Viral Chip Technology in Genomic Medicine

Földes-Papp, Zeno

doi:10.1016/b978-0-12-369420-1.00048-2

Cited by 3 publications

(5 citation statements)

References 180 publications

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“…(4B-C) shows that we could clearly distinguish between the dark (non-fluorescent) unbound probes in the extracellular solution and the bright (fluorescent) bound miRNA-122 target probes within the single Huh-7D12 cells. This high quenching efficiency is a consequence of improved absorption extinction coefficient of the quencher BHQ and increased dipole-dipole interaction between the fluorophore and quencher molecule in the closed unbound conformation of the molecular beacon probe [15,31]. Because we also optically knocked out the nuclei membranes, we observed a fluorescence steady-state across the whole cells within 5 min after optoporations of cell and nuclei membranes (Fig.…”

Section: Resultsmentioning

confidence: 90%

“…The sensitivity of a molecular beacon probe depends on the brightness of the fluorescent donor, the efficiency of the fluorescence resonance energy transfer (FRET) to the quencher (acceptor), and properties of the quencher, i.e. the quencher was a dark molecule that is not fluorescent under the experiment conditions used [15,31]. Because the efficiency of extracellular probe delivery to a single live cell is still a limiting factor for basic single-cell research, we circumvented the use of electroshocks in bulk suspensions, the so-called electroporation, or chemical means for perforation of cell membranes in bulk suspensions.…”

Section: Resultsmentioning

confidence: 99%

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Trafficking of Mature miRNA-122 into the Nucleus of Live Liver Cells

Földes-Papp¹,

König²,

Studier³

et al. 2009

CPB

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The binding of superquencher molecular beacon (SQMB) probes to human single-stranded cellular miRNA-122 targets was detected in various single live cells with femtosecond laser microscopy. For delivery of the SQMB-probes, 3D-nanoprocessing of single cells with sub-15 femtosecond 85 MHz near-infrared laser pulses was applied. Transient nanopores were formed by focusing the laser beam for some milliseconds on the membrane of a single cell in order to import of SQMB-probes into the cells. In single cells of the human liver cell lines Huh-7D12 and IHH that expressed miRNA-122, we measured target binding in the cytoplasm by two-photon fluorescence imaging. We found increased fluorescence with time in a nonlinear manner up to the point where steady state saturation was reached. We also studied the intracellular distribution of target SQMB and provide for the first time strong experimental evidence that cytoplasmic miRNA travels into the cell nucleus. To interpret nonlinear binding, a number of individual miRNA-122 positive cells (Huh-7D12 and IHH) and negative control cells, human VA13 fibroblasts and Caco-2 cells were analyzed. Our experimental data are consistent with the cytoplasmic assembly of nuclear miRNA and provide further mechanistic insight in the regulatory function of miRNAs in cellular physiology. An open issue in the regulation of gene expression by miRNA is whether miRNA can activate gene expression in addition to the well-known inhibitory effect. A first step for such a regulatory role could be the travelling of miRNA-RISC into the nucleus.

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Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Trafficking of Mature miRNA-122 into the Nucleus of Live Liver Cells

Földes-Papp¹,

König²,

Studier³

et al. 2009

CPB

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“…48.2 in ref. [ 39 ]: Synopsis of a new physically grounded technology of fluorescence fluctuation spectroscopy for observing single molecules at longer time scales than currently available). The corresponding discussion of Baumann and Földes-Papp [ 17 ] also applies here.…”

Section: Resultsmentioning

confidence: 99%

Fluorescence Molecule Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity

Földes-Papp¹,

Baumann

2011

CPB

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We present a new approach to distinguish between non-ergodic and ergodic behavior. Performing ensemble averaging in a subpopulation of individual molecules leads to a mean value that can be similar to the mean value obtained in an ergodic system. The averaging is carried out by minimizing the variation between the sum of the temporal averaged mean square deviation of the simulated data with respect to the logarithmic scaling behavior of the subpopulation. For this reason, we first introduce a kind of Continuous Time Random Walks (CTRW), which we call Limited Continuous Time Random Walks (LCTRW) on fractal support. The random waiting time distributions are sampled at points which fulfill the condition N < 1, where N is the Poisson probability of finding a single molecule in the femtoliter-sized observation volume ΔV at the single-molecule level. Given a subpopulation of different single molecules of the same kind, the ratio T/ Tm between the measurement time T and the meaningful time Tm, which is the time for observing just one and the same single molecule, is the experimentally accessible quantity that allows to compare different molecule numbers in the subpopulation. In addition, the mean square displacement traveled by the molecule during the time t is determined by an upper limit of the geometric dimension of the living cell or its nucleus.

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“…The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or dual color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The concept of fluctuations due to the same molecule was theoretically and experimentally developed in recent years [55,57,8,9,18,35,36,27,12,13,62,63,1,2]. Under conditions in which only one molecule is present in the observation volume being sampled, we have shown that it is possible to measure correlations due to the re-entry of the same molecule in the volume of illumination.…”

Section: Resultsmentioning

confidence: 95%

“…Under conditions in which only one molecule is present in the observation volume being sampled, we have shown that it is possible to measure correlations due to the re-entry of the same molecule in the volume of illumination. The meaningful time [57,55,35,36,9] is the physical parameter that enables to learn some characteristic signatures of the same single molecule in solution and live cells. …”

Section: Resultsmentioning

confidence: 99%

Anomalous Subdiffusive Measurements by Fluorescence Correlations Spectroscopy and Simulations of Translational Diffusive Behavior in Live Cells

2014

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Using a combination of optical experiments and computer simulations, we found that live cells act as traps to produce anomalous subdiffusive translational motion of molecules exemplified by glucocorticoid receptor α. Glucocorticoid receptor α was expressed as a fusion protein with the green fluorescent protein in living mammalian U2OS cells. The measurements were carried out by fluorescence correlation spectroscopy. The GFP-tagged glucocorticoid receptor α was either a homodimer or a monomer in the nucleoplasm depending on the presence or absence of the stimulus dexamethasone. Our simulations showed that the experimentally measured rates of translational diffusion could be attributed to spatial and temporal traps. Thus, traps acted by spatial and temporal heterogeneity and randomness, respectively. As we prove here for the first time, that anomalous translational diffusion caused by spatial randomness was different from anomalous translational diffusion caused by heterogeneous temporal randomness. For this purpose, we explored two classes of transformations in the time domain for which the assumption that they are stable and have probability density functions was satisfied. The first one was the Inverse Gamma distribution and the second class was a stable Levy distribution. The spatial exponent α that was extracted from time series expressed scale invariance in the space domain. The time exponent γ measured limited time scaling of the embedding complex dynamics. Hence, both exponents must not be mixed up by a single exponent. Fluorescence correlation spectroscopy is the method of choice for measuring the exponents of anomalous subdiffusive translational motion of molecules in live cells.

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Viral Chip Technology in Genomic Medicine

Cited by 3 publications

References 180 publications

Trafficking of Mature miRNA-122 into the Nucleus of Live Liver Cells

Trafficking of Mature miRNA-122 into the Nucleus of Live Liver Cells

Fluorescence Molecule Counting for Single-Molecule Studies in Crowded Environment of Living Cells without and with Broken Ergodicity

Anomalous Subdiffusive Measurements by Fluorescence Correlations Spectroscopy and Simulations of Translational Diffusive Behavior in Live Cells

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