R-loops are stable nucleic acid structures that have important physiological functions, but which also pose a significant threat to genomic stability. Increased R-loops cause replication stress and ...chromosome fragility and have been associated with diseases such as neurodegeneration and cancer. Although excessive R-loops are a feature of cells that are defective in RNA processing, what causes them to form is unclear. Here, we demonstrate that DHX9 (RNA helicase A) promotes the formation of pathological and non-pathological R-loops. In the absence of splicing factors, formation of R-loops correlates with the prolonged association of DHX9 with RNA Polymerase II (RNA Pol II). This leads to the production of DNA-RNA hybrid, which traps RNA Pol II on chromatin with the potential to block DNA replication. Our data provide a molecular mechanism for the formation of R-loops that is relevant to neurodegenerative diseases and cancers in which deregulated RNA processing is a feature.
Sputum neutrophil elastase and serum desmosine, which is a linked marker of endogenous elastin degradation, are possible biomarkers of disease severity and progression in bronchiectasis. This study ...aimed to determine the association of elastase activity and desmosine with exacerbations and lung function decline in bronchiectasis.
This was a single-center prospective cohort study using the TAYBRIDGE (Tayside Bronchiectasis Registry Integrating Datasets, Genomics, and Enrolment into Clinical Trials) registry in Dundee, UK. A total of 433 patients with high-resolution computed tomography-confirmed bronchiectasis provided blood samples for desmosine measurement, and 381 provided sputum for baseline elastase activity measurements using an activity-based immunosassay and fluorometric substrate assay. Candidate biomarkers were tested for their relationship with cross-sectional markers of disease severity, and with future exacerbations, mortality and lung function decline over 3 years.
Elastase activity in sputum was associated with the bronchiectasis severity index (r = 0.49; P < 0.0001) and was also correlated with the Medical Research Council dyspnea score (r = 0.34; P < 0.0001), FEV
% predicted (r = -0.33; P < 0.0001), and the radiological extent of bronchiectasis (r = 0.29; P < 0.0001). During a 3-year follow-up, elevated sputum elastase activity was associated with a higher frequency of exacerbations (P < 0.0001) but was not independently associated with mortality. Sputum elastase activity was independently associated with FEV
decline (β coefficient, -0.139; P = 0.001). Elastase showed good discrimination for severe exacerbations with an area under the curve of 0.75 (95% confidence interval CI, 0.72-0.79) and all-cause mortality (area under the curve, 0.70; 95% CI, 0.67-0.73). Sputum elastase activity increased at exacerbations (P = 0.001) and was responsive to treatment with antibiotics. Desmosine was correlated with sputum elastase (r = 0.42; P < 0.0001) and was associated with risk of severe exacerbations (hazard ratio 2.7; 95% CI, 1.42-5.29; P = 0.003) but not lung function decline.
Sputum neutrophil elastase activity is a biomarker of disease severity and future risk in adults with bronchiectasis.
Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk
. More than 60% of this effect is attributable to the ability of ...metformin to lower body weight in a sustained manner
. The molecular mechanisms by which metformin lowers body weight are unknown. Here we show-in two independent randomized controlled clinical trials-that metformin increases circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15), which has been shown to reduce food intake and lower body weight through a brain-stem-restricted receptor. In wild-type mice, oral metformin increased circulating GDF15, with GDF15 expression increasing predominantly in the distal intestine and the kidney. Metformin prevented weight gain in response to a high-fat diet in wild-type mice but not in mice lacking GDF15 or its receptor GDNF family receptor α-like (GFRAL). In obese mice on a high-fat diet, the effects of metformin to reduce body weight were reversed by a GFRAL-antagonist antibody. Metformin had effects on both energy intake and energy expenditure that were dependent on GDF15, but retained its ability to lower circulating glucose levels in the absence of GDF15 activity. In summary, metformin elevates circulating levels of GDF15, which is necessary to obtain its beneficial effects on energy balance and body weight, major contributors to its action as a chemopreventive agent.
Osimertinib is a third-generation inhibitor of the epidermal growth factor receptor used in treatment of non-small cell lung cancer. A full understanding of its disposition and capacity for ...interaction with other medications will facilitate its effective use as a single agent and in combination therapy.
Recombinant cytochrome P450s and liver microsomal preparations were used to identify novel pathways of osimertinib metabolism
A panel of knockout and mouse lines humanized for pathways of drug metabolism were used to establish the relevance of these pathways
Although some osimertinib metabolites were similar in mouse and human liver samples there were several significant differences, in particular a marked species difference in the P450s involved. The murine Cyp2d gene cluster played a predominant role in mouse, whereas CYP3A4 was the major human enzyme responsible for osimertinib metabolism. Induction of this enzyme in CYP3A4 humanized mice substantially decreased circulating osimertinib exposure. Importantly, we discovered a further novel pathway of osimertinib disposition involving CPY1A1. Modulation of CYP1A1/CYP1A2 levels markedly reduced parent drug concentrations, significantly altering metabolite pharmacokinetics (PK) in humanized mice
We demonstrate that a P450 enzyme expressed in smokers' lungs and lung tumors has the capacity to metabolise osimertinib. This could be a significant factor in defining the outcome of osimertinib treatment. This work also illustrates how P450-humanized mice can be used to identify and mitigate species differences in drug metabolism and thereby model the
effect of critical metabolic pathways on anti-tumor response.
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The sputum microbiome has a potential role in disease phenotyping and risk stratification in chronic obstructive pulmonary disease (COPD), but few large longitudinal cohort studies exist.
Our aim was ...to investigate the COPD sputum microbiome and its association with inflammatory phenotypes and mortality.
16S ribosomal RNA gene sequencing was performed on sputum from 253 clinically stable COPD patients (4-year median follow-up). Samples were classified as Proteobacteria or Firmicutes (phylum level) and Haemophilus or Streptococcus (genus level) dominant. Alpha diversity was measured by using Shannon-Wiener diversity and Berger-Parker dominance indices. Survival was modeled by using Cox proportional hazards regression. A subset of 78 patients had label-free liquid chromatography with tandem mass spectrometry performed, with partial least square discriminant analysis integrating clinical, microbiome, and proteomics data.
Proteobacteria dominance and lower diversity was associated with more severe COPD according to the Global Initiative for Chronic Obstructive Lung Disease classification system (P = .0015), more frequent exacerbations (P = .0042), blood eosinophil level less than or equal to 100 cells/μL (P < .0001), and lower FEV1 (P = .026). Blood eosinophil counts showed a positive relationship with percent of Firmicutes and Streptococcus and a negative association with percent Proteobacteria and Haemophilus. Proteobacteria dominance was associated with increased mortality compared with Firmicutes-dominated or balanced microbiome profiles (hazard ratio = 2.58; 95% CI = 1.43-4.66; P = .0017 and hazard ratio = 7.47; 95% CI = 1.02-54.86; P = .048, respectively). Integrated omics analysis showed significant associations between Proteobacteria dominance and the neutrophil activation pathway in sputum.
The sputum microbiome is associated with clinical and inflammatory phenotypes in COPD. Reduced microbiome diversity, associated with Proteobacteria (predominantly Haemophilus) dominance, is associated with neutrophil-associated protein profiles and an increased risk of mortality.
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PZP (pregnancy zone protein) is a broad-spectrum immunosuppressive protein believed to suppress T-cell function during pregnancy to prevent fetal rejection. It has not previously been reported in the ...airway.
To characterize PZP in the bronchiectasis airway, including its relationship with disease severity.
Label-free liquid chromatography/mass spectrometry was performed for sputum protein profiling of patients with bronchiectasis confirmed by high-resolution computed tomography. Results for patients with and without
infection were compared. Sputum and serum PZP was measured by validated ELISA. Airway infection status was established by culture and 16S ribosomal RNA sequencing. Immunofluorescence, ELISA, and electron microscopy were used to identify the cellular source of PZP in neutrophils treated with multiple stimuli.
Elevated PZP was identified by label-free liquid chromatography/mass spectrometry as being associated with
infection. In a validation study of 124 patients, sputum but not serum concentrations of PZP were significantly associated with the Bronchiectasis Severity Index, the frequency of exacerbations, and symptoms. Airway infection with Proteobacteria such as
was associated with higher concentrations of PZP. PZP in sputum was directly related to airway bacterial load. Neutrophils induced to form neutrophil extracellular traps (NETs) with phorbol myristate acetate released high concentrations of PZP
, and fluorescence microscopy confirmed the presence of PZP in NETs, whereas fluorescence and electron microscopy localized PZP to the cytoplasm and nuclei of neutrophils. Effective antibiotic therapy reduced sputum PZP.
PZP is released into NETs. We report a novel link between airway infection, NET formation, and disease severity in bronchiectasis during chronic airway inflammation.
The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3) ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA ...satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.
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•BRCA1/BARD1 is a histone-H2A-specific ubiquitin ligase•BRCA1/BARD1 ubiquitylates histone H2A on lysines 127 and 129•Specificity for H2A is provided within in a nucleosomal context•Heterodimeric RING domains of BRCA1/BARD1 are sufficient for E3 function in vivo
The ubiquitin ligase (E3) activity of BRCA1 is its only known biochemical activity in vitro. Kalb et al. report that the heterodimeric complex formed by BRCA1/BARD1 ubiquitylates histone H2A in nucleosomes specifically at its C-terminal tail. Moreover, in vitro and in vivo assays identify H2A lysines127 and 129 as the target lysines. These results help to explain the localization and activity of BRCA1/BARD1 on chromatin in cells.
There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and ...food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.
Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because little is understood of the underlying ...mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a method of host defence that has been implicated in multiple inflammatory diseases. We aimed to investigate the role of NETs in disease severity and treatment response in bronchiectasis.
In this observational study, we did a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. First, we used liquid chromatography-tandem mass spectrometry to identify proteomic biomarkers associated with disease severity, defined using the bronchiectasis severity index, in patients with bronchiectasis (n=40) in Dundee, UK. Second, we validated these biomarkers in two cohorts of patients with bronchiectasis, the first comprising 175 patients from the TAYBRIDGE study in the UK and the second comprising 275 patients from the BRIDGE cohort study from centres in Italy, Spain, and UK, using an immunoassay to measure NETs. Third, we investigated whether pathogenic bacteria had a role in NET concentrations in patients with severe bronchiectasis. In a separate study, we enrolled patients with acute exacerbations of bronchiectasis (n=20) in Dundee, treated with intravenous antibiotics for 14 days and proteomics were used to identify proteins associated with treatment response. Findings from this cohort were validated in an independent cohort of patients who were admitted to the same hospital (n=20). Fourth, to assess the potential use of macrolides to reduce NETs in patients with bronchiectasis, we examined two studies of long-term macrolide treatment, one in patients with bronchiectasis (n=52 from the UK) in which patients were given 250 mg of azithromycin three times a week for a year, and a post-hoc analysis of the Australian AMAZES trial in patients with asthma (n=47) who were given 500 mg of azithromycin 3 times per week for a year.
Sputum proteomics identified that NET-associated proteins were the most abundant and were the proteins most strongly associated with disease severity. This finding was validated in two observational cohorts, in which sputum NETs were associated with bronchiectasis severity index, quality of life, future risk of hospital admission, and mortality. In a subgroup of 20 patients with acute exacerbations, clinical response to intravenous antibiotic treatment was associated with successfully reducing NETs in sputum. Patients with Pseudomonas aeruginosa infection had a lessened proteomic and clinical response to intravenous antibiotic treatment compared with those without Pseudomonas infections, but responded to macrolide therapy. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma.
We identified NETs as a key marker of disease severity and treatment response in bronchiectasis. These data support the concept of targeting neutrophilic inflammation with existing and novel therapies.
Scottish Government, British Lung Foundation, and European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC).
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