Highlights


Live high-resolution imaging of nanoparticle-cell interactions

Using our newly installed high-resolution STED microscope (Abberior Instruments), the interaction dynamic between nanoparticles and the biological world can now be studied with great precision. On this 50 megapixel composite image with spatial resolution of 30 nm, TiO2 nanotubes (seen in red) can clearly be seen stealing lipids from the plasma membrane of lung epithelial cells (labelled green). Yellow and orange-coloured areas in the image offer a direct proof of the existence of lipid wraps around TiO2 nanoparticles.

See more in the art gallery of the 2017 Liquids conference


Correlation-corrected conformational entropy

Fast intramolecular motion greatly affects the structure and function of macromolecules and supramolecular assemblies, which can be described by conformational entropy. We have developed a method to measure motional correlations from temperature-dependent entropy data to obtain thermodynamically relevant corrected total entropy. The approach was demonstrated by EPR data of various spin probes in model lipid membranes and validated against basic biophysical phenomena and interactions: membrane formation and stability (hydrophobic and van der Waals), phase transitions (diffusion) and fatty acid dissociation (electrostatics).

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Importance of molecular mobility of biopolymers on cell growth – exploring biocompatibility

Understanding biocompatibility of hydrogels and scaffolds is one of the main challenges in the field of tissue engineering in regenerative medicine. The complexity of cell-biomaterial interaction which results from the interplay between various properties of materials and their effects on triggering specific cell responses still raises many questions. In our study more comprehensive approach was applied, where different scale biomaterial properties were correlated with cell growth. Molecular mobility of polymers investigated by site-directed spin labeling and EPR spectroscopy was found to be the most decisive which suggests that it could play an important role in cell response to medical devices, reflecting a new aspect of the biocompatibility concept.

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Bleaching-corrected FMS with nm λMAX resolution

Fluorescence microspectroscopy (FMS) localizes spectroscopic information with microscopic spatial resolution. To achieve nanometer peak position (λMAX) resolution, characteristic for spectroscopic measurements of bulk samples, also at microscopic objects, we introduced spectral fitting, which greatly extends the range of FMS-applicable environment-sensitive probes. By stochastic wavelength sampling and careful data analysis we overcame the spectral distortions due to photosensitivity of the dyes. Moreover, the spatially dependent bleaching rates, provided during spectral correction, can be used to monitor characteristics of molecular environment.

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Probe conformation coexistence, revealed by pFMS

We used bleaching-corrected fluorescence microspectroscopy (FMS) with nanometer peak-position resolution to investigate the behavior of two commonly used phospholipid-based NBD fluorescent probes in various model membranes. The combination of polarized and spectral detection (polarized FMS - pFMS) unambiguously revealed a coexistence of molecular conformations at distances below microscopic spatial resolution, which was further corroborated by a numerical model. Since the conformations were the most affected by high concentrations of cholesterol, the approach could be exploited to study lipid rafts and biomembrane nanodomains.

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Nanoparticle tracking by FMS

An experimental setup for fluorescence microspectroscopy (FMS) was developed which enables acquisition of fluorescence emission spectra in each voxel of confocal fluorescence images. Spectral contrasting of the images allows (i) (co)localization of fluorescent probes with considerably overlapping emission spectra, (ii) identification and tracking of fluorescent nanoparticles that are smaller than the optical diffraction limit, and (iii) characterization of very small differences in local environment that can be detected by environment sensitive probes. A shift in spectral maximum of down to 1 nm can be resolved with our FMS system, as was demonstrated in the case of interaction of cancer cells with cancerostatic liposomes.

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Synthesis of double fluorescent-spin probes

New, dual fluorescent spin-probes and their fluorescent amine analogues, as potential spin traps, have been synthesized and characterized. Dual probes allow real-time localization of the spin probes in the investigated system, revealing the origin of acquired EPR signal by fluorescence microscopy. Moreover, we use environment-sensitive dyes, such as NBD or coumarin, that can provide additional information about their molecular surroundings, using fluorescence microspectroscopy. We believe that such double probes will be particularly useful for studies of plasma membrane heterogeneity and associated cellular processes.

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Conformational Analysis of NTAIL-XD Complex

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (NTAIL) both free and in complex with the X domain (XD, aa 459–507) of the viral phosphoprotein.

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Molecular Dynamics GPU Computing

We upgraded our cluster with 2 GTX 560 TI GPUs per board and managed to speed up our MD simulations 6-times.


fXa forms inactive dimers on membranes

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Proteolytic activity experiments of human FXa activity toward human Pre2 as a substrate suggested the existence of a membrane-bound inactive form of FXa. The fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.

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Cholesterol influence on liposome-cell interaction

Interaction of alkyphospholipid liposomes with human MT-3 breast-cancer cells depends critically on cholesterol (CH) concentration of the liposomes. Faster release of hydrophilic material from liposomes with low CH content coincides with the presence of domains with highly disordered alkyl chain motion, which disappear at around 50 mol% of CH. Micelles are not the only reason for cytotoxic effect of liposome formulations. Liposomes, containing around 50 mol% CH or less, also contribute to the cytotoxic effect, due to their fast interaction with cells

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