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The prevalence and determinants of anti-DFS70 autoantibodies in an international inception cohort of systemic lupus erythematosus patients

Autoantibodies to dense fine speckles 70 (DFS70) are purported to rule out the diagnosis of SLE when they occur in the absence of other SLE-related autoantibodies. This study is the first to report the prevalence of anti-DFS70 in an early, multinational inception SLE cohort and examine demographic, clinical, and autoantibody associations. Patients were enrolled in the Systemic Lupus International

Robust Phase-Based Positioning Using Massive MIMO with Limited Bandwidth

This paper presents a robust phase-based positioningframework using a massive multiple-input multiple-output(MIMO) system. The phase-based distance estimates of MPCstogether with other parameters are tracked with an ExtendedKalman Filter (EKF), the state dimension of which varies withthe birth-death processes of paths. The iterative maximumlikelihoodestimation algorithm (RIMAX) and the modeling of

Polynomial Solvers for Saturated Ideals

In this paper we present a new method for creating polynomialsolvers for problems where a (possibly infinite) subsetof the solutions are undesirable or uninteresting. Thesesolutions typically arise from simplifications made duringmodeling, but can also come from degeneracies which areinherent to the geometry of the original problem.The proposed approach extends the standard action matrixmethod to

Initiation of T cell signaling by CD45 segregation at 'close contacts'

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying t

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and

Label-free measurements of the diffusivity of molecules in lipid membranes

An important and characteristic property of a cell membrane is the lateral mobility of protein molecules in the lipid bilayer. This has conventionally been measured by labeling the molecules with fluorescent markers and monitoring their mobility by different fluorescence-based techniques. However, adding the label to the studied molecule may affect the system, so it is an assumption in almost all

Local delivery of molecules from a nanopipette for quantitative receptor mapping on live cells

Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level of single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nano

Single-molecule measurements of transient biomolecular complexes through microfluidic dilution

Single-molecule confocal microscopy experiments require concentrations which are low enough to guarantee that, on average, less than one single molecule resides in the probe volume at any given time. Such concentrations are, however, significantly lower than the dissociation constants of many biological complexes which can therefore dissociate under single-molecule conditions. To address the chall

Time-resolved surface-enhanced ellipsometric contrast imaging for label-free analysis of biomolecular recognition reactions on glycolipid domains

We have applied surface-enhanced ellipsometry contrast (SEEC) imaging for time-resolved label-free visualization of biomolecular recognition events on spatially heterogeneous supported lipid bilayers (SLB). Using a conventional inverted microscope equipped with total internal reflection (TIR) illumination, biomolecular binding events were monitored with a lateral resolution near the optical diffra

Continuous lipid bilayers derived from cell membranes for spatial molecular manipulation

Progress with respect to enrichment and separation of native membrane components in complex lipid environments, such as native cell membranes, has so far been very limited. The reason for the slow progress can be related to the lack of efficient means to generate continuous and laterally fluid supported lipid bilayers (SLBs) made from real cell membranes. We show in this work how the edge of a hyd

Accumulation and separation of membrane-bound proteins using hydrodynamic forces

The separation of molecules residing in the cell membrane remains a largely unsolved problem in the fields of bioscience and biotechnology. We demonstrate how hydrodynamic forces can be used to both accumulate and separate membrane-bound proteins in their native state. A supported lipid bilayer (SLB) was formed inside a microfluidic channel with the two proteins streptavidin (SA) and cholera toxin

Effects of surface pressure and internal friction on the dynamics of shear-driven supported lipid bilayers

Supported lipid bilayers (SLBs) are one of the most common model systems for cell membrane studies. We have previously found that when applying a bulk flow of liquid above an SLB the lipid bilayer and its constituents move in the direction of the bulk flow in a rolling type of motion, with the lower monolayer being essentially stationary. In this study, a theoretical platform is developed to model

Sealing of submicrometer wells by a shear-driven lipid bilayer

A supported lipid bilayer (SLB) was formed in a microfluidic channel by vesicle fusion. The SLB, formed on a flat part of the surface, was driven by the shear forces of a bulk flow above the SLB to a part of the surface with embedded submicrometer wells. When using a bulk solution with a pH of 9.5 the advancing lipid bilayer sealed the wells, creating free-spanning membranes, whereas at a pH of 8.

Improving the limit of detection of nanoscale sensors by directed binding to high-sensitivity areas

The revelation of protein-protein interactions is one of the main preoccupations in the field of proteomics. Nanoplasmonics has emerged as an attractive surface-based technique because of its ability to sense protein binding under physiological conditions in a label-free manner. Here, we use short-range ordered holes with a diameter of approximately 150 nm and a depth of approximately 50 nm as a n

Site-specific DNA-controlled fusion of single lipid vesicles to supported lipid bilayers

We investigate the Ca(2+)-triggered fusion of lipid vesicles site-selectively tethered to a DNA-modified supported lipid bilayer array, with the DNA strands designed such that hybridization occurs in a zipperlike fashion. Prior to the addition of Ca(2+), which is observed to induce docking and subsequent fusion (within 200 ms), the vesicles display lateral mobility determined by the number of teth

Self-assembly formation of multiple DNA-tethered lipid bilayers

Inspired by natural cell-cell junctions, where membrane-residing proteins control the separation between two or more membranes without interfering with their integrity, we report a new self-assembly route for formation of multiple highly fluid tethered lipid bilayers with the inter-membrane volume geometrically confined by membrane-anchored DNA duplexes. The formation of multiple planar membrane-m

Kinetic and thermodynamic characterization of single-mismatch discrimination using single-molecule imaging

A single-molecule detection setup based on total internal reflection fluorescence (TIRF) microscopy has been used to investigate association and dissociation kinetics of unlabeled 30mer DNA strands. Single-molecule sensitivity was accomplished by letting unlabeled DNA target strands mediate the binding of DNA-modified and fluorescently labeled liposomes to a DNA-modified surface. The liposomes, ac

Hydrodynamic separation of proteins in supported lipid bilayers confined by gold barriers

Hydrodynamic drag forces generated by liquid flow above a supported lipid bilayer (SLB) can be used to induce lateral movement of molecules protruding from the SLB. Since the velocity of the individual molecules depends on their size and coupling to the lipid bilayer, these forces can also be used to enrich and separate different types of membrane-bound molecules. To improve and better quantify hy