whose 15N chemical shifts differ most from the natively folded protein for strands ß5, its preceding turn, and the two strands that pair with ß5 in the native structure.

Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell.

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15N and 13C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15N relaxation data show evidence for rapid exchange, on a ∼100-µs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 µM, oligomeric off-pathway intermediates compete with folding of the native state.

Resonance assignments of an α-synuclein fibril prepared in Tris buffer at moderate ionic strength.

Fibrils of the protein α-synuclein (α-syn) are implicated in the pathogenesis of Parkinson's disease and related neurodegenerative disorders. We have reported a high-resolution structure (PDB 2N0A) of an α-syn fibril form prepared by in vitro incubation of monomeric protein in 50 mM sodium phosphate buffer pH 7.4 with 0.1 mM EDTA and 0.01% sodium azide. In parallel with this structure determination, ongoing studies of small molecule ligands binding to α-syn fibrils, prepared in 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer, have been in progress, and it is therefore of interest to determine the structural similarity of these forms. Here we report the 13C and 15N resonance assignments for α-syn fibrils prepared with Tris-HCl buffer (pH 7.7 at 37 °C) and 100 mM NaCl. These fibrillization conditions yield a form with fibril core chemical shifts highly similar to those we reported (BMRB 16939) in the course of determining the high-resolution 2N0A structure, with the exception of some small perturbations from T44 to V55, including two sets of peaks observed for residues T44-V48. Additional differences occur in the patterns of observed residues in the primarily unstructured N-terminus. These results demonstrate a common fold of the fibril core for α-syn fibrils prepared in phosphate or Tris-HCl buffer at moderate ionic strength.

Solid-State NMR of highly 13C-enriched cholesterol in lipid bilayers.

Cholesterol (Chol) is vital for cell function as it is essential to a myriad of biochemical and biophysical processes. The atomistic details of Chol's interactions with phospholipids and proteins is therefore of fundamental interest, and NMR offers unique opportunities to interrogate these properties at high resolution. Towards this end, here we describe approaches for examining the structure and dynamics of Chol in lipid bilayers using high levels of 13C enrichment in combination with magic-angle spinning (MAS) methods. We quantify the incorporation levels and demonstrate high sensitivity and resolution in 2D 13C-13C and 1H-13C spectra, enabling de novo assignments and site-resolved order parameter measurements obtained in a fraction of the time required for experiments with natural abundance sterols. We envision many potential future applications of these methods to study sterol interactions with drugs, lipids and proteins.

Efficient dipolar double quantum filtering under magic angle spinning without a (1)H decoupling field.

We present a systematic study of dipolar double quantum (DQ) filtering in (13)C-labeled organic solids over a range of magic-angle spinning rates, using the SPC-n recoupling sequence element with a range of n symmetry values from 3 to 11. We find that efficient recoupling can be achieved for values n⩾7, provided that the (13)C nutation frequency is on the order of 100kHz or greater. The decoupling-field dependence was investigated and explicit heteronuclear decoupling interference conditions identified. The major determinant of DQ filtering efficiency is the decoupling interference between (13)C and (1)H fields. For (13)C nutation frequencies greater than 75kHz, optimal performance is observed without an applied (1)H field. At spinning rates exceeding 20kHz, symmetry conditions as low as n=3 were found to perform adequately.

Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein.

Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson's disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register ß-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.