Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Towards Legal Knowledge Management Systems for Regulatory Compliance

Maintaining regulatory compliance is an increasing concern. Legal Knowledge Management systems could support the work of compliance managers. However, there are challenges to overcome, of interpreting legal knowledge and mapping it onto business processes. In this discussion paper we determine requirements for Legal Knowledge Management systems that can handle these challenges. We argue that an approach to Legal Knowledge Management should consist of four pillars: (1) a legal ontology to express the legal concepts that are central to the interpretation of rules and regulations in practice, (2) natural language processing techniques, to semi-automatically populate the ontology, (3) a systematic method for mapping the legal concepts onto the actual data and business processes of a company, and (4) a method to construct an audit trail as part of the process design, providing legally acceptable evidence of compliance. The challenges are illustrated by an example of MIFID, a set of European regulations in the financial domain.Infrastructures, Systems and ServicesTechnology, Policy and Managemen

CENP-A and H3 Nucleosomes Display a Similar Stability to Force-Mediated Disassembly

Centromere-specific nucleosomes are a central feature of the kinetochore complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test this hypothesis by subjecting CENP-A and H3 octameric nucleosomes, assembled on random or on centromeric DNA sequences, to varying amounts of applied force by using single-molecule magnetic tweezers. We monitor individual disassembly events of CENP-A and H3 nucleosomes. Regardless of the DNA sequence, the force-mediated disassembly experiments for CENP-A and H3 nucleosomes demonstrate similar rupture forces, life time residency and disassembly steps. From these experiments, we conclude that CENP-A does not, by itself, contribute unique structural features to the nucleosome that lead to a significant resistance against force-mediated disruption. The data present insights into the mechanistic basis for how CENP-A nucleosomes might contribute to the structural foundation of the centromere in vivo.BN/Cees Dekker LabBN/Technici en Analiste

Single-molecule visualization of twin-supercoiled domains generated during transcription

Transcription-coupled supercoiling of DNA is a key factor in chromosome compaction and the regulation of genetic processes in all domains of life. It has become common knowledge that, during transcription, the DNA-dependent RNA polymerase (RNAP) induces positive supercoiling ahead of it (downstream) and negative supercoils in its wake (upstream), as rotation of RNAP around the DNA axis upon tracking its helical groove gets constrained due to drag on its RNA transcript. Here, we experimentally validate this so-called twin-supercoiled-domain model with in vitro real-time visualization at the single-molecule scale. Upon binding to the promoter site on a supercoiled DNA molecule, RNAP merges all DNA supercoils into one large pinned plectoneme with RNAP residing at its apex. Transcription by RNAP in real time demonstrates that up- and downstream supercoils are generated simultaneously and in equal portions, in agreement with the twin-supercoiled-domain model. Experiments carried out in the presence of RNases A and H, revealed that an additional viscous drag of the RNA transcript is not necessary for the RNAP to induce supercoils. The latter results contrast the current consensus and simulations on the origin of the twin-supercoiled domains, pointing at an additional mechanistic cause underlying supercoil generation by RNAP in transcription.BN/BionanoscienceBN/Cees Dekker La

Condensin-driven loop extrusion on supercoiled DNA

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, for example, the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping are stimulated by positively supercoiled DNA, and condensin preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.Accepted Author ManuscriptBN/Cees Dekker La

CRISPR-dCas9 based DNA detection scheme for diagnostics in resource-limited settings

Nucleic-acid detection is crucial for basic research as well as for applications in medicine such as diagnostics. In resource-limited settings, however, most DNA-detection diagnostic schemes are inapplicable since they rely on expensive machinery, electricity, and trained personnel. Here, we present an isothermal DNA detection scheme for the diagnosis of pathogenic DNA in resource-limited settings. DNA was extracted from urine and blood samples using two different instrument-free methods, and amplified using Recombinase Polymerase Amplification with a sensitivity of <10 copies of DNA within 15 minutes. Target DNA was bound by dCas9/sgRNA that was labelled with a DNA oligomer to subsequently induce Rolling Circle Amplification. This second amplification step produced many copies of a G-quadruplex DNA structure that facilitates a colorimetric readout that is visible to the naked eye. This isothermal DNA-detection scheme can be performed at temperatures between 20-45 °C. As an example of the applicability of the approach, we isothermally (23 °C) detected DNA from a parasite causing visceral leishmaniasis that was spiked into buffer and resulted in a sensitivity of at least 1 zeptomole. For proof of principle, DNA spiked into blood was coupled to the CRISPR-dCas9-based detection scheme yielding a colorimetric readout visible to the naked eye. Given the versatility of the guide-RNA programmability of targets, we envision that this DNA detection scheme can be adapted to detect any DNA with minimal means, which facilitates applications such as point-of-care diagnostics in resource-limited settings.BN/Cees Dekker LabScience Centre & Programmerin

Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores

Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.BN/Cees Dekker LabBN/Chirlmin Joo LabBN/Technici en Analiste

SMC complexes can traverse physical roadblocks bigger than their ring size

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.BN/Cees Dekker LabBN/BionanoscienceBN/Nynke Dekker La

Author Correction: Testing pseudotopological and nontopological models for SMC-driven DNA loop extrusion against roadblock-traversal experiments (Scientific Reports, (2023), 13, 1, (8100), 10.1038/s41598-023-35359-2)

Correction to: Scientific Reports, published online 19 May 2023 The original version of this Article contained an error in Figure 1b-1, where the fore- and background order of the strands “DNA” (in black) and “Brn1 Kleisin” (in green), were switched. The original Figure 1 and accompanying legend appear below. (Figure presented.) Description of the mechanism postulated by Shaltiel et al. for roadblock passage into an extruded loop on the DNA and a potential nontopological model. (a) The steps through the proposed DNA loop extrusion cycle are commented in more detail in steps 1–6 within the figure. Adapted from Ref.11. (b) Potential nontopological model which is closely analogous to the pseudotopological model, but with a slight variation in the DNA-SMC topology which allows particle bypass. The original Article has been corrected.author correction DOI 10.1038/s41598-023-35359-2BN/Cees Dekker La

Extracting and characterizing protein-free megabase-pair DNA for in vitro experiments

Chromosome structure and function is studied using various cell-based methods as well as with a range of in vitro single-molecule techniques on short DNA substrates. Here, we present a method to obtain megabase-pair-length deproteinated DNA for in vitro studies. We isolated chromosomes from bacterial cells and enzymatically digested the native proteins. Mass spectrometry indicated that 97%–100% of DNA-binding proteins are removed from the sample. Fluorescence microscopy analysis showed an increase in the radius of gyration of the DNA polymers, while the DNA length remained megabase-pair sized. In proof-of-concept experiments using these deproteinated long DNA molecules, we observed DNA compaction upon adding the DNA-binding protein Fis or PEG crowding agents and showed that it is possible to track the motion of a fluorescently labeled DNA locus. These results indicate the practical feasibility of a “genome-in-a-box” approach to study chromosome organization from the bottom up.BN/Cees Dekker LabDynamics of Micro and Nano SystemsBT/Environmental Biotechnolog