Detecting nanoscale distribution of protein pairs by proximity dependent super-resolution microscopy

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively

Membrane Scaffolds Enhance the Responsiveness and Stability of DNA-Based Sensing Circuits.

Target-induced DNA strand displacement is an excellent candidate for developing analyte-responsive DNA circuitry to be used in clinical diagnostics and synthetic biology. While most available technologies rely on DNA circuitry free to diffuse in bulk, here we explore the use of liposomes as scaffolds for DNA-based sensing nanodevices. Our proof-of-concept sensing circuit responds to the presence of a model target analyte by releasing a DNA strand, which in turn activates a fluorescent reporter. Through a combination of experiments and coarse-grained Monte Carlo simulations, we demonstrate that the presence of the membrane scaffold accelerates the process of oligonucleotide release and suppresses undesired leakage reactions, making the sensor both more responsive and robust

Flexibility defines structure in crystals of amphiphilic DNA nanostars.

DNA nanostructures with programmable shape and interactions can be used as building blocks for the self-assembly of crystalline materials with prescribed nanoscale features, holding a vast technological potential. Structural rigidity and bond directionality have been recognised as key design features for DNA motifs to sustain long-range order in 3D, but the practical challenges associated with prescribing building-block geometry with sufficient accuracy have limited the variety of available designs. We have recently introduced a novel platform for the one-pot preparation of crystalline DNA frameworks supported by a combination of Watson-Crick base pairing and hydrophobic forces (Brady et al 2017 Nano Lett. 17 3276-81). Here we use small angle x-ray scattering and coarse-grained molecular simulations to demonstrate that, as opposed to available all-DNA approaches, amphiphilic motifs do not rely on structural rigidity to support long-range order. Instead, the flexibility of amphiphilic DNA building-blocks is a crucial feature for successful crystallisation

New graduate doctors' preparedness for practice: A multistakeholder, multicentre narrative study

This is the final version. Available on open access from BMJ Publishing Group via the link in this recordData sharing statement The raw data for this research consist of audio-recordings of narrative interviews and audio diaries. The principal investigator (Professor Lynn V Monrouxe) has access to this specific data set, including audio-recordings of interviews and interview transcripts, in addition to participant contact details and signed consent forms. All authors have access to anonymised data from this set. All data are stored securely on password-protected and encrypted computers. Participants have not given their permission for data sharing outside the research group. Thus, no additional data are available.Objective While previous studies have begun to explore newly graduated junior doctors' preparedness for practice, findings are largely based on simplistic survey data or perceptions of newly graduated junior doctors and their clinical supervisors alone. This study explores, in a deeper manner, multiple stakeholders' conceptualisations of what it means to be prepared for practice and their perceptions about newly graduated junior doctors' preparedness (or unpreparedness) using innovative qualitative methods. Design A multistakeholder, multicentre qualitative study including narrative interviews and longitudinal audio diaries. Setting Four UK settings: England, Northern Ireland, Scotland and Wales. Participants Eight stakeholder groups comprising n=185 participants engaged in 101 narrative interviews (27 group and 84 individual). Twenty-six junior doctors in their first year postgraduation also provided audio diaries over a 3-month period. Results We identified 2186 narratives across all participants (506 classified as 'prepared', 663 as 'unprepared', 951 as 'general'). Seven themes were identified; this paper focuses on two themes pertinent to our research questions: (1) explicit conceptualisations of preparedness for practice; and (2) newly graduated junior doctors' preparedness for the General Medical Council's (GMC) outcomes for graduates. Stakeholders' conceptualisations of preparedness for practice included short-term (hitting the ground running) and long-term preparedness, alongside being prepared for practical and emotional aspects. Stakeholders' perceptions of medical graduates' preparedness for practice varied across different GMC outcomes for graduates (eg, Doctor as Scholar and Scientist, as Practitioner, as Professional) and across stakeholders (eg, newly graduated doctors sometimes perceived themselves as prepared but others did not). Conclusion Our narrative findings highlight the complexities and nuances surrounding new medical graduates' preparedness for practice. We encourage stakeholders to develop a shared understanding (and realistic expectations) of new medical graduates' preparedness. We invite medical school leaders to increase the proportion of time that medical students spend participating meaningfully in multiprofessional teams during workplace learning.General Medical Counci

Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment.

Biology has evolved a variety of agents capable of permeabilizing and disrupting lipid membranes, from amyloid aggregates, to antimicrobial peptides, to venom compounds. While often associated with disease or toxicity, these agents are also central to many biosensing and therapeutic technologies. Here, we introduce a class of synthetic, DNA-based particles capable of disrupting lipid membranes. The particles have finely programmable size, and self-assemble from all-DNA and cholesterol-DNA nanostructures, the latter forming a membrane-adhesive core and the former a protective hydrophilic corona. We show that the corona can be selectively displaced with a molecular cue, exposing the 'sticky' core. Unprotected particles adhere to synthetic lipid vesicles, which in turn enhances membrane permeability and leads to vesicle collapse. Furthermore, particle-particle coalescence leads to the formation of gel-like DNA aggregates that envelop surviving vesicles. This response is reminiscent of pathogen immobilisation through immune cells secretion of DNA networks, as we demonstrate by trapping E. coli bacteria

Stationary solutions for the parity-even sector of the CPT-even and Lorentz-covariance-violating term of the standard model extension

In this work, we focus on some properties of the parity-even sector of the CPT-even electrodynamics of the standard model extension. We analyze how the six non-birefringent terms belonging to this sector modify the static and stationary classical solutions of the usual Maxwell theory. We observe that the parity-even terms do not couple the electric and magnetic sectors (at least in the stationary regime). The Green's method is used to obtain solutions for the field strengths E and B at first order in the Lorentz- covariance-violating parameters. Explicit solutions are attained for point-like and spatially extended sources, for which a dipolar expansion is achieved. Finally, it is presented an Earth-based experiment that can lead (in principle) to an upper bound on the anisotropic coefficients as stringent as $(\widetilde{\kappa}_{e-}) ^{ij}<2.9\times10^{-20}.$Comment: 8 pages, revtex style, revised published version, to appear in EPJC (2009

Consistency analysis of a nonbirefringent Lorentz-violating planar model

In this work analyze the physical consistency of a nonbirefringent Lorentz-violating planar model via the analysis of the pole structure of its Feynman propagators. The nonbirefringent planar model, obtained from the dimensional reduction of the CPT-even gauge sector of the standard model extension, is composed of a gauge and a scalar fields, being affected by Lorentz-violating (LIV) coefficients encoded in the symmetric tensor $\kappa_{\mu\nu}$. The propagator of the gauge field is explicitly evaluated and expressed in terms of linear independent symmetric tensors, presenting only one physical mode. The same holds for the scalar propagator. A consistency analysis is performed based on the poles of the propagators. The isotropic parity-even sector is stable, causal and unitary mode for $0\leq\kappa_{00}<1$. On the other hand, the anisotropic sector is stable and unitary but in general noncausal. Finally, it is shown that this planar model interacting with a $\lambda|\varphi|^{4}-$Higgs field supports compactlike vortex configurations.Comment: 11 pages, revtex style, final revised versio