Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained ...challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.
The kinematics and stellar populations of the low-mass nearby S0 galaxy NGC 5102 are studied from integral field spectra taken with the Multi-Unit Spectroscopic Explorer. The kinematic maps reveal ...for the first time that NGC 5102 has the characteristic 2 sigma peaks indicative of galaxies with counter-rotating discs. This interpretation is quantitatively confirmed by fitting two kinematic components to the observed spectra. Through stellar population analysis, we confirm the known young stellar population in the centre and find steep age and metallicity gradients. We construct axisymmetric Jeans anisotropic models of the stellar dynamics to investigate the initial mass function (IMF) and the dark matter halo of the galaxy. The models show that this galaxy is quite different from all galaxies previously studied with a similar approach: even within the half-light radius, it cannot be approximated with the self-consistent mass-follows-light assumption. Including a Navarro, Frenk & White dark matter halo, we need a heavy IMF and a dark matter fraction of 0.37 plus or minus 0.04 within a sphere of one Re radius to describe the stellar kinematics. The more general model with a free slope of the dark matter halo shows that slope and IMF are degenerate, but indicates that a light weight IMF (Chabrier-like) and a higher dark matter fraction, with a steeper (contracted) halo, fit the data better. Regardless of the assumptions about the halo profile, we measure the slope of the total mass density to be -1.75 plus or minus 0.04. This is shallower than the slope of -2 of an isothermal halo and shallower than published slopes for more massive early-type galaxies.
Replacing lead in halide perovskites is of great interest due to concerns about stability and toxicity. Recently, lead free double perovskites in which the unit cell is doubled and two divalent lead ...cations are substituted by a combination of mono- and trivalent cations have been synthesized as bulk single crystals and as thin films. Here, we study stability and optical properties of all-inorganic cesium silver(I) bismuth(III) chloride and bromide nanocrystals with the double perovskite crystal structure. The cube-shaped nanocrystals are monodisperse in size with typical side lengths of 8 to 15 nm. The absorption spectrum of the nanocrystals presents a sharp peak, which we assign to a direct bismuth s–p transition and not to a quantum confined excitonic transition. Using this spectroscopic handle combined with high-resolution transmission electron microscopy (TEM) based elemental analysis, we conduct stoichiometric studies at the single nanocrystal level as well as decomposition assays in solution and observe that Ag+ diffusion and coalescence is one of the pathways by which this material degrades. Drying the nanocrystals leads to self-assembly into ordered nanocrystal solids, and these exhibit less degradation than nanocrystals in solution. Our results demonstrate that Cs2AgBiX6 (X = Cl, Br) nanocrystals are a useful model system to study structure–function relationships in the search for stable nontoxic halide perovskites.
Lead toxicity has sparked interest into alternative halide nanomaterials with properties similar to CsPbX3 perovskites. A promising alternative suggested from bulk studies is the family of double ...perovskites of the form Cs2AgMX6. Here, we report the synthesis of colloidal Cs2AgInCl6 and Cs2AgSbCl6 nanocrystals via injection of acyl halides into a metal acetate solution under atmospheric conditions and relatively mild temperatures. We demonstrate the synthesis of single-crystalline cubic nanocrystals of ca. 10 nm side length and their morphological similarities to other double perovskite nanostructures in terms of their 200 facet termination and decoration with Ag(0) smaller nanocrystallites. To compare the stabilities of the synthesized materials, we develop a titration assay based on the degradation of nanocrystals with amines as a proxy for degradation by humidity, which provides a quantifiable stability metric. This measurement shows that Cs2AgSbCl6 releases more than twice the decomposition energy compared to Cs2AgInCl6 or CsPbCl3 and degrades in the presence of approximately one molar equivalent of amine, whereas the other two materials require more than a 100-fold excess. Using facile chemical titration to quantitatively determine chemical stability provides an additional tool to aid in the basic understanding of what makes some of these materials more environmentally stable than others.
Fluorescent labeling of specific cell-surface proteins enables a manifold of techniques to study their function in health and disease. A frequently cited family of methods employs phosphopantetheinyl ...transferases (PPTases) to attach probes, provided as conjugates of Coenzyme A. This method appears attractive, as only short peptide tags genetically fused to the protein of interest are needed as conjugation sites. Here, we describe observations we made when evaluating such protocols for delicate single-molecule applications where we require a particular combination of dyes, low background binding or low labeling of other proteins, and a high degree of labeling.
When we tested a PPTase-acceptor peptide couple with several experimental protocols and various CoA conjugates for labeling of a protein on the cell surface, we noticed substantial non-specific labeling. For the first time, we provide here a quantification of the non-specific fraction of the signals obtained using appropriate controls. We further present evidence that this background is due to CoA-dye conjugates entering the cell, where they may be covalently attached to endogenous proteins. However, when studying cell-surface proteins, most fluorescent readouts require that labeling is strictly limited to the protein of interest located at the cell surface. While such data have so far been missing in the literature, they suggest that for applications where labeling of unwanted molecules would affect the conclusions, researchers need to be aware of this potential non-specificity of PPTase methods when selecting a labeling strategy. We show, again by quantitative comparison, that the HaloTag is a viable alternative.
Cell surface proteins are key regulators of fundamental cellular processes and, therefore, often at the root of human diseases. Thus, a large number of targeted drugs which are approved or under ...development act upon cell surface proteins. Although down-regulation of surface proteins by many natural ligands is well-established, the ability of drug candidates to cause internalization or degradation of the target is only recently moving into focus. This property is important both for the pharmacokinetics and pharmacodynamics of the drug but may also constitute a potential resistance mechanism. The enormous numbers of drug candidates targeting cell surface molecules, comprising small molecules, antibodies, or alternative protein scaffolds, necessitate methods for the investigation of internalization and degradation in high throughput. Here, we present a generic high-throughput assay protocol, which allows the simultaneous and independent quantification of internalization and degradation of surface proteins on a single-cell level. Because we fuse a HaloTag to the cell surface protein of interest and exploit the differential cell permeability of two fluorescent HaloTag ligands, no labeling of the molecules to be screened is required. In contrast to previously described approaches, our homogeneous assay is performed with adherent live cells in a 96-well format. Through channel rescaling, we are furthermore able to obtain true relative abundances of surface and internal protein. We demonstrate the applicability of our procedure to three major drug targets, EGFR, HER2, and EpCAM, examining a selection of well-investigated but also novel small molecule ligands and protein affinity reagents.
Optical applications of lanthanide‐doped nanoparticles require materials with low phonon energies to minimize nonradiative relaxation and promote nonlinear processes like upconversion. Heavy halide ...hosts offer low phonon energies but are challenging to synthesize as nanocrystals. Here, we demonstrate the size‐controlled synthesis of low‐phonon‐energy KPb2X5 (X=Cl, Br) nanoparticles and the ability to tune nanocrystal phonon energies as low as 128 cm−1. KPb2Cl5 nanoparticles are moisture resistant and can be efficiently doped with lighter lanthanides. The low phonon energies of KPb2X5 nanoparticles promote upconversion luminescence from higher lanthanide excited states and enable highly nonlinear, avalanche‐like emission from KPb2Cl5 : Nd3+ nanoparticles. The realization of nanoparticles with tunable, ultra‐low phonon energies facilitates the discovery of nanomaterials with phonon‐dependent properties, precisely engineered for applications in nanoscale imaging, sensing, luminescence thermometry and energy conversion.
We develop phonon‐engineered, lanthanide‐doped upconverting nanoparticles with the lowest phonon energies available to date. Low‐phonon‐energy KPb2X5 (X=Cl, Br) upconverting nanoparticles exhibit dramatically suppressed multiphonon relaxation, enhancing upconversion emission from higher lanthanide excited states and enabling the first room‐temperature observation of avalanche‐like upconversion by Nd3+ ions.
Vision plays a central role in maintaining balance. When humans perceive their body as moving, they trigger counter movements. This results in body sway, which has typically been investigated by ...measuring the body’s center of pressure (COP). Here, we aimed to induce visually evoked postural responses (VEPR) by simulating self-motion in virtual reality (VR) using a sinusoidally oscillating “moving room” paradigm. Ten healthy subjects participated in the experiment. Stimulation consisted of a 3D-cloud of random dots, presented through a VR headset, which oscillated sinusoidally in the anterior–posterior direction at different frequencies. We used a force platform to measure subjects’ COP over time and quantified the resulting trajectory by wavelet analyses including inter-trial phase coherence (ITPC). Subjects exhibited significant coupling of their COP to the respective stimulus. Even when spectral analysis of postural sway showed only small responses in the expected frequency bands (power), ITPC revealed an almost constant strength of coupling to the stimulus within but also across subjects and presented frequencies. Remarkably, ITPC even revealed a strong phase coupling to stimulation at 1.5 Hz, which exceeds the frequency range that has generally been attributed to the coupling of human postural sway to an oscillatory visual scenery. These findings suggest phase-locking to be an essential feature of visuomotor control.
A great deal of emphasis, clinical and financial, is placed on limb salvage efforts in diabetic patients suffering from lower extremity ulceration. This is because of the impression that amputation ...in such patients may be a proximal cause of death. While amputation is certainly a negative clinical outcome, it is not entirely clear that it causes death.
In this systematic review, we examine the available literature to attempt to understand the role that the ulceration itself may play in mortality. In brief, we searched for human studies in OVID, CINAHL and the COCHRANE CENTRAL DATABASE from 1980 to 2013, looking for articles related to ulcer or wound of the foot, in patients with diabetes or peripheral vascular disease, and death. We looked for articles with 5 years of follow‐up, or Kaplan–Meier estimates of 5‐year mortality, and excluded reviews and letters. Articles were assessed for quality and potential bias using the Newcastle–Ottawa scale.
We find that while the patient populations studied varied widely in terms of demographics and comorbidities, limiting generalisability, 5‐year mortality rates after ulceration were around 40%. Risk factors for death commonly identified were increased age, male gender, peripheral vascular disease and renal disease.
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