Recombinant Adeno-associated Viral Vectors Serotypes 6 and 9 are Able to Transduce Human Tracheal Epithelial Cells but Not Human Induced Pluripotent Stem Cells.

Recombinant adeno-associated viruses (rAAVs) may be useful for the development of gene therapy for hereditary diseases. Patient-specific human induced pluripotent stem cells (hiPSCs) can be differentiated into a variety of cells which are difficult or impossible to obtain by biopsy. To date, few research on the efficiency of rAAV transduction of hiPSCs has been published, but the obtained data are very contradictory and do not answer the actual question: how effective are rAAVs for the delivery of transgenes into hiPSCs. In this work, we used rAAV serotypes 5, 6, and 9 carrying the GFP transgene. The transduction efficiency of rAAV2/9-GFP and rAAV2/6-GFP for the immortalized tracheal epithelial cell line derived from a patient with cystic fibrosis (CFTE29o-) was relatively high. At the same time, the efficiency of transduction of iPSCs from a healthy donor and a cystic fibrosis (CF) donor was extremely low. Thus, our results show that the efficiency of hiPSC transduction by rAAV serotypes 5, 6, and 9 is not suitable for the delivery of transgenes.

Minimally Manipulated Bone Marrow-Derived Cells Can Be Used for Tissue Engineering In Situ and Simultaneous Formation of Personalized Tissue Models.

Red bone marrow and autologous bone tissue (bone fragments and bone chips) of the donor were harvested intraoperatively during autoplasty of talus bone defect. Titanium chips were obtained by grinding a fragment of a microporous titanium-coated hip arthroplasty (Zimmer). Bone marrow mononuclear cells were isolated in the operating room, and bone and titanium fragments were incubated with a suspension of mononuclear cells. The quality of revitalization was assessed by fluorescence microscopy and histological examination after culturing of adherent cells on the bone and titanium fragments. During culturing on bone chips, bone marrow mononuclear fraction cells demonstrated significantly higher metabolic activity than bone marrow cells (p=0.04). Mononuclear fraction cells were also capable of stable colonization of titanium fragments with the formation of composite tissue model.

Biological Activity of Agaricinic Acid Nanoparticles against Human Hepatoma HepG2 Cells.

A stable preparation of agaricinic acid nanoparticles was obtained. The mean hydrodynamic size of nanoparticles according to photon correlation spectroscopy was 200 nm and zeta potential was -57 mV. Cytotoxic activity of agaricinic acid nanoparticles against human HepG2 hepatoma cells was evaluated. Nanoparticles with a low concentration of agaricinic acid stimulated and with high concentration - suppressed metabolic activity and viability of hepatoma cells. The EC50 for the stimulating effect was 32.8 µg/ml, and the IC50=602.1 mg/ml. The preparation of agaricinic acid nanoparticles can be used in medicine as a potential antitumor agent.

ANTIOXIDATIVE EFFICACY OF NEUROPROTECTIVE THERAPY IN PATIENTS WITH CHRONIC CEREBRAL ISCHEMIA.

The aim of the research is to estimate the molecular and biochemical efficacy of Tiocetam administration to patients with ChCI, with regard of the state of glutathione system in plasma and of erythrocytes hemolysate. 60 patients with ChCI, averagely aged 54,70±7,71 years, were examined and divided into two groups of 30 people each, depending on the treatment regimen. The patients from the control group were intravenously administered infusions of Tiocetam with the dosage of 20 ml (for 10 days, then changed for Tiocetam forte (400 mg of Piracetam and 100 mg of Tiotriazoline) with the dosage of 1 pill thrice a day during 30 days. The patients of the experimental group were taking basic treatment, without administration of neuroprotective drugs. Concentration of renewed glutathione (RG) in blood plasma and erythrocytes hemolysate of patients was defined according to the reaction with ortho-phthalic anhydride; the contents of SH-group thioles in blood plasma, the activity of glutathione-dependent enzymes were defined spectrophotometrically. Treating patients with ChCI with Tiocetam has revealed probable (by Friedman test) decreased activity of glutathione transferase (GT) (p=0,003), and increased activity of glutathione reductase (GR) (p=0,002) and glutathione peroxidase (GPx) (p=0,002) in blood plasma, as well as decreased activity of GT (p=0,004), increased activity of GR (p=0,007) and GPx (p<0,001), as well as an increase of RG (p<0,001) in erythrocytes hemolysate of the patients with ChCI. Obtained results prove pathogenetically substantiated administration of Tiocetam for patients with ChCI, due to its positive impact on the condition of glutathione antioxidative system in blood plasma and erythrocytes hemolysate, by means of suppression of main mechanisms of oxidative stress, which make preconditions for clinical manifestations of ChCI.

Spread and morphological-structural properties of plant rhabdoviruses and similar pathogens in Basidiomycetes.

Long-term studies of spread of rhabdoviruses which indicated their harmfulness in different plant species under conditions of environmental factors were first discussed. Their harmfulness to different plant species under environmental conditions was shown. A comparative description of rhabdoviruses with similar pathogens of the mushrooms is carried out. Thus the main focus was on the morphology and structure of the pathogens. These data are extremely important in the study of distribution of the rhabdovirus on crops in different regions.

Cholesterol induces uneven curvature of asymmetric lipid bilayers.

A remarkable flexibility is observed in biological membranes, which allows them to form the structures of different curvatures. We addressed the question of intrinsic ability of phospholipid membranes to form highly curved structures and the role of cholesterol in this process. The distribution of cholesterol in the highly curved asymmetric DOPC/DOPS lipid bilayer was investigated by the coarse-grained molecular dynamics simulations in the membrane patches with large aspect ratio. It is shown that cholesterol induces uneven membrane curvature promoting the formation of extended flattened regions of the membrane interleaved by sharp bends. It is shown that the affinity of cholesterol to anionic DOPS or neutral DOPC lipids is curvature dependent. The cholesterol prefers DOPS to DOPC in either planar or highly curved parts of the membrane. In contrast, in the narrow interval of moderate membrane curvatures this preference is inverted. Our data suggest that there is a complex self-consistent interplay between the membrane curvature and cholesterol distribution in the asymmetric lipid bilayers. The suggested new function of cholesterol may have a biological relevance.

[Physical-chemical properties of the mutant (protein) form of D-glucose/D-galactose-binding protein GGBP/H152C with an attached fluorescent dye BADAN].

The influence of various factors on the physico-chemical characteristics and complexation of glucose with a mutant form of D-glucose/D-galactose-binding protein which can be regarded as a sensor of the glucometer, namely the protein GGBP/H152C with solvatochromic dye BADAN attached to the cysteine residue Cys 152, has been investigated. The point mutation His 152Cys and attaching BADAN reduced the affinity of the mutant form GGBP/H152C to glucose more than 8-fold compared to the wild type protein. This allows using this mutant for the determination of sugar content in biological fluids extracted by transdermal technologies. Sufficiently rapid complexation of GGBP/H152C with glucose (the time of protein-glucose complex formation is not more than three seconds even in solutions with a viscosity of 4 cP) provides timely monitoring changes in the concentration of sugar. The changes of ionic strength and pH within the physiological range of values of these variables do not have significant influence on fluorescent characteristics of GGBP/H152C-BADAN. At acidic pH, (see symbol) some of the molecules GGBP/H152C is in the unfolded state. It has been shown that mutant form GGBP/H152C has relatively low resistance to guanidine hydrochloride denaturing effects. This result indicates the need for more stable proteins to create a sensor for glucose biosensor system.

The change of cellular membranes on apoptosis: fluorescence detection.

The strong plasma membrane asymmetry existing in living cells is lost on apoptosis, and it is commonly detected with the probes interacting strongly and specifically with phosphatidylserine (PS). This phospholipid becomes exposed to the cell surface, and the labeled annexin V is used for its detection. The requirement for early and Ca(2+)-independent detection of apoptosis in the formats of spectroscopy of cell suspensions, flow cytometry, microarray technology and confocal or two-photon microscopy stimulated efforts for the development of new methods. Since the PS exposure must produce integrated changes of electrostatic potential and hydration in the outer leaflet of cell membrane, its detection can be provided by direct response of smart fluorescence probes. This review is focused on basic mechanisms underlying the loss of membrane asymmetry during apoptosis and the principles lying in the background of new methods that demonstrate essential advantages over the annexin V-binding assay. The convenient wavelength-ratiometric technique based on fluorescent probe F2N12S is described in detail. It incorporates spontaneously into outer leaflet of cell membrane and the color change of its fluorescent emission associated with apoptosis can be easily detected. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

Glycosyl Thioimidates as Versatile Building Blocks for Organic Synthesis.

This review discusses the synthesis and application of glycosyl thioimidates in chemical glycosylation and oligosaccharide assembly. Although glycosyl thioimidates include a broad range of compounds, the discussion herein centers on S-benzothiazolyl (SBaz), S-benzoxazolyl (SBox), S-thiazolinyl (STaz), and S-benzimidazolyl (SBiz) glycosides. These heterocyclic moieties have recently emerged as excellent anomeric leaving groups that express unique characteristics for highly diastereoselective glycosylation and help to provide the streamlined access to oligosaccharides.

Thermal biosensor for detecting nonylphenol in the environment.

The main goal of the research was the development of thermal immune biosensor for highly sensitive and specific determination of nonylphenol (NPh), based on measuring the heat released as a result of the interaction between hapten and specific antibodies. As it was shown previously, in case of SPR based immune biosensor a number of algorithms of analysis was realized, including "competitive" (with the sensitivity on the level of about 7-10 ng/ml), "direct" (10 ng/ml) ways, and the so called algorithm "to saturation" (about 2-5 ng/ml). The time of analysis by immune SPR biosensor is about 10 min (on the previously prepared transducer surface, including immobilization of sensitive structures). The developed thermal biosensor provides direct detection of NPh with the sensitivity of about 1 microg/ml and the overall time of analysis of about 20-30 min. In spite of a lower sensitivity of the thermal biosensor, it is less sensitive to admixtures in real samples and simpler in use than the biosensor based on SPR and, consequently, the thermal biosensor is more applicable in the field conditions.

Hierarchical clustering of the correlation patterns: new method of domain identification in proteins.

New method of identification of dynamical domains in proteins - Hierarchical Clustering of the Correlation Patterns (HCCP) is proposed. HCCP allows to identify the domains using single three-dimensional structure of the studied proteins and does not require any adjustable parameters that can influence the results. The method is based on hierarchical clustering performed on the matrices of correlation patterns, which are obtained by the transformation of ordinary pairwise correlation matrices. This approach allows to extract additional information from the correlation matrices, which increases reliability of domain identification. It is shown that HCCP is insensitive to small variations of the pairwise correlation matrices. Particularly it produces identical results if the data obtained for the same protein crystallized with different spatial positions of domains are used for analysis. HCCP can utilize correlation matrices obtained by any method such as normal mode or essential dynamics analysis, Gaussian network or anisotropic network models, etc. These features make HCCP an attractive method for domain identification in proteins.

Antibody immobilisation on the metal and silicon surfaces. The use of self-assembled layers and specific receptors.

The use of Staphylococcal protein A and lectins as intermediate immobilising agents allows operators to orient antibodies (Ab) towards the solution due to the presence of a specific binding sites of immunoglobulin (Ig) molecules. Antibodies of different species of animals have unequal affinities to individual lectins. The effective thickness of immobilised Ab's depends on the type of substrates used and increases in the following sequence: bare gold or silicon surface, the surface treated with self-assembled polyelectrolytes (PESA) or with protein A or some lectins deposited on the preliminary formed polyelectrolyte layer. The glycolysated protein of jp51 may be selectively immobilised from the mixture of retroviral proteins (p24 and jp51), if it is necessary to distinguish infected animals from preliminarily immunised ones by means of a vaccine based on p24 protein. It was shown that the use of Staphylococcal protein A, instead of some lectins as intermediate layer for the Ab immobilisation, does not lead to a more sensitive determination of such low-weight toxins as 2,4-dichlorophenoxyacetic acid (2,4-D). The above-mentioned results were obtained with surface plasmon resonance (SPR) technique.

Heat perturbation of bovine eye lens alpha-crystallin probed by covalently attached ratiometric fluorescent dye 4'-diethylamino-3-hydroxyflavone.

Bovine eye lens alpha-crystallin was covalently labeled with 6-bromomethyl-4'-diethylamino-3-hydroxyflavone and studied under native-like conditions and at the elevated temperature (60 degrees C) that is known to facilitate alpha-crystallin chaperone-like activity. This novel SH-reactive two-band ratiometric fluorescent probe is characterized by two highly emissive N*- and T*-bands; the latter appears due to excited state intramolecular proton transfer reaction. The positions of these bands and the ratio of their intensities for the alpha-crystallin-dye conjugate are the sensitive indicators of polarity of the dye environment and its participation in intermolecular hydrogen bonding. Although we found that the dye labels both the SH and the NH2 groups in alpha-crystallin, a recently developed procedure allowed us to distinguish between the heat-induced spectral changes of the dye molecules attached to SH and NH2 groups. We observed that at elevated temperature the environment of the SH-attached dye becomes more polar and flexible. The number of H-bond acceptor groups in the vicinity of the dye decreases. Since alpha-crystallin contains a single Cys residue within the C-terminal domain of its (alpha)A subunit (the (alpha)B subunit contains none), we can attribute the observed effects to temperature-induced changes in the C-terminal domain of this protein.

[Immunoenzyme analysis of non-ionic surfactants in water].

The approaches for high sensitive and specific determination of nonylphenol (NP) with the help of immune enzymatic (ELISA) method have been developed. The process of preparation of conjugates of NP with proteins, antiserum obtaining, purification of immunoglobulin (Ig) fractions and study of specificity of the obtained antibodies were described in detail. It was shown that the antiserum and total Ig fraction do not differ in respect of specificity and binding abilities. The developed algorithm of ELISA method fulfillment is able to provide NP revealing with the sensitivity in the range of 20-50 ng/ml and working controlled concentrations--from 20-50 to 1000 ng/ml.

Towards realistic description of collective motions in the lattice protein folding models.

Collective motions and the formation of clusters of residues play an important role in the folding of real proteins. However, existing Monte Carlo (MC) techniques of the protein folding simulations based on highly popular lattice models provide only a schematic representation of collective motions, which is rather far from physical reality. The Clustering Monte Carlo (CMC) algorithm was developed with particular aim to provide a realistic description of collective motions on the lattice. CMC allows modeling the cluster dynamics and the effects of the solvent viscosity, which is impossible in conventional algorithms. In this study two 2D lattice peptides, with the ground states of hierarchical and non-hierarchical design, were investigated comparatively using three methods: Metropolis MC with the local move set, Metropolis MC with unspecific rigid rotations and the CMC algorithm. We present evidence that the folding pathways and kinetics of hierarchically folding clustered sequence are not adequately described in conventional MC simulations, and the account for cluster dynamics provided by CMC allows to capture essential features of the folding process. Our data suggest that the methods, which enable specific cluster motions, such as CMC, should be used for a more realistic description of protein folding.

Neutral fluorescence probe with strong ratiometric response to surface charge of phospholipid membranes.

We report on dramatic differences in fluorescence spectra of 4'-dimethylamino-3-hydroxyflavone (probe F) studied in phospholipid membranes of different charge (phosphatidyl glycerol, phosphatidylcholine (PC), their mixture and the mixture of PC with a cationic lipid). The effect consists in variations of relative intensities at two well-separated band maxima at 520 and 570 nm belonging to normal (N*) and tautomer (T*) excited states of flavone chromophore. Based on these studies we propose a new approach to measure electrostatic potential at the surface layer of phospholipid membranes, which is based on potential-dependent changes of bilayer hydration and involves very sensitive and convenient ratiometric measurements in fluorescence emission.

A stereoselective approach for the synthesis of alpha-sialosides.

A highly efficient synthesis of the human melanoma associated antigen GD(3) derivative has been described. A key feature of the synthetic approach was the use of sialyl donors that were protected with a C-5 trifluoroacetamide moiety. These sialyl donors gave high yields and excellent alpha-anomeric selectivities in direct glycosylations with a wide variety of glycosyl acceptors ranging from C-8 hydroxyls of sialic acids and C-3 hydroxyls of galactosides to reactive primary alcohols.

On the excited-state energy transfer between tryptophan residues in proteins: the case of penicillin acylase.

The problem, whether excited-state energy transfer occurs between Trp residues in a multi-tryptophan proteins and if it does, what kind of changes it induces in different parameters of protein fluorescence, is currently under active investigation. In our previous paper [Biophys. Chem. 72 (1998) 265], the energy transfer was found and studied in detail for Na,K-ATPase. It was shown that this transfer influences all parameters of fluorescence emission, which is detected at site-selective conditions (red-edge of excitation, blue and red edges of emission). Present experiments were performed on unusually tryptophan-rich protein, bacterial penicillin acylase (28 Trp per dimer of 82 kDa) and were aimed to extend these observations. They demonstrate substantial heterogeneity in the environments of tryptophan residues within the protein structure. This suggests, that in the present case, if the energy transfer exists, it should be directed from short-wavelength-emitting to long-wavelength-emitting tryptophan residues and thus could be easily observed by a number of time-resolved and steady-state fluorescence techniques. Unexpectedly, no signature of inter-tryptophan energy transfer was found.

A highly convergent synthesis of a complex oligosaccharide derived from group B type III Streptococcus.

An efficient synthesis of a heptasaccharide derived from group B type III Streptococcus carrying an artificial spacer (1) is described. Rapid assembly of a protected heptasaccharide (16a) is accomplished from readily available building blocks 2-5 without a single protecting group manipulation between glycosylation steps. The synthetic strategy may be applied to the assembly of other branched complex oligosaccharides. The deprotected heptasaccharide 1 was coupled to a poly[N-(acryloyloxy)succinimide, and the resulting material will be used for the development of an ELISA assay to detect antibodies against GBS, type III in pregnant women.