11 research outputs found
Calibration of optical tweezers with positional detection in the back-focal-plane
We explain and demonstrate a new method of force- and position-calibration
for optical tweezers with back-focal-plane photo detection. The method combines
power spectral measurements of thermal motion and the response to a sinusoidal
motion of a translation stage. It consequently does not use the drag
coefficient of the trapped ob ject as an input. Thus, neither the viscosity,
nor the size of the trapped ob ject, nor its distance to nearby surfaces need
to be known. The method requires only a low level of instrumentation and can be
applied in situ in all spatial dimensions. It is both accurate and precise:
true values are returned, with small error-bars. We tested this experimentally,
near and far from surfaces. Both position- and force-calibration were accurate
to within 3%. To calibrate, we moved the sample with a piezo-electric
translation stage, but the laser beam could be moved instead, e.g. by
acousto-optic deflectors. Near surfaces, this precision requires an improved
formula for the hydrodynamical interaction between an infinite plane and a
micro-sphere in non-constant motion parallel to it. We give such a formula.Comment: Submitted to: Review of Scientific Instruments. 13 pages, 5 figures.
Appendix added (hydrodynamically correct calibration
Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome
Background: The low-density lipoprotein receptor (LDLR) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease.Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of datasets on gene expression and variants in human populations.Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in non-transfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in non-alcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and three rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared to overexpression of wild type RBM25, overexpression of the three rare RBM25 mutants in Huh-7 cells led to lower LDL uptake.Conclusions: We identified a novel mechanism of post-transcriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.</p
Harmonic oscillator in heat bath: Exact simulation of time-lapse-recorded data and exact analytical benchmark statistics
The stochastic dynamics of the damped harmonic oscillator in a heat bath is
simulated with an algorithm that is exact for time steps of arbitrary size.
Exact analytical results are given for correlation functions and power spectra
in the form they acquire when computed from experimental time-lapse recordings.
Three applications are discussed: (i) Effects of finite sampling-rate and
-time, described exactly here, are similar for other stochastic dynamical
systems-e.g. motile micro-organisms and their time-lapse recorded trajectories.
(ii) The same statistics is satisfied by any experimental system to the extent
it is interpreted as a damped harmonic oscillator at finite temperature-such as
an AFM cantilever. (iii) Three other models of fundamental interest are
limiting cases of the damped harmonic oscillator at finite temperature; it
consequently bridges their differences and describes effects of finite sampling
rate and sampling time for these models as well. Finally, we give a brief
discussion of nondimensionalization.Comment: 12 pages, 8 figure
Cell motility as random motion: A review
The historical co-evolution of biological motility models with models of Brownian motion is outlined.
Recent results for how to derive cell-type-specific motility models from experimental cell trajectories are reviewed.
Experimental work in progress, which tests the generality of this phenomenological model building is reported.
So is theoretical work in progress, which explains the characteristic time scales and correlations
of phenomenological models in terms of the dynamics of cytoskeleton, lamellipodia, and pseudopodia
Detecting variation in starch granule size and morphology by high-throughput microscopy and flow cytometry
Starch forms semi-crystalline, water-insoluble granules, the size and morphology of which vary according to biological origin. These traits, together with polymer composition and structure, determine the physicochemical properties of starch. However, screening methods to identify differences in starch granule size and shape are lacking. Here, we present two approaches for high-throughput starch granule extraction and size determination using flow cytometry and automated, high-throughput light microscopy. We evaluated the practicality of both methods using starch from different species and tissues and demonstrated their effectiveness by screening for induced variation in starch extracted from over 10,000 barley lines, yielding four with heritable changes in the ratio of large A-granules to small B-granules. Analysis of Arabidopsis lines altered in starch biosynthesis further demonstrates the applicability of these approaches. Identifying variation in starch granule size and shape will enable identification of trait-controlling genes for developing crops with desired properties, and could help optimise starch processing.ISSN:0144-8617ISSN:1879-134
VEGF-A regulates cellular localization of SR-BI as well as transendothelial transport of HDL but not LDL
OBJECTIVE: Low- and high-density lipoproteins (LDL and HDL) must pass the endothelial layer to exert pro- and antiatherogenic activities, respectively, within the vascular wall. However, the rate-limiting factors that mediate transendothelial transport of lipoproteins are yet little known. Therefore, we performed a high-throughput screen with kinase drug inhibitors to identify modulators of transendothelial LDL and HDL transport.
APPROACH AND RESULTS: Microscopy-based high-content screening was performed by incubating human aortic endothelial cells with 141 kinase-inhibiting drugs and fluorescent-labeled LDL or HDL. Inhibitors of vascular endothelial growth factor (VEGF) receptors (VEGFR) significantly decreased the uptake of HDL but not LDL. Silencing of VEGF receptor 2 significantly decreased cellular binding, association, and transendothelial transport of (125)I-HDL but not (125)I-LDL. RNA interference with VEGF receptor 1 or VEGF receptor 3 had no effect. Binding, uptake, and transport of HDL but not LDL were strongly reduced in the absence of VEGF-A from the cell culture medium and were restored by the addition of VEGF-A. The restoring effect of VEGF-A on endothelial binding, uptake, and transport of HDL was abrogated by pharmacological inhibition of phosphatidyl-inositol 3 kinase/protein kinase B or p38 mitogen-activated protein kinase, as well as silencing of scavenger receptor BI. Moreover, the presence of VEGF-A was found to be a prerequisite for the localization of scavenger receptor BI in the plasma membrane of endothelial cells.
CONCLUSIONS: The identification of VEGF as a regulatory factor of transendothelial transport of HDL but not LDL supports the concept that the endothelium is a specific and, hence, druggable barrier for the entry of lipoproteins into the vascular wall