Effective scheme of photolysis of GFP in live cell as revealed with confocal fluorescence microscopy.

We proposed an effective kinetics scheme of photolysis of green fluorescent protein (GFP) observed in live cells with a commercial confocal fluorescence microscope. We investigated the photolysis of GFP-tagged heterochromatin protein, HP1ß-GFP, in live nucleus with the pulse position modulation approach, which has several advantages over the classical pump-and-probe method. At the basis of the proposed scheme lies a process of photoswitching from the native fluorescence state to the intermediate fluorescence state, which has a lower fluorescence yield and recovers back to native state in the dark. This kinetics scheme includes four effective parameters (photoswitching, reverse switching, photodegradation rate constants, and relative brightness of the intermediate state) and covers the time scale from dozens of milliseconds to minutes of the experimental fluorescence kinetics. Additionally, the applicability of the scheme was demonstrated in the cases of continuous irradiation and the classical pump-and-probe approach using numerical calculations and analytical solutions. An interesting finding of experimental data analysis was that the overall photodegradation of GFP proceeds dominantly from the intermediate state, and demonstrated approximately the second-order reaction versus irradiation power. As a practical example, the proposed scheme elucidates the artifacts of fluorescence recovery after the photobleaching method, and allows us to propose some suggestions on how to diminish them.

Discovery of Novel Isatin-Based p53 Inducers.

A series of isatin Schiff base derivatives were identified during in silico screening of the small molecule library for novel activators of p53. The compounds selected based on molecular docking results were further validated by a high-content screening assay using U2OS human osteosarcoma cells with an integrated EGFP-expressing p53-dependent reporter. The hit compounds activated and stabilized p53, as shown by Western blotting, at higher rates than the well-known positive control Nutlin-3. Thus, the p53-activating compounds identified by this approach represent useful molecular probes for various cancer studies.

[The role of mitochondrial dynamics in cell death].

Mitochondria are dynamic organelles whose homeostasis is defined by two opposite processes: fission (or fragmentation), or fusion. Fission of mitochondria results in generation of smaller organelles and fusion is when they produce tubular or net-like structures. Although a number of proteins are already known to control the process of fission/fusion additional regulators controlling these processes are being found. The Bcl-2 family members take part in the regulation of apoptosis and according to the current view are involved in the mitochondrial net-like structure maintenance. In this review we will discuss mechanisms of mitochondrial fission/fusion regulation and summarize the available information on the role of Bcl-2 family members in the regulation of mitochondrial fission/fusion dynamics.

Dynamic quantification of antigen molecules with flow cytometry.

Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

Chlorin e6-ZnSe/ZnS quantum dots based system as reagent for photodynamic therapy.

Stable water-soluble complexes of Cd-free ZnSe/ZnS quantum dots (QDs) and chlorin e6 complexes have been prepared. These complexes have shown approximately 50% intracomplex fluorescence resonance energy transfer from QDs to chlorin e6. The photodynamic therapy (PDT) test of the complexes against the Erlich acsite carcinoma cell culture demonstrated a two-fold enhancement of the cancer cell photodynamic destruction as compared to that of free chlorin e6 molecules. It was shown that the PDT effect was significantly increased due to two factors: the efficient QD-chlorin e6 photoexcitation energy transfer and the improvement of cellular uptake of the photosensitizer in the presence of ZnSe/ZnS QDs.

Dynamic quantification of antigen molecules with flow cytometry.

Traditional methods for estimating the number of expressed molecules, based on the detection of target antigens bound with fluorescently labeled antibodies, assume that the antigen-antibody reaction reaches equilibrium. A calibration procedure is used to convert the intensity of the fluorescence signal to the number of target molecules. Along with the different limitations of every calibration system, this substantially limits the applicability of the traditional approaches especially in the case of low affinity antibodies. We address this problem here with studies in which we demonstrate a new approach to the antigen molecule quantification problem. Instead of using a static calibration system, we analyzed mean fluorescence values over time by flow cytometry during antibody-antigen binding. Experimental data obtained with an LSRII cytometer were fitted by a diffusion-reaction mathematical model using the Levenberg-Marquardt nonlinear least squares curve-fitting algorithm in order to obtain the number of target antigen molecules per cell. Results were compared with the Quanti-BRITE calibration system. We conclude that, instead of using experiment-specific calibration, the value of the binding rate constant for each particular antibody-antigen reaction can be used to quantify antigen molecules with flow cytometry. The radius of CD8 antibody molecule binding site was found, that allows recalculating the binding rate constant for other conditions (different sizes of reagent molecules, fluorescent label, medium viscosity and temperature). This approach is independent of specially prepared calibration beads, antibody reagents and the specific dye and can be applied to both low and high affinity antibodies, under both saturating and non-saturating binding conditions. The method was demonstrated on a human blood sample dataset investigating CD8α antigen on T cells in stable binding conditions.

[The diagnostic significance of testing cerebral natriuretic propeptide in patients with hemorrhagic fever and renal syndrome].

The study of content of cerebral natriuretic propeptide (NT-proBNP) in patients with hemorrhagic fever and renal syndrome was carried out considering severity degree, period of disease, presence of clinical roentgenological signs of lungs affection. It is demonstrated that in patients with mild and severe degree of disease the content of NT-proBNP is reliably higher than in the control group and gets its peak values in oliguric period The indices of NT-proBNP are reliably higher in patients with clinical roentgenological signs of lungs affection as compared with patients without such signs. The correlation analysis revealed direct dependence between the content of NT-proBNP and systolic blood pressure in pulmonary artery in in patients with hemorrhagic fever and renal syndrome of mild severity

Surface structure of sterically stabilized ferrofluids in a normal magnetic field: grazing-incidence x-ray study.

We studied the internal structure of sterically stabilized water- and oil-based ferrofluids in the vicinity of the free interface with a gas by means of x-ray reflectometry and grazing-incidence x-ray diffraction. It was found that in-depth distribution of the magnetic nanoparticles in the layer close to the interface is essentially inhomogeneous. In the case of water-based ferrofluids an enhanced concentration of surfactant and subsequent reduced concentration of the particles were detected in the 100-200-A -thick interface-adjacent layer. Scattering patterns possessing characteristic features of powder diffraction revealed partial ordering of the surfactant in a multilamellar structure. External magnetic fields applied perpendicular to the interface effectively reduced thickness of the depleted layer bringing the particles from the bulk to the surface. However no field-induced correlations between the particles were detected. In the top 500-A -thick layer of an oil-based ferrofluid depletion of the particles density was also present; however, no special arrangement of the surfactant molecules was manifested by the experimental data. Interestingly, for all samples we observed wavy surface deformation appearing in the normal magnetic field of a strength H much smaller than the critical values H_{c} calculated according to the conventional theory of ferrofluid surface instability. This deformation with lateral periodicity of a few millimeters has an amplitude smoothly increasing up to a few microns at H=0.5H_{c} .

Magnetic field dependent ordering in ferrofluids at SiO2 interfaces.

We report pronounced smecticlike ordering in a ferrofluid adjacent to a SiO2 wall. In the presence of small magnetic fields perpendicular to the interface, ordered layers of magnetite nanoparticles form that can extend up to 30 layers. We also show that short ranged ordered structures emerge when the magnetic field direction is parallel to the interface; however, the layering is strongly perturbed. These results have been obtained by in situ neutron reflectometry which gives a detailed microscopic picture of these ordering phenomena. They also reveal the formation of a wetting double-layer which forms the magnetic template for the observed ordering sheets. The implications of these findings are discussed.

[Selenium content in the internal organs, musculature and blood of broiler chickens]. / Sudurzhanie na selen v niakoi vutreshni organi, muskulatura i kruv ot pileta-broileri.

Experiments were carried out to establish the presence of selenium in broiler birds at different age, that had been given prophylactically selenium preparations. At the single application of such preparations selenium was found to drop in the liver, kidneys, brain, and blood, and to rise in the breast and femoral muscles. By the end of the fattening period highest was the level of selenium in the organs and tissues with the intake of sodium selenite in the drinking water; it was lowest at the injection of sodium selenite in effective amounts.