Explore the vast range of titles available.

Nucleosomes play a dual role in compacting the genome and regulating the access to DNA. To unravel the underlying mechanism, we study fluorescently labeled mononucleosomes by multi-parameter FRET measurements and characterize their structural and dynamic heterogeneity upon NaCl-induced destabilization. Species-selective fluorescence lifetime analysis and dynamic photon distribution analysis reveal intermediates during nucleosome opening and lead to a coherent structural and kinetic model. In dynamic octasomes and hexasomes the interface between the H2A-H2B dimers and the (H3-H4) 2 tetramer opens asymmetrically by an angle of ≈20° on a 50 and 15 µs time scale, respectively. This is followed by a slower stepwise release of the dimers coupled with DNA unwrapping. A mutation (H2A-R81A) at the interface between H2A and H3 facilitates initial opening, confirming the central role of the dimer:tetramer interface for nucleosome stability. Partially opened states such as those described here might serve as convenient nucleation sites for DNA-recognizing proteins. Nucleosomes compact the genome and regulate access to specific DNA sequences. Here the authors employ single-molecule FRET studies to characterize nucleosome dynamics at different salt concentrations and dissect nucleosome disassembly into elementary steps.

The nucleosome has a central role in the compaction of genomic DNA and the control of DNA accessibility for transcription and replication. To help understanding the mechanism of nucleosome opening and closing in these processes, we studied the disassembly of mononucleosomes by quantitative single-molecule FRET with high spatial resolution, using the SELEX-generated \"Widom 601\" positioning sequence labeled with donor and acceptor flu orophores. Reversible dissociation was induced by increasing NaCI concentration. At least 3 species with different FRET were identified and assigned to structures: (i) the most stable high-FRET species corresponding to the intact nucleosome, (ii) a less stable mid-FRET species that we attribute to a first intermediate with a partially unwrapped DNA and less hi stones, and (iii) a low-FRET species characterized by a very broad FRET distribution, representing highly unwrapped structures and free DNA formed at the expense of the other 2 species. Selective FCS analysis indicates that even in the low-FRET state, some histones are still bound to the DNA. The interdye distance of 54.0 Å measured for the high-FRET species corresponds to a compact conformation close to the known crystallographic structure. The coexistence and interconversion of these species is first demonstrated under non-invasive conditions. A geometric model of the DNA unwinding predicts the presence of the observed FRET species. The different structures of these species in the disassembly pathway map the energy landscape indicating major barriers for 10-bp and minor ones for 5-bp DNA unwinding steps.

Nucleosomes are important for chromatin compaction and gene regulation; their integrity depends crucially on the structural properties of the histone tails. Recent all-atom molecular dynamics simulations revealed that removal of the N-terminal tails of histone H3, known to destabilize nucleosomes, causes a rearrangement of two arginines of histone H2A, namely R81 and R88 by altering the electrostatic environment of the H2A α3 domain. Whether this rearrangement is the cause or the effect of decreased stability, is unclear. Here, we emulate the altered electrostatic environment that was found after H3 tail clipping through charge-modifying mutations to decouple its impact on intranucleosomal interactions from that of the histone tails. Förster resonance energy transfer experiments on recombinant nucleosomes and all-atom molecular dynamics simulations reveal a compensatory role of those amino acids in nucleosome stability. The simulations indicate a weakened interface between H2A-H2B dimers and the (H3-H4) 2 tetramer, as well as between dimers and DNA. These findings agree with the experimental observations of position and charge dependent decreased nucleosome stability induced by the introduced mutations. This work highlights the importance of the H2A α3 domain and suggests allosteric effects between this domain and the outer DNA gyre as well as the H3 N-terminal tail.

Nucleosome structure and stability affect genetic accessibility by altering the local chromatin morphology. Recent FRET experiments on nucleosomes have given valuable insight into the structural transformations they can adopt. Yet, even if performed under seemingly identical conditions, experiments performed in bulk and at the single molecule level have given mixed answers due to the limitations of each technique. To compare such experiments, however, they must be performed under identical conditions. Here we develop an experimental framework that overcomes the conventional limitations of each method: single molecule FRET experiments are carried out at bulk concentrations by adding unlabeled nucleosomes, while bulk FRET experiments are performed in microplates at concentrations near those used for single molecule detection. Additionally, the microplate can probe many conditions simultaneously before expending valuable instrument time for single molecule experiments. We highlight this experimental strategy by exploring the role of selective acetylation of histone H3 on nucleosome structure and stability; in bulk, H3-acetylated nucleosomes were significantly less stable than non-acetylated nucleosomes. Single molecule FRET analysis further revealed that acetylation of histone H3 promoted the formation of an additional conformational state, which is suppressed at higher nucleosome concentrations and which could be an important structural intermediate in nucleosome regulation.

Background With the passing of Jörg Langowski 6 May 2017 in a sailplane accident, the scientific community was deprived of a strident and effective voice for DNA and chromatin molecular and computational biophysics, for open access publishing and for the creation of effective scientific research networks. Methods Here, after reviewing some of Jörg’s key research contributions and ideas, we offer through the personal remembrance of his closest collaborators, a deep analysis of the major results of his research and the future directions they have engendered. Conclusions The legacy of Jörg Langowski has been to propel a way of viewing biological function that considers living systems as dynamic and in three dimensions. This physical view of biology that he pioneered is now, finally, becoming established also because of his great effort.

Purpose To evaluate the impact of an AI-based software trained to detect cerebral aneurysms on TOF-MRA on the diagnostic performance and reading times across readers with varying experience levels. Methods One hundred eighty-six MRI studies were reviewed by six readers to detect cerebral aneurysms. Initially, readings were assisted by the CNN-based software mdbrain. After 6 weeks, a second reading was conducted without software assistance. The results were compared to the consensus reading of two neuroradiological specialists and sensitivity (lesion and patient level), specificity (patient level), and false positives per case were calculated for the group of all readers, for the subgroup of physicians, and for each individual reader. Also, reading times for each reader were measured. Results The dataset contained 54 aneurysms. The readers had no experience (three medical students), 2 years experience (resident in neuroradiology), 6 years experience (radiologist), and 12 years (neuroradiologist). Significant improvements of overall specificity and the overall number of false positives per case were observed in the reading with AI support. For the physicians, we found significant improvements of sensitivity on lesion and patient level and false positives per case. Four readers experienced reduced reading times with the software, while two encountered increased times. Conclusion In the reading with the AI-based software, we observed significant improvements in terms of specificity and false positives per case for the group of all readers and significant improvements of sensitivity and false positives per case for the physicians. Further studies are needed to investigate the effects of the AI-based software in a prospective setting.

IntroductionAI is increasingly used in clinical practice to support radiologists when reading imaging studies.Aim of StudyTo evaluate the impact of an, AI based software trained to detect cerebral aneurysms on TOF-MRA on the diagnostic performance of multiple readers with different amounts of experience in diagnostic neuroimaging.Methods186 MRI studies were evaluated by six readers (three medical students, one radiology resident, one radiologist and one neuroradiologist) for the presence of cerebral aneurysms. First, the reading was done with the support of the software. After six weeks, the reading was repeated without the support of the software. The results were compared to the consensus reading of two neuroradiological specialists. Sensitivity (patient level and aneurysm level), specificity (patient level), and false positives/case were calculated.ResultsSensitivities (aneurysm level) ranged from 66.7%-87.0% with and 57.7%-87.0% without AI, sensitivities (patient level) were 63.4%-81.8% with and 52.3%-75.0% without AI. Specificities ranged from 93.7%-97.2% with and 89.4%-98.6% without AI. False positive findings/case ranged from 0.03–0.12 with and 0.02–0.17 without AI (differences not statistically significant, p-values 0.05–1). Four readers showed a significant decrease of reading times with the software, the remaining two readers showed a significant increase of reading times.ConclusionWe found equivocal results for the diagnostic performance of six different readers for the detection of cerebral aneurysms with and without the use of an AI software. Although we found a tendency towards better diagnostic performances, these differences were not statistically significant. The majority of readers showed a significant decrease of reading times.Disclosure of InterestNothing to disclose