Protocol for an observational study to identify potential predictors of an acute exacerbation in patients with chronic obstructive pulmonary disease (the PACE Study).

INTRODUCTION: Acute exacerbations of chronic obstructive pulmonary disease (AECOPD) are the most critical events for patients with COPD that have a negative impact on patients' quality of life, accelerate disease progression, and can result in hospital admissions and death. Although there is no distinct definition or detailed knowledge about AECOPD, it is commonly used as primary outcome in clinical studies. Furthermore, it may be difficult in clinical practice to differentiate the worsening of symptoms due to an AECOPD or to the development of heart failure. Therefore, it is of major clinical importance to investigate the underlying pathophysiology, and if possible, predictors of an AECOPD and thus to identify patients who are at high risk for developing an acute exacerbation. METHODS AND ANALYSIS: In total, 355 patients with COPD will be included prospectively to this study during a 3-week inpatient pulmonary rehabilitation programme at the Schoen Klinik Berchtesgadener Land, Schoenau am Koenigssee (Germany). All patients will be closely monitored from admission to discharge. Lung function, exercise tests, clinical parameters, quality of life, physical activity and symptoms will be recorded, and blood samples and exhaled air will be collected. If a patient develops an AECOPD, there will be additional comprehensive diagnostic assessments to differentiate between cardiac, pulmonary or cardiopulmonary causes of worsening. Follow-up measures will be performed at 6, 12 and 24 months.Exploratory data analyses methods will be used for the primary research question (screening and identification of possible factors to predict an AECOPD). Regression analyses and a generalised linear model with a binomial outcome (AECOPD) will be applied to test if predictors are significant. ETHICS AND DISSEMINATION: This study has been approved by the Ethical Committee of the Philipps University Marburg, Germany (No. 61/19). The results will be presented in conferences and published in a peer-reviewed journal. TRIAL REGISTRATION NUMBER: NCT04140097

Selection platforms for directed evolution in synthetic biology

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules – gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function – be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code

Plug-and-Play Approach for Preparing Chromatin Containing Site-Specific DNA Modifications: The Influence of Chromatin Structure on Base Excision Repair

The genomic DNA of eukaryotic cells exists in the form of chromatin, the structure of which controls the biochemical accessibility of the underlying DNA to effector proteins. In order to gain an in depth molecular understanding of how chromatin structure regulates DNA repair, detailed <i>in vitro</i> biochemical and biophysical studies are required. However, because of challenges associated with reconstituting nucleosome arrays containing site-specifically positioned DNA modifications, such studies have been limited to the use of mono- and dinucleosomes as model <i>in vitro</i> substrates, which are incapable of folding into native chromatin structures. To address this issue, we developed a straightforward and general approach for assembling chemically defined oligonucleosome arrays (i.e., designer chromatin) containing site-specifically modified DNA. Our method takes advantage of nicking endonucleases to excise short fragments of unmodified DNA, which are subsequently replaced with synthetic oligonucleotides containing the desired modification. Using this approach, we prepared several oligonucleosome substrates containing precisely positioned 2′-deoxyuridine (dU) residues and examined the efficiency of base excision repair (BER) within several distinct chromatin architectures. We show that, depending on the translational position of the lesion, the combined catalytic activities of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) can be either inhibited by as much as 20-fold or accelerated by more than 5-fold within compact chromatin (i.e., the 30 nm fiber) relative to naked DNA. Moreover, we demonstrate that digestion of dU by UDG/APE1 proceeds much more rapidly in mononucleosomes than in compacted nucleosome arrays, thereby providing the first direct evidence that internucleosome interactions play an important role in regulating BER within higher-order chromatin structures. Overall, this work highlights the value of performing detailed biochemical studies on precisely modified chromatin substrates <i>in vitro</i> and provides a robust platform for investigating DNA modifications in chromatin biology