The disordered N-terminal tail of SARS-CoV-2 Nucleocapsid protein forms a dynamic complex with RNA.

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA binding domain (RBD) and its flanking disordered N-terminal domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.

Reweighting of molecular simulations with explicit-solvent SAXS restraints elucidates ion-dependent RNA ensembles.

Small-angle X-ray scattering (SAXS) experiments are increasingly used to probe RNA structure. A number of forward models that relate measured SAXS intensities and structural features, and that are suitable to model either explicit-solvent effects or solute dynamics, have been proposed in the past years. Here, we introduce an approach that integrates atomistic molecular dynamics simulations and SAXS experiments to reconstruct RNA structural ensembles while simultaneously accounting for both RNA conformational dynamics and explicit-solvent effects. Our protocol exploits SAXS pure-solute forward models and enhanced sampling methods to sample an heterogenous ensemble of structures, with no information towards the experiments provided on-the-fly. The generated structural ensemble is then reweighted through the maximum entropy principle so as to match reference SAXS experimental data at multiple ionic conditions. Importantly, accurate explicit-solvent forward models are used at this reweighting stage. We apply this framework to the GTPase-associated center, a relevant RNA molecule involved in protein translation, in order to elucidate its ion-dependent conformational ensembles. We show that (a) both solvent and dynamics are crucial to reproduce experimental SAXS data and (b) the resulting dynamical ensembles contain an ion-dependent fraction of extended structures.

Physiotherapy service provision in a specialist adult cystic fibrosis service: A pre-post design study with the inclusion of an allied health assistant.

What is the impact of including an allied health assistant (AHA) role on physiotherapy service delivery in an acute respiratory service? A pragmatic pre-post design study examined physiotherapy services across two 3-month periods: current service delivery [P1] and current service delivery plus AHA [P2]. Clinical and non-clinical activity quantified as number, type and duration (per day) of all staff activity categorised for skill level (AHA, junior, senior). Physiotherapy service delivery increased in P2 compared to P1 (n = 4730 vs n = 3048). Physiotherapists undertook fewer respiratory (p < 0.001) and exercise treatments (p < 0.001) but increased reviews for inpatients (p < 0.001) and at multidisciplinary clinics in P2 (56% vs 76%, p < 0.01). The AHA accounted for 20% of all service provision. AHA activity comprised mainly non-direct clinical care including oversight of respiratory equipment use (e.g. supply, set-up, cleaning, loan audits) and other patient-related administrative tasks associated with delegation handovers, supervision and clinical documentation (72%), delegated supervision of established respiratory (5%) and exercise treatments (10%) and delegated exercise tests (3%). The AHA completed most of the exercise tests (n = 25). AHA non-direct clinical tasks included departmental management activities (11%). No adverse events were reported. AHA inclusion in an acute respiratory care service changed physiotherapy service provision. The AHA completed delegated routine clinical and non-clinical tasks. Physiotherapists increased clinic activity and annual reviews. Including an AHA role offers sustainable options for enhancing physiotherapy service provision in acute respiratory care.

Divalent ions tune the kinetics of a bacterial GTPase center rRNA folding transition from secondary to tertiary structure.

Folding of an RNA from secondary to tertiary structure often depends on divalent ions for efficient electrostatic charge screening (nonspecific association) or binding (specific association). To measure how different divalent cations modify folding kinetics of the 60 nucleotide Ecoli rRNA GTPase center, we combined stopped-flow fluorescence in the presence of Mg2+, Ca2+, or Sr2+ together with time-resolved small angle X-ray scattering (SAXS) in the presence of Mg2+ to observe the folding process. Immediately upon addition of each divalent ion, the RNA undergoes a transition from an extended state with secondary structure to a more compact structure. Subsequently, specific divalent ions modulate populations of intermediates in conformational ensembles along the folding pathway with transition times longer than 10 msec. Rate constants for the five folding transitions act on timescales from submillisecond to tens of seconds. The sensitivity of RNA tertiary structure to divalent cation identity affects all but the fastest events in RNA folding, and allowed us to identify those states that prefer Mg2+ The GTPase center RNA appears to have optimized its folding trajectory to specifically utilize this most abundant intracellular divalent ion.

Gas exchange responses during 6-min walk test in patients with pulmonary arterial hypertension.

BACKGROUND AND OBJECTIVE: The 6-min walk test (6MWT) is the most widely utilized method of assessing exercise capacity in pulmonary arterial hypertension (PAH). Cardiopulmonary exercise testing has the advantage of providing additional physiological information over 6MWT. The goals of our study were to describe the addition of gas exchange measurements to 6MWT and to determine how these parameters were related to the severity of PAH in three major subgroups of PAH (idiopathic (IPAH), connective tissue disease-related (CTPAH) and congenital heart disease-related (CHPAH)). METHODS: Seventy-six PAH patients (IPAH, n = 28; CTPAH, n = 24; CHPAH, n = 24) completed the 6MWT with simultaneous gas exchange measurements. The 6-min walk distance (6MWD), oxygen uptake ( V ˙ O2 ), carbon dioxide production ( V ˙ CO2 ), oxygen saturation, minute ventilation to carbon dioxide output ( V ˙ E / V ˙ CO2 ) and end-tidal partial pressure for carbon dioxide (PET CO2 ) were compared between subgroups, different functional classes (FCs) and pharmacotherapy. RESULTS: Whilst no significant difference in 6MWT was observed, absolute V ˙ O2 and V ˙ CO2 were higher for IPAH (P < 0.05). Differences were removed when V ˙ O2 and V ˙ CO2 were expressed relative to body mass (i.e. mL/kg/min). CHPAH had the most significant desaturation during 6MWT (CPAH: 73 ± 15%; CTPAH: 90 ± 8%, IPAH: 92 ± 8%, P < 0.01). There was no difference in V ˙ E / V ˙ CO2 and PET CO2 between groups; however, New York Health Association (NYHA) FC II performed better than FC III subjects in 6MWT with lower V ˙ E / V ˙ CO2 and higher end-exercise PET CO2 . Similarly, individuals on more advanced pharmacotherapy (triple therapy vs monotherapy) had poorer gas exchange during exercise. CONCLUSION: Whilst 6MWT and gas exchange did not differentiate between PAH groups, individuals with more severe disease and on more advanced pharmacotherapy had poorer gas exchange during exercise.

Two novel loci, COBL and SLC10A2, for Alzheimer's disease in African Americans.

INTRODUCTION: African Americans' (AAs) late-onset Alzheimer's disease (LOAD) genetic risk profile is incompletely understood. Including clinical covariates in genetic analyses using informed conditioning might improve study power. METHODS: We conducted a genome-wide association study (GWAS) in AAs employing informed conditioning in 1825 LOAD cases and 3784 cognitively normal controls. We derived a posterior liability conditioned on age, sex, diabetes status, current smoking status, educational attainment, and affection status, with parameters informed by external prevalence information. We assessed association between the posterior liability and a genome-wide set of single-nucleotide polymorphisms (SNPs), controlling for APOE and ABCA7, identified previously in a LOAD GWAS of AAs. RESULTS: Two SNPs at novel loci, rs112404845 (P = 3.8 × 10-8), upstream of COBL, and rs16961023 (P = 4.6 × 10-8), downstream of SLC10A2, obtained genome-wide significant evidence of association with the posterior liability. DISCUSSION: An informed conditioning approach can detect LOAD genetic associations in AAs not identified by traditional GWAS.

Transethnic genome-wide scan identifies novel Alzheimer's disease loci.

INTRODUCTION: Genetic loci for Alzheimer's disease (AD) have been identified in whites of European ancestry, but the genetic architecture of AD among other populations is less understood. METHODS: We conducted a transethnic genome-wide association study (GWAS) for late-onset AD in Stage 1 sample including whites of European Ancestry, African-Americans, Japanese, and Israeli-Arabs assembled by the Alzheimer's Disease Genetics Consortium. Suggestive results from Stage 1 from novel loci were followed up using summarized results in the International Genomics Alzheimer's Project GWAS dataset. RESULTS: Genome-wide significant (GWS) associations in single-nucleotide polymorphism (SNP)-based tests (P < 5 × 10-8) were identified for SNPs in PFDN1/HBEGF, USP6NL/ECHDC3, and BZRAP1-AS1 and for the interaction of the (apolipoprotein E) APOE ε4 allele with NFIC SNP. We also obtained GWS evidence (P < 2.7 × 10-6) for gene-based association in the total sample with a novel locus, TPBG (P = 1.8 × 10-6). DISCUSSION: Our findings highlight the value of transethnic studies for identifying novel AD susceptibility loci.

Climbing the vertebrate branch of U1A/U2B″ protein evolution.

In the vertebrate lineage of the U1A/U2B″/SNF protein family, the U1A and U2B″ proteins bind to RNA stem-loops in the U1 or U2 snRNPs, respectively. However, their specialization is fairly recent, as they evolved from a single ancestral protein. The progress of their specialization (subfunctionalization) can be monitored by the amino acid sequence changes that give rise to their modern RNA-binding specificity. Using ancestral sequence reconstruction to predict the intermediates on the evolutionary branch, a probable path of sequential changes is defined for U1A and U2B″. The RNA-binding affinity for U1A/U2B″ protein ancestors was measured using modern U1 and U2 snRNA stem-loops and RNA stem-loop variants to understand how the proteins' RNA specificities evolved.

Physiotherapy for cystic fibrosis in Australia and New Zealand: A clinical practice guideline.

Physiotherapy management is a key element of care for people with cystic fibrosis (CF) throughout the lifespan. Although considerable evidence exists to support physiotherapy management of CF, there is documented variation in practice. The aim of this guideline is to optimize the physiotherapy management of people with CF in Australia and New Zealand. A systematic review of the literature in key areas of physiotherapy practice for CF was undertaken. Recommendations were formulated based on National Health and Medical Research Council (Australia) guidelines and considered the quality, quantity and level of the evidence; the consistency of the body of evidence; the likely clinical impact; and applicability to physiotherapy practice in Australia and New Zealand. A total of 30 recommendations were made for airway clearance therapy, inhalation therapy, exercise assessment and training, musculoskeletal management, management of urinary incontinence, managing the newly diagnosed patient with CF, delivery of non-invasive ventilation, and physiotherapy management before and after lung transplantation. These recommendations can be used to underpin the provision of evidence-based physiotherapy care to people with CF in Australia and New Zealand.

Dementia incidence declined in African-Americans but not in Yoruba.

INTRODUCTION: To compare dementia incidence of African-American and Yoruba cohorts aged ≥70 years enrolled in 1992 and 2001. METHODS: African-Americans residing in Indianapolis and Yoruba in Ibadan, Nigeria without dementia were enrolled in 1992 and 2001 and evaluated every 2-3 years until 2009. The cohorts consist of 1440 African-Americans, 1774 Yoruba in 1992 and 1835 African-Americans and 1895 Yoruba in the 2001 cohorts aged ≥70 years. RESULTS: In African-Americans, dementia and Alzheimer's disease (AD) incidence rates were significantly lower in 2001 than 1992 for all age groups except the oldest group. The overall standardized annual dementia incidence rates were 3.6% (95% confidence interval [CI], 3.2%-4.1%) in the 1992 cohort and 1.4% (95% CI, 1.2%-1.7%) in the 2001 cohort. There was no significant difference in dementia or AD incidence between the Yoruba cohorts. DISCUSSION: Future research is needed to explore the reasons for the differential changes in incidence rates in these two populations.

Global and local ancestry in African-Americans: Implications for Alzheimer's disease risk.

INTRODUCTION: African-American (AA) individuals have a higher risk for late-onset Alzheimer's disease (LOAD) than Americans of primarily European ancestry (EA). Recently, the largest genome-wide association study in AAs to date confirmed that six of the Alzheimer's disease (AD)-related genetic variants originally discovered in EA cohorts are also risk variants in AA; however, the risk attributable to many of the loci (e.g., APOE, ABCA7) differed substantially from previous studies in EA. There likely are risk variants of higher frequency in AAs that have not been discovered. METHODS: We performed a comprehensive analysis of genetically determined local and global ancestry in AAs with regard to LOAD status. RESULTS: Compared to controls, LOAD cases showed higher levels of African ancestry, both globally and at several LOAD relevant loci, which explained risk for AD beyond global differences. DISCUSSION: Exploratory post hoc analyses highlight regions with greatest differences in ancestry as potential candidate regions for future genetic analyses.

Effect of loop composition on the stability and folding kinetics of RNA hairpins with large loops.

RNA hairpins are ubiquitous structural elements in biological RNAs, where they have the potential to regulate RNA folding and interactions with other molecules. There are established methods for predicting the thermodynamic stability of an RNA hairpin, but there are still relatively few detailed examinations of the kinetics of folding. Nonetheless, several recent studies indicate that hairpin folding does not proceed via a simple two-state model. Here, we monitor fluorescence from hairpins constructed as molecular beacons in ensemble, fluorescence correlation spectroscopy, and stopped-flow experiments to describe the folding of RNA hairpins with long (15 nucleotide) loops. Our results show that folding of these hairpins occurs through more than two states and that the mechanism of folding includes a fast intermediate phase observed on the tens of microseconds time scale and a slow phase, attributed to formation of the native folded hairpin loop and stem, observed on the milliseconds time scale. The composition of the RNA loop determines the time scale of intermediate and native folded states. Hairpins with a polyuracil loop sequence exhibit slower relaxation of the intermediate state and faster relaxation of the native folded state when compared to that of hairpins with cytosine or adenine in the loop. We hypothesize this composition dependence could be attributed to nucleobase stacking in cytosine and adenine containing regions of the loop, which would be absent in hairpins containing polyuracil loops. Such base stacking could destabilize the intermediate folds, thereby speeding the relaxation of the intermediate relative to similar sized hairpins with no base stacking in the loop. Likewise, the lower intermediate stability could prolong the relaxation of the native folded state.

Statin Use, Incident Dementia and Alzheimer Disease in Elderly African Americans.

OBJECTIVE: To investigate the association between statin use, incident dementia, and Alzheimer disease (AD) in a prospective elderly African American cohort. DESIGN: Two stage design with a screening interview followed by a comprehensive in-home assessment conducted over an eight-year period. Diagnoses of incident AD and dementia were made by consensus. Statin use was collected at each evaluation. Measurements of low-density lipoprotein cholesterol (LDL), C-reactive protein (CRP) and APOE genotype were obtained from baseline blood samples. Logistic regression models were used to test the association of statin use on incident dementia and AD and its possible association with lipid and CRP levels. SETTING: Indianapolis, Indiana. PARTICIPANTS: From an original cohort of 2629 participants, a subsample of 974 African Americans aged >70 years with normal cognition, at least one follow up evaluation, complete statin information, and biomarker availability were included. MAIN OUTCOME MEASURES: Incident dementia and incident AD. RESULTS: After controlling for age at diagnosis, sex, education level, presence of the APOE ε4 allele and history of stroke for the incident dementia model, baseline use of statins was associated with a significantly decreased risk of incident dementia (OR=.44, P=.029) and incident AD (OR=.40, P=.029). The significant effect of statin use on reduced AD risk and trend for dementia risk was found only for those participants who reported consistent use over the observational period (incident AD: P=.034; incident dementia: P=.061). Additional models found no significant interaction between baseline statin use, baseline LDL, or CRP level and incident dementia/AD. CONCLUSIONS: Consistent use of statin medications during eight years of follow-up resulted in significantly reduced risk for incident AD and a trend toward reduced risk for incident dementia.

Pesticide sorption and leaching potential on three Hawaiian soils.

On the Hawaiian Islands, groundwater is the principal source of potable water and contamination of this key resource by pesticides is of great concern. To evaluate the leaching potential of four weak acid herbicides [aminocyclopyrachlor, picloram, metsulfuron-methyl, biologically active diketonitrile degradate of isoxaflutole (DKN)] and two neutral non-ionizable herbicides [oxyfluorfen, alachlor], their sorption coefficients were determined on three prevalent soils from the island of Oahu. Metsulfuron-methyl, aminocylcopyrachlor, picloram, and DKN were relatively low sorbing herbicides (K(oc) = 3-53 mL g(-1)), alachlor was intermediate (K(oc) = 120-150 mL g(-1)), and oxyfluorfen sorbed very strongly to the three soils (K(oc) > 12,000 mL g(-1)). Following determination of K(oc) values, the groundwater ubiquity score (GUS) indices for these compounds were calculated to predicted their behavior with the Comprehensive Leaching Risk Assessment System (CLEARS; Tier-1 methodology for Hawaii). Metsulfuron-methyl, aminocyclopyrachlor, picloram, and DKN would be categorized as likely leachers in all three Hawaiian soils, indicating a high risk of groundwater contamination across the island of Oahu. In contrast, oxyfluorfen, regardless of the degradation rate, would possess a low and acceptable leaching risk due to its high sorption on all three soils. The leaching potential of alachlor was more difficult to classify, with a GUS value between 1.8 and 2.8. In addition, four different biochar amendments to these soils did not significantly alter their sorption capacities for aminocyclopyrachlor, indicating a relatively low impact of black carbon additions from geologic volcanic inputs of black carbon. Due to the fact that pesticide environmental risks are chiefly dependent on local soil characteristics, this work has demonstrated that once soil specific sorption parameters are known one can assess the potential pesticide leaching risks.

A compare-and-contrast NMR dynamics study of two related RRMs: U1A and SNF.

The U1A/U2B″/SNF family of small nuclear ribonucleoproteins uses a phylogenetically conserved RNA recognition motif (RRM1) to bind RNA stemloops in U1 and/or U2 small nuclear RNA (snRNA). RRMs are characterized by their α/ß sandwich topology, and these RRMs use their ß-sheet as the RNA binding surface. Unique to this RRM family is the tyrosine-glutamine-phenylalanine (YQF) triad of solvent-exposed residues that are displayed on the ß-sheet surface; the aromatic residues form a platform for RNA nucleobases to stack. U1A, U2B″, and SNF have very different patterns of RNA binding affinity and specificity, however, so here we ask how YQF in Drosophila SNF RRM1 contributes to RNA binding, as well as to domain stability and dynamics. Thermodynamic double-mutant cycles using tyrosine and phenylalanine substitutions probe the communication between those two residues in the free and bound states of the RRM. NMR experiments follow corresponding changes in the glutamine side-chain amide in both U1A and SNF, providing a physical picture of the RRM1 ß-sheet surface. NMR relaxation and dispersion experiments compare fast (picosecond to nanosecond) and intermediate (microsecond-to-millisecond) dynamics of U1A and SNF RRM1. We conclude that there is a network of amino acid interactions involving Tyr-Gln-Phe in both SNF and U1A RRM1, but whereas mutations of the Tyr-Gln-Phe triad result in small local responses in U1A, they produce extensive microsecond-to-millisecond global motions throughout SNF that alter the conformational states of the RRM.

Binding affinity and cooperativity control U2B″/snRNA/U2A' RNP formation.

The U1A and U2B″ proteins are components of the U1 and U2 snRNPs, respectively, where they bind to snRNA stemloops. While localization of U1A and U2B″ to their respective snRNP is a well-known phenomenon, binding of U2B″ to U2 snRNA is typically thought to be accompanied by the U2A' protein. The molecular mechanisms that lead to formation of the RNA/U2B″/U2A' complex and its localization to the U2 snRNP are investigated here, using a combination of in vitro RNA-protein and protein-protein fluorescence and isothermal titration calorimetry binding experiments. We find that U2A' protein binds to U2B″ with nanomolar affinity but binds to U1A with only micromolar affinity. In addition, there is RNA-dependent cooperativity (linkage) between protein-protein and protein-RNA binding. The unique combination of tight binding and cooperativity ensures that the U2A'/U2B″ complex is partitioned only to the U2 snRNP.

Linkage and allostery in snRNP protein/RNA complexes.

Drosophila SNF is a member of the U1A/U2B″/SNF protein family that is found in U1 and U2 snRNPs, where it binds to Stemloop II and Stemloop IV of U1 and U2 snRNA, respectively. SNF also binds to the U2A' protein, but only in the U2 snRNP. Although previous reports have implicated U2A' as a necessary auxiliary protein for the binding of SNF to Stemloop IV, there are no mechanisms that explain the partitioning of U2A' to the U2 snRNP and its absence from the U1 snRNP. Using in vitro RNA binding isotherms and isothermal titration calorimetry, the thermodynamics of SNF/RNA/U2A' ternary complex formation have now been characterized. There is a very large binding cooperativity unique to Stemloop IV that favors formation of the SLIV/SNF/U2A' complex. The binding cooperativity, or heterotropic linkage, is interpreted with respect to linked conformational equilibria of both SNF and its RNA ligand and so represents an example of protein-RNA allostery.

Intrinsic flexibility of snRNA hairpin loops facilitates protein binding.

Stem-loop II of U1 snRNA and Stem-loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops.