Static mucus impairs bacterial clearance and allows chronic infection with Pseudomonas aeruginosa in the cystic fibrosis rat.

Cystic fibrosis (CF) airway disease is characterised by chronic Pseudomonas aeruginosa infection. Successful eradication strategies have been hampered by a poor understanding of the mechanisms underlying conversion to chronicity. The CFTR-knockout (KO) rat harbors a progressive defect in mucociliary transport and viscosity. KO rats were infected before and after the appearance of the mucus defect, using a clinical, mucoid-isolate of P. aeruginosa embedded in agarose beads. Young KO rats that were exposed to bacteria before the development of mucociliary transport defects resolved the infection and subsequent tissue damage. However, older KO rats that were infected in the presence of hyperviscous and static mucus were unable to eradicate bacteria, but instead had bacterial persistence through 28 days post-infection that was accompanied by airway mucus occlusion and lingering inflammation. Normal rats responded to infection with increased mucociliary transport to supernormal rates, which reduced the severity of a second bacterial exposure. We therefore conclude that the aberrant mucus present in the CF airway permits persistence of P. aeruginosa in the lung.

Ivacaftor partially corrects airway inflammation in a humanized G551D rat.

Animal models have been highly informative for understanding the pathogenesis and progression of cystic fibrosis (CF) lung disease. In particular, the CF rat models recently developed have addressed mechanistic causes of the airway mucus defect characteristic of CF, and how these may change when cystic fibrosis transmembrane conductance regulator (CFTR) activity is restored using new modulator therapies. We hypothesized that inflammatory changes to the airway would develop spontaneously and progressively, and that these changes would be resolved with modulator therapy. To test this, we used a humanized-CFTR rat expressing the G551D variant that responds to the CFTR modulator ivacaftor. Markers typically found in the CF lung were assessed, including neutrophil influx, small airway histopathology, and inflammatory cytokine concentration. Young hG551D rats did not express inflammatory cytokines at baseline but did upregulate these in response to inflammatory trigger. As the hG551D rats aged, histopathology worsened, accompanied by neutrophil influx into the airway and increasing concentrations of TNF-α, IL-1α, and IL-6 in the airways. Ivacaftor administration reduced concentrations of these cytokines when administered to the rats at baseline but was less effective in the rats that had also received inflammatory stimulus. Therefore, we conclude that administration of ivacaftor resulted in an incomplete resolution of inflammation when rats received an external trigger, suggesting that CFTR activation may not be enough to resolve inflammation in the lungs of patients with CF.

Sex-specific associations of asthma acquisition with changes in DNA methylation during adolescence.

BACKGROUND: Underlying biological mechanisms involved in sex differences in asthma status changes from pre- to post-adolescence are unclear. DNA methylation (DNAm) has been shown to be associated with the risk of asthma. OBJECTIVE: We hypothesized that asthma acquisition from pre- to post-adolescence was associated with changes in DNAm during this period at asthma-associated cytosine-phosphate-guanine (CpG) sites and such an association was sex-specific. METHODS: Subjects from the Isle of Wight birth cohort (IOWBC) with DNAm in blood at ages 10 and 18 years (n = 124 females, 151 males) were studied. Using a training-testing approach, epigenome-wide CpGs associated with asthma were identified. Logistic regression was used to examine sex-specific associations of DNAm changes with asthma acquisition between ages 10 and 18 at asthma-associated CpGs. The ALSPAC birth cohort was used for independent replication. To assess functional relevance of identified CpGs, association of DNAm with gene expression in blood was assessed. RESULTS: We identified 535 CpGs potentially associated with asthma. Significant interaction effects of DNAm changes and sex on asthma acquisition in adolescence were found at 13 of the 535 CpGs in IOWBC (P-values <1.0 × 10-3 ). In the replication cohort, consistent interaction effects were observed at 10 of the 13 CpGs. At 7 of these 10 CpGs, opposite DNAm changes across adolescence were observed between sexes in both cohorts. In both cohorts, cg20891917, located on IFRD1 linked to asthma, shows strong sex-specific effects on asthma transition (P-values <.01 in both cohorts). CONCLUSION AND CLINICAL RELEVANCE: Gender reversal in asthma acquisition is associated with opposite changes in DNAm (males vs females) from pre- to post-adolescence at asthma-associated CpGs. These CpGs are potential biomarkers of sex-specific asthma acquisition in adolescence.

Ivacaftor Reverses Airway Mucus Abnormalities in a Rat Model Harboring a Humanized G551D-CFTR.

Rationale: Animal models have been highly informative for understanding the characteristics, onset, and progression of cystic fibrosis (CF) lung disease. In particular, the CFTR-/- rat has revealed insights into the airway mucus defect characteristic of CF but does not replicate a human-relevant CFTR (cystic fibrosis transmembrane conductance regulator) variant.Objectives: We hypothesized that a rat expressing a humanized version of CFTR and harboring the ivacaftor-sensitive variant G551D could be used to test the impact of CFTR modulators on pathophysiologic development and correction.Methods: In this study, we describe a humanized-CFTR rat expressing the G551D variant obtained by zinc finger nuclease editing of a human complementary DNA superexon, spanning exon 2-27, with a 5' insertion site into the rat gene just beyond intron 1. This targeted insertion takes advantage of the endogenous rat promoter, resulting in appropriate expression compared with wild-type animals.Measurements and Main Results: The bioelectric phenotype of the epithelia recapitulates the expected absence of CFTR activity, which was restored with ivacaftor. Large airway defects, including depleted airway surface liquid and periciliary layers, delayed mucus transport rates, and increased mucus viscosity, were normalized after the administration of ivacaftor.Conclusions: This model is useful to understand the mechanisms of disease and the extent of pathology reversal with CFTR modulators.

Pseudo-enantiomeric carbohydrate-based N-heterocyclic carbenes as promising chiral ligands for enantiotopic discrimination.

The practical synthesis of carbohydrate-based NHC-Rh complexes bearing C1 or C3 sterically differentiated positions, accessed by glycosylation or SNAr strategies, is reported. These catalysts exhibit pseudo-enantiomeric behaviour in the hydrosilylation of acetophenone. We show that steric bulk at C1 gives preference for (S)-phenyl-1-ethanol, while bulk at C3 leads to the (R)-enantiomer. These results represent the first example of pseudo-enantiomeric carbohydrate-based NHC ligands leading to enantiotopic discrimination.

Epigenome-wide association study of lung function level and its change.

Previous reports link differential DNA methylation (DNAme) to environmental exposures that are associated with lung function. Direct evidence on lung function DNAme is, however, limited. We undertook an agnostic epigenome-wide association study (EWAS) on pre-bronchodilation lung function and its change in adults.In a discovery-replication EWAS design, DNAme in blood and spirometry were measured twice, 6-15 years apart, in the same participants of three adult population-based discovery cohorts (n=2043). Associated DNAme markers (p<5×10-7) were tested in seven replication cohorts (adult: n=3327; childhood: n=420). Technical bias-adjusted residuals of a regression of the normalised absolute ß-values on control probe-derived principle components were regressed on level and change of forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC) and their ratio (FEV1/FVC) in the covariate-adjusted discovery EWAS. Inverse-variance-weighted meta-analyses were performed on results from discovery and replication samples in all participants and never-smokers.EWAS signals were enriched for smoking-related DNAme. We replicated 57 lung function DNAme markers in adult, but not childhood samples, all previously associated with smoking. Markers not previously associated with smoking failed replication. cg05575921 (AHRR (aryl hydrocarbon receptor repressor)) showed the statistically most significant association with cross-sectional lung function (FEV1/FVC: pdiscovery=3.96×10-21 and pcombined=7.22×10-50). A score combining 10 DNAme markers previously reported to mediate the effect of smoking on lung function was associated with lung function (FEV1/FVC: p=2.65×10-20).Our results reveal that lung function-associated methylation signals in adults are predominantly smoking related, and possibly of clinical utility in identifying poor lung function and accelerated decline. Larger studies with more repeat time-points are needed to identify lung function DNAme in never-smokers and in children.

Associations of atopic dermatitis and asthma with child behaviour: Results from the PROBIT cohort.

BACKGROUND: Conflicting findings from studies evaluating associations of allergic disease with child behaviour require longitudinal studies to resolve. OBJECTIVE: To estimate the magnitude of associations of atopic dermatitis (AD) in infancy, and symptoms of asthma and AD at 6.5 years, with child behaviour at 6.5 years. METHODS: Secondary cohort analysis of the Promotion of Breastfeeding Intervention Trial (PROBIT). PROBIT enrolled 17 046 infants at birth and followed them up at 6.5 years (n = 13 889). Study paediatricians collected data on infantile AD at repeated follow-up examinations during the first year of life. At 6.5 years, paediatricians performed skin prick tests and parents reported asthma and AD symptoms during the prior year. In addition, parents and teachers completed the Strength and Difficulties Questionnaire, which includes scales on hyperactivity/inattention, emotional problems, conduct problems, peer problems and prosocial behaviours. RESULTS: Physician-diagnosed AD in the first year of life was not associated with increased risk for behavioural problems at 6.5 years. Emotional problems at 6.5 years were more common among children with AD symptoms (OR: 2.24, 95% CI: 1.62-3.12) and asthma symptoms (OR: 1.45; 95% CI: 1.07-1.96) during the past year at 6.5 years and ORs for children with symptoms of more severe AD and asthma were also higher. AD in the past year was also associated with probable hyperactivity/inattention disorder at 6.5 years (OR: 2.05; 95% CI: 1.09-3.84). Other subscales of the SDQ were not related to asthma or AD symptoms during the past year. CONCLUSIONS AND CLINICAL RELEVANCE: Children with AD symptoms were at higher risk for concomitant hyperactivity/inattention and emotional disorder, and children with asthma symptoms were at higher risk of having concomitant emotional problems. However, AD during infancy did not predict childhood behaviours.

SERPINA1 methylation and lung function in tobacco-smoke exposed European children and adults: a meta-analysis of ALEC population-based cohorts.

BACKGROUND: The pathophysiological role of SERPINA1 in respiratory health may be more strongly determined by the regulation of its expression than by common genetic variants. A family based study of predominantly smoking adults found methylation at two Cytosine-phosphate-Guanine sites (CpGs) in SERPINA1 gene to be associated with chronic obstructive pulmonary disease risk. The objective of this study was to confirm the association of lung function with SERPINA1 methylation in general population samples by testing a comprehensive set of CpGs in the SERPINA gene cluster. We considered lung function level and decline in adult smokers from three European population-based cohorts and lung function level and growth in tobacco-smoke exposed children from a birth cohort. METHODS: DNA methylation using Illumina Infinium Human Methylation 450 k and EPIC beadchips and lung function were measured at two time points in 1076 SAPALDIA, ECRHS and NFBC adult cohort participants and 259 ALSPAC children. Associations of methylation at 119 CpG sites in the SERPINA gene cluster (PP4R4-SERPINA13P) with lung functions and circulating alpha-1-antitripsin (AAT) were assessed using multivariable cross-sectional and longitudinal regression models. RESULTS: Methylation at cg08257009 in the SERPINA gene cluster, located 32 kb downstream of SERPINA1, not annotated to a gene, was associated with FEV1/FVC at the Bonferroni corrected level in adults, but not in children. None of the methylation signals in the SERPINA1 gene showed associations with lung function after correcting for multiple testing. CONCLUSIONS: The results do not support a role of SERPINA1 gene methylation as determinant of lung function across the life course in the tobacco smoke exposed general population exposed.

Carbohydrates as enantioinduction components in stereoselective catalysis.

Carbohydrate derivatives are readily available chiral molecules, yet they are infrequently employed as enantioinduction components in stereoselective catalysis. In this review, synthetic approaches to carbohydrate-based ligands and catalysts are outlined, along with example applications in enantioselective catalysis. A wide range of carbohydrate-based functionality is covered, and key trends and future opportunities are identified.

Identification of sister chromatids by DNA template strand sequences.

It is generally assumed that sister chromatids are genetically and functionally identical and that segregation to daughter cells is a random process. However, functional differences between sister chromatids regulate daughter cell fate in yeast and sister chromatid segregation is not random in Escherichia coli. Differentiated sister chromatids, coupled with non-random segregation, have been proposed to regulate cell fate during the development of multicellular organisms. This hypothesis has not been tested because molecular features to reliably distinguish between sister chromatids are not obvious. Here we show that parental 'Watson' and 'Crick' DNA template strands can be identified in sister chromatids of murine metaphase chromosomes using CO-FISH (chromosome orientation fluorescence in situ hybridization) with unidirectional probes specific for centromeric and telomeric repeats. All chromosomes were found to have a uniform orientation with the 5' end of the short arm on the same strand as T-rich major satellite repeats. The invariable orientation of repetitive DNA was used to differentially label sister chromatids and directly study mitotic segregation patterns in different cell types. Whereas sister chromatids appeared to be randomly distributed between daughter cells in cultured lung fibroblasts and embryonic stem cells, significant non-random sister chromatid segregation was observed in a subset of colon crypt epithelial cells, including cells outside positions reported for colon stem cells. Our results establish that DNA template sequences can be used to distinguish sister chromatids and follow their mitotic segregation in vivo.

Detection of G-quadruplex DNA in mammalian cells.

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.

Ultraviolet Absorption Induces Hydrogen-Atom Transfer in G⋠C Watson-Crick DNA Base Pairs in Solution.

Ultrafast deactivation pathways bestow photostability on nucleobases and hence preserve the structural integrity of DNA following absorption of ultraviolet (UV) radiation. One controversial recovery mechanism proposed to account for this photostability involves electron-driven proton transfer (EDPT) in Watson-Crick base pairs. The first direct observation is reported of the EDPT process after UV excitation of individual guanine-cytosine (G⋅C) Watson-Crick base pairs by ultrafast time-resolved UV/visible and mid-infrared spectroscopy. The formation of an intermediate biradical species (G[-H]⋅C[+H]) with a lifetime of 2.9 ps was tracked. The majority of these biradicals return to the original G⋅C Watson-Crick pairs, but up to 10% of the initially excited molecules instead form a stable photoproduct G*⋅C* that has undergone double hydrogen-atom transfer. The observation of these sequential EDPT mechanisms across intermolecular hydrogen bonds confirms an important and long debated pathway for the deactivation of photoexcited base pairs, with possible implications for the UV photochemistry of DNA.

Carbohydrate-based N-heterocyclic carbenes for enantioselective catalysis.

Versatile syntheses of C2-linked and C2-symmetric carbohydrate-based imidazol(in)ium salts from functionalised amino-carbohydrate derivatives are reported. The novel NHCs were ligated to [Rh(COD)Cl]2 and evaluated in Rh-catalysed asymmetric hydrosilylation of ketones with good yields and promising enantioselectivities.

Higher systolic blood pressure with normal vascular function measurements in preterm-born children.

UNLABELLED: Preterm birth, low birth weight and poor foetal nutrition have been linked to cardiovascular disease, but the underlying mechanisms remain unclear. We explored prematurity and vascular function by studying a UK cohort of 14 049 children and conducting a systematic review. CONCLUSION: Systolic blood pressure was higher in subjects born preterm than term, but there were no differences in endothelial dysfunction or arterial stiffness. The systematic review revealed no clear association between prematurity and vascular function.

Deep sequencing of proteotoxicity modifier genes uncovers a Presenilin-2/beta-amyloid-actin genetic risk module shared among alpha-synucleinopathies.

Whether neurodegenerative diseases linked to misfolding of the same protein share genetic risk drivers or whether different protein-aggregation pathologies in neurodegeneration are mechanistically related remains uncertain. Conventional genetic analyses are underpowered to address these questions. Through careful selection of patients based on protein aggregation phenotype (rather than clinical diagnosis) we can increase statistical power to detect associated variants in a targeted set of genes that modify proteotoxicities. Genetic modifiers of alpha-synuclein (ɑS) and beta-amyloid (Aß) cytotoxicity in yeast are enriched in risk factors for Parkinson's disease (PD) and Alzheimer's disease (AD), respectively. Here, along with known AD/PD risk genes, we deeply sequenced exomes of 430 ɑS/Aß modifier genes in patients across alpha-synucleinopathies (PD, Lewy body dementia and multiple system atrophy). Beyond known PD genes GBA1 and LRRK2, rare variants AD genes (CD33, CR1 and PSEN2) and Aß toxicity modifiers involved in RhoA/actin cytoskeleton regulation (ARGHEF1, ARHGEF28, MICAL3, PASK, PKN2, PSEN2) were shared risk factors across synucleinopathies. Actin pathology occurred in iPSC synucleinopathy models and RhoA downregulation exacerbated ɑS pathology. Even in sporadic PD, the expression of these genes was altered across CNS cell types. Genome-wide CRISPR screens revealed the essentiality of PSEN2 in both human cortical and dopaminergic neurons, and PSEN2 mutation carriers exhibited diffuse brainstem and cortical synucleinopathy independent of AD pathology. PSEN2 contributes to a common-risk signal in PD GWAS and regulates ɑS expression in neurons. Our results identify convergent mechanisms across synucleinopathies, some shared with AD.

Patient assessment in emergency medical services: complexity and uncertainty in street-level patient processing.

Though still a relatively nascent public function, emergency medical services (EMS) agencies have come to constitute a core local government service. The potentially life-saving interventional and palliative treatments provided by EMS personnel are inarguably vital for positive short- and long-term patient outcomes, yet our understanding of the behavior of these individuals during service interactions is limited. This exploratory research begins to narrow this gap in our understanding of street-level bureaucrats in EMS organizations by analyzing semi-structured interviews of paramedics recounting uncertain and complex accounts of street-level patient processing. Results indicate that factors such as paramedic communication skills, influential bystanders with key incident information, and patient identity serve to shape street-level interactions with patients.

Muc5b Contributes to Mucus Abnormality in Rat Models of Cystic Fibrosis.

Cystic fibrosis (CF) airway disease is characterized by excessive and accumulative mucus in the airways. Mucociliary clearance becomes defective as mucus secretions become hyperconcentrated and viscosity increases. The CFTR-knockout (KO) rat has been previously shown to progressively develop delayed mucociliary transport, secondary to increased viscoelasticity of airway secretions. The humanized-G551D CFTR rat model has demonstrated that abnormal mucociliary clearance and hyperviscosity is reversed by ivacaftor treatment. In this study, we sought to identify the components of mucus that changes as the rat ages to contribute to these abnormalities. We found that Muc5b concentrations, and to a lesser extent Muc5ac, in the airway were increased in the KO rat compared to WT, and that Muc5b concentration was directly related to the viscosity of the mucus. Additionally, we found that methacholine administration to the airway exacerbates these characteristics of disease in the KO, but not WT rat trachea. Lastly we determined that at 6 months of age, CF rats had mucus that was adherent to the airway epithelium, a process that is reversed by ivacaftor therapy in the hG551D rat. Overall, these data indicate that accumulation of Muc5b initiates the muco-obstructive process in the CF lung prior to infection.

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering.

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.

Is UV-Induced Electron-Driven Proton Transfer Active in a Chemically Modified A·T DNA Base Pair?

Transient electronic and vibrational absorption spectroscopies have been used to investigate whether UV-induced electron-driven proton transfer (EDPT) mechanisms are active in a chemically modified adenine-thymine (A·T) DNA base pair. To enhance the fraction of biologically relevant Watson-Crick (WC) hydrogen-bonding motifs and eliminate undesired Hoogsteen structures, a chemically modified derivative of A was synthesized, 8-(tert-butyl)-9-ethyladenine (8tBA). Equimolar solutions of 8tBA and silyl-protected T nucleosides in chloroform yield a mixture of WC pairs, reverse WC pairs, and residual monomers. Unlike previous transient absorption studies of WC guanine-cytosine (G·C) pairs, no clear spectroscopic or kinetic evidence was identified for the participation of EDPT in the excited-state relaxation dynamics of 8tBA·T pairs, although ultrafast (sub-100 fs) EDPT cannot be discounted. Monomer-like dynamics are proposed to dominate in 8tBA·T.