Raised sputum extracellular DNA confers lung function impairment and poor symptom control in an exacerbation-susceptible phenotype of neutrophilic asthma

Background Extracellular DNA (e-DNA) and neutrophil extracellular traps (NETs) are linked to asthmatics airway inflammation. However, data demonstrating the characterization of airway inflammation associated with excessive e-DNA production and its impact on asthma outcomes are limited. Objective To characterize the airway inflammation associated with excessive e-DNA production and its association with asthma control, severe exacerbations and pulmonary function, particularly, air trapping and small airway dysfunction. Methods We measured e-DNA concentrations in induced sputum from 134 asthma patients and 28 healthy controls. We studied the correlation of e-DNA concentrations with sputum neutrophils, eosinophils and macrophages and the fractional exhaled nitric oxide (FeNO). Lung function was evaluated using spirometry, body plethysmography, impulse oscillometry and inert gas multiple breath washout. We stratified patients with asthma into low-DNA and high-DNA to compare lung function impairments and asthma outcomes. Results Patients with severe asthma had higher e-DNA concentration (54.2 ± 42.4 ng/µl) than patients with mild-moderate asthma (41.0 ± 44.1 ng/µl) or healthy controls (26.1 ± 16.5 ng/µl), (all p values < 0.05). E-DNA concentrations correlated directly with sputum neutrophils (R = 0.49, p < 0.0001) and negatively with sputum macrophages (R = − 0.36, p < 0.0001), but neither with sputum eosinophils (R = 0.10, p = 0.26), nor with FeNO (R = − 0.10, p = 0.22). We found that 29% of asthma patients (n = 39) had high e-DNA concentrations above the upper 95th percentile value in healthy controls (55.6 ng /μl). High-DNA was associated with broad lung function impairments including: airflow obstruction of the large (FEV1) and small airways (FEF50%, FEF25–75), increased air trapping (RV, RV/TLC), increased small airway resistance (R5-20, sReff), decreased lung elasticity (X5Hz) and increased ventilation heterogeneity (LCI), (all P values < 0.05). We also found that high e-DNA was associated with nearly three-fold greater risk of severe exacerbations (OR 2·93 [95% CI 1.2–7.5]; p = 0·012), worse asthma control test (p = 0.03), worse asthma control questionnaire scores (p = 0.01) and higher doses of inhaled corticosteroids (p = 0.026). Conclusion Increased production of extracellular DNA in the airway characterizes a subset of neutrophilic asthma patients who have broad lung function impairments, poor symptom control and increased risk of severe exacerbations.

A widespread pathway for substitution of adenine by diaminopurine in phage genomes

DNA modifications vary in form and function but generally do not alter Watson-Crick base pairing. Diaminopurine (Z) is an exception because it completely replaces adenine and forms three hydrogen bonds with thymine in cyanophage S-2L genomic DNA. However, the biosynthesis, prevalence, and importance of Z genomes remain unexplored. Here, we report a multienzyme system that supports Z-genome synthesis. We identified dozens of globally widespread phages harboring such enzymes, and we further verified the Z genome in one of these phages, Acinetobacter phage SH-Ab 15497, by using liquid chromatography with ultraviolet and mass spectrometry. The Z genome endows phages with evolutionary advantages for evading the attack of host restriction enzymes, and the characterization of its biosynthetic pathway enables Z-DNA production on a large scale for a diverse range of applications.

Improved architectures for flexible DNA production using retrons across kingdoms of life

Exogenous DNA is a critical molecular tool for biology. This is particularly true for gene editing, where exogenous DNA can be used as a template to introduce precise changes to the sequence of a cell's genome. This DNA is typically synthesized or assembled in vitro and then delivered to target cells. However, delivery can be inefficient, and low abundance of template DNA may be one reason that precise editing typically occurs at a low rate. It has recently been shown that producing DNA inside cells can using reverse transcriptases can increase the efficiency of genome editing. One tool to produce that DNA is a retron, a bacterial retroelement that has an endogenous role in phage defense. However, little effort has been directed at optimizing the retron for production of designed sequences when used as a component of biotechnology. Here, we identify modifications to the retron non-coding RNA that result in more abundant reverse transcribed DNA. We also test architectures of the retron operon that enable efficient reverse transcription across kingdoms of life from bacteria, to yeast, to cultured human cells. We find that gains in DNA production using modified retrons are portable from prokaryotic to eukaryotic cells. Finally, we demonstrate that increased production of RT-DNA results in more efficient genome editing in both prokaryotic and eukaryotic cells. These experiments provide a general framework for production of DNA using a retron for biotechnological applications.

A Roadblock-and-Kill Mechanism of Action Model for the DNA-Targeting Antibiotic Ciprofloxacin

ABSTRACT Fluoroquinolones, antibiotics that cause DNA damage by inhibiting DNA topoisomerases, are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here, we investigated the growth inhibition of the bacterium Escherichia coli by the fluoroquinolone ciprofloxacin at low concentrations. We measured the long-term and short-term dynamical response of the growth rate and DNA production rate to ciprofloxacin at both the population and single-cell levels. We show that, despite the molecular complexity of DNA metabolism, a simple roadblock-and-kill model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates in the presence of ciprofloxacin. The model also predicts dynamical changes in the DNA production rate in wild-type E. coli and in a recombination-deficient mutant following a step-up of ciprofloxacin. Our work highlights that bacterial cells show a delayed growth rate response following fluoroquinolone exposure. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. We also show that the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response, challenging the accepted view. Our work highlights the importance of including detailed biophysical processes in biochemical-systems models to quantitatively predict the bacterial response to antibiotics.

Human Endogenous Retroviruses (HERVs): Shaping the Innate Immune Response in Cancers

Human Endogenous Retroviruses (HERVs) are accounting for 8% of the human genome. These sequences are remnants from ancient germline infections by exogenous retroviruses. After million years of evolution and multiple integrations, HERVs have acquired many damages rendering them defective. At steady state, HERVs are mostly localized in the heterochromatin and silenced by methylation. Multiple conditions have been described to induce their reactivation, including auto-immune diseases and cancers. HERVs re-expression leads to RNA (simple and double-stranded) and DNA production (by reverse transcription), modulating the innate immune response. Some studies also argue for a role of HERVs in shaping the evolution of innate immunity, notably in the development of the interferon response. However, their exact role in the innate immune response, particularly in cancer, remains to be defined. In this review, we see how HERVs could be key-players in mounting an antitumor immune response. After a brief introduction on HERVs characteristics and biology, we review the different mechanisms by which HERVs can interact with the immune system, with a focus on the innate response. We then discuss the potential impact of HERVs expression on the innate immune response in cancer.

A roadblock-and-kill model explains the dynamical response to the DNA-targeting antibiotic ciprofloxacin

Fluoroquinolones - antibiotics that cause DNA damage by inhibiting DNA topoisomerases - are clinically important, but their mechanism of action is not yet fully understood. In particular, the dynamical response of bacterial cells to fluoroquinolone exposure has hardly been investigated, although the SOS response, triggered by DNA damage, is often thought to play a key role. Here we investigate growth inhibition of the bacterium Escherichia coli by the fluoroquinolone ciprofloxacin at low doses (up to 5x the minimum inhibitory concentration). We measure the long-term and short-term (dynamic) response of the growth rate and DNA production rate to ciprofloxacin, at both population- and single-cell level. We show that despite the molecular complexity of DNA metabolism, a simple `roadblock-and-kill’ model focusing on replication fork blockage and DNA damage by ciprofloxacin-poisoned DNA topoisomerase II (gyrase) quantitatively reproduces long-term growth rates. The model also predicts dynamical changes in DNA production rate in wild type E. coli and in an SOS-deficient mutant, following a step-up of ciprofloxacin. Our work reveals new insights into the dynamics of fluoroquinolone action, with important implications for predicting the rate of resistance evolution. Most importantly, our model explains why the response is delayed: it takes many doubling times to fragment the DNA sufficiently to inhibit gene expression. Our model also challenges the view that the SOS response plays a central role: the dynamical response is controlled by the timescale of DNA replication and gyrase binding/unbinding to the DNA rather than by the SOS response. More generally, our work highlights the importance of including biophysical processes in biochemical-systems models to fully understand bacterial response to antibiotics.