Experimental Models for Studying Structural and Functional State of the Pathological Liver (Review).

Liver pathologies remain one of the leading causes of mortality worldwide. Despite a high prevalence of liver diseases, the possibilities of diagnosing, prognosing, and treating non-alcoholic and alcoholic liver diseases still have a number of limitations and require the development of new methods and approaches. In laboratory studies, various models are used to reconstitute the pathological conditions of the liver, including cell cultures, spheroids, organoids, microfluidic systems, tissue slices. We reviewed the most commonly used in vivo, in vitro, and ex vivo models for studying non-alcoholic fatty liver disease and alcoholic liver disease, toxic liver injury, and fibrosis, described their advantages, limitations, and prospects for use. Great emphasis was placed on the mechanisms of development of pathological conditions in each model, as well as the assessment of the possibility of reconstructing various key aspects of pathogenesis for all these pathologies. There is currently no consensus on the choice of the most adequate model for studying liver pathology. The choice of a certain effective research model is determined by the specific purpose and objectives of the experiment.

Magnetic hysteresis in self-assembled monolayers of Dy-fullerene single molecule magnets on gold.

Fullerene single molecule magnets (SMMs) DySc2N@C80 and Dy2ScN@C80 are functionalized via a 1,3-dipolar cycloaddition with surface-anchoring thioether groups. The SMM properties of Dy-fullerenes are substantially affected by the cycloaddition. Submonolayers of the physisorbed derivatives exhibit magnetic hysteresis on an Au(111) surface at 2 K as revealed by X-ray magnetic circular dichroism.

Magnetization relaxation in the single-ion magnet DySc2N@C80: quantum tunneling, magnetic dilution, and unconventional temperature dependence.

Relaxation of magnetization in endohedral metallofullerenes DySc2N@C80 is studied at different temperatures, in different magnetic fields, and in different molecular arrangements. Magnetization behavior and relaxation are analyzed for powder sample, and for DySc2N@C80 diluted in non-magnetic fullerene Lu3N@C80, adsorbed in voids of a metal-organic framework, and dispersed in a polymer. The magnetic field dependence and zero-field relaxation are also studied for single-crystals of DySc2N@C80 co-crystallized with Ni(ii) octaethylporphyrin, as well as for the single crystal diluted with Lu3N@C80. Landau-Zener theory is applied to analyze quantum tunneling of magnetization in the crystals. The field dependence of relaxation rates revealed a dramatic dependence of the zero-field tunneling resonance width on the dilution and is explained with the help of an analysis of dipolar field distributions. AC magnetometry is used then to get access to the relaxation of magnetization in a broader temperature range, from 2 to 87 K. Finally, a theoretical framework describing the spin dynamics with dissipation is proposed to study magnetization relaxation phenomena in single molecule magnets.

Giant exchange coupling and field-induced slow relaxation of magnetization in Gd2@C79N with a single-electron Gd-Gd bond.

Magnetic properties of the azafullerene Gd2@C79N are studied by SQUID magnetometry. The effective exchange coupling constant jGd,e between the Gd spins and the spin of unpaired electron residing on the single-electron Gd-Gd bond is determined to be 170 ± 10 cm-1. Low temperature AC measurements revealed field-induced millisecond-long relaxation of magnetization.

Record-high thermal barrier of the relaxation of magnetization in the nitride clusterfullerene Dy2ScN@C80-Ih.

The Dy-Sc nitride clusterfullerene Dy2ScN@C80-Ih exhibits slow relaxation of magnetization up to 76 K. Above 60 K, thermally-activated relaxation proceeds via the fifth-excited Kramers doublet with the energy of 1735 ± 21 K, which is the highest barrier ever reported for dinuclear lanthanide single molecule magnets.

Magnetic anisotropy of endohedral lanthanide ions: paramagnetic NMR study of MSc2N@C80-Ih with M running through the whole 4f row.

Paramagnetic and variable temperature 13C and 45Sc nuclear magnetic resonance studies are performed for nitride clusterfullerenes MSc2N@C80 with icosahedral Ih(7) carbon cage, where M runs through all lanthanides forming nitride clusters. The influence of the endohedral lanthanide ions on the NMR spectral pattern is carefully followed, and dramatic differences are found in peak positions and line widths. Thus, 13C lines broaden from 0.01-0.02 ppm in diamagnetic MSc2N@C80 molecules (M = La, Y, Lu) to several ppm in TbSc2N@C80 and DySc2N@C80. Direction of the paramagnetic shift depends on the shape of the 4f electron density in corresponding lanthanide ions. In TmSc2N@C80 and ErSc2N@C80 with prolate 4f-density of lanthanide ions, 13C signals are shifted down-field, whereas 45Sc peaks are shifted up-field versus diamagnetic values. In all other MSc2N@C80 molecules lanthanide ions have oblate-shaped 4f electron density, and the lanthanide-induced shift is negative for 13C and positive for 45Sc peaks. Analysis of the pseudocontact and contact contributions to chemical shifts revealed that the pseudocontact term dominates both in 13C and 45Sc NMR spectra, although contact shifts for 13C signals are also considerable. Point charge computations of the ligand field splitting are performed to explain experimental results, and showed reasonable agreement with experimental pseudocontact shifts. Nitrogen atom bearing large negative charge and located close to the lanthanide ion results in large magnetic anisotropy of lanthanide ions in nitride clusterfullerenes with quasi-uniaxial ligand field.

[Efficacy of Venarus in treatment of patients with post-thrombotic disease of lower limbs].

The authors studied efficacy of Venarus in comprehensive treatment of patients presenting with post-thrombotic disease. An open multicenter retrospective study included a total of 110 patients subdivided into two groups. Group One (Study Group) consisted of 51 patients with post-thrombotic syndrome, undergoing comprehensive medical treatment with the use of phlebotonic agent Venarus. Group Two (Control Group) comprised 59 patients with post-thrombotic syndrome undergoing similar conservative treatment but without taking phlebotonics. It was proved that Venarus included into comprehensive treatment of patients with post-thrombotic syndrome led to a statistically significant increase of both psychological and social activity and improved patients' quality of life. During the standard term of administration (2 months) Venarus levelled subjective symptoms and certain objective symptoms (according to the Villalta Scale) of post-thrombotic syndrome. After 2-month use Venarus demonstrated the highest efficacy in treating patients with mild-to-moderate post-thrombotic syndrome. The maximal efficacy was observed after 3 months of administration in standard doses. No side effects were noted during the whole period of the study.

Cluster-size dependent internal dynamics and magnetic anisotropy of Ho ions in HoM2N@C80 and Ho2MN@C80 families (M = Sc, Lu, Y).

The paramagnetic NMR study of HoM2N@C80-Ih and Ho2MN@C80-Ih nitride cluster fullerenes (M = Sc, Lu, Y) reveals strong dependence of Ho-induced paramagnetic shifts (δ(para)) in (13)C NMR spectra on the size of the diamagnetic metal in the cluster. In particular, the δ(para) value in HoY2N@C80 is almost doubled in comparison to that in HoSc2N@C80. X-ray magnetic circular dichroism studies show that all Ho-nitride cluster fullerenes have the same magnetic ground state of Ho(3+). Point-charge ligand-field splitting calculations show that the increase of the M(3+) radius in going from Sc to Y results in a considerable increase of the energy splitting between different Jz states. This leads to a 19% higher magnetic anisotropy of Ho(3+) in HoY2N@C80 than in HoSc2N@C80 at 300 K. Variations of the molecular geometry and cluster dynamics with the size of the cluster are found to have even greater influence on δ(para) values. This work shows that the magnetic properties of the species confined inside the fullerene cages can be tuned using the geometrical factors such as the cluster and the cage size.

[First experience with minilaparotomy for Leriche syndrome].

The authors present the first experience with reconstruction of the terminal part of the infrarenal aorta from minilaparotomy. There were 11 aortofemoral bifurcation shunts in patients with Leriche syndrome. In two cases conversion (general calcification of the aorta, visceral obesity) was necessary. Regression of extremity ischemia was obtained in all the patients.

Identification of proximate regions in a complex of retinal guanylyl cyclase 1 and guanylyl cyclase-activating protein-1 by a novel mass spectrometry-based method.

A key challenge in studying protein/protein interactions is to accurately identify contact surfaces, i.e. regions of two proteins that are in direct physical contact. Aside from x-ray crystallography and NMR spectroscopy few methods are available that address this problem. Although x-ray crystallography often provides detailed information about contact surfaces, it is limited to situations when a co-crystal of proteins is available. NMR circumvents this requirement but is limited to small protein complexes. Other methods, for instance protection from proteolysis, are less direct and therefore less informative. Here we describe a new method that identifies candidate contact surfaces in protein complexes. The complexes are first stabilized by cross-linking. They are then digested with a protease, and the cross-linked fragments are analyzed by mass spectrometry. We applied this method, referred to as COSUMAS (contact surfaces by mass spectrometry), to two proteins, retinal guanylyl cyclase 1 (RetGC1) and guanylyl cyclase-activating protein-1 (GCAP-1), that regulate cGMP synthesis in photoreceptors. Two regions in GCAP-1 and three in RetGC1 were identified as possible contact sites. The two regions of RetGC1 that are in the vicinities of Cys(741) and Cys(780) map to a kinase homology domain in RetGC1. Their identities as contact sites were independently evaluated by peptide inhibition analysis. Peptides with sequences from these regions block GCAP-1-mediated regulation of guanylyl cyclase at both high and low Ca2+ concentrations. The two regions of GCAP-1 cross-linked to these peptides were in the vicinities of Cys(17) and Cys(105) of GCAP-1. Peptides with sequences derived from these regions inhibit guanylyl cyclase activity directly. These results support a model in which GCAP-1 binds constitutively to RetGC1 and regulates cyclase activity by structural changes caused by the binding or dissociation of Ca2+.

Cantor set fractals from solitons

We show how a nonlinear system that supports solitons can be driven to generate exact (regular) Cantor set fractals. As an example, we use numerical simulations to demonstrate the formation of Cantor set fractals by temporal optical solitons. This fractal formation occurs in a cascade of nonlinear optical fibers through the dynamical evolution from a single input soliton.

Purification of myristoylated and nonmyristoylated neuronal calcium sensor-1 using single-step hydrophobic interaction chromatography.

Neuronal calcium sensors (NCSs) belong to a family of Ca(2+)-binding proteins, which serve important functions in neurotransmission, and are highly conserved from yeast to humans. Overexpression of the neuronal calcium sensor-1, called frequenin in the fruit fly and in frog, increases the release of neurotransmitters. Studying the functional role of frequenin in mammals and understanding its structural dynamics is critically dependent on the availability of active purified protein. Neuronal calcium sensors like other members of the family share common structural features: they contain four EF-hands as potential binding sites for Ca(2+) and an N-terminal consensus sequence for myristoylation. Previously, recoverin, distantly related to NCSs, has been expressed and purified from Escherichia coli, involving a combination of different chromatographic steps. NCS-1 has earlier been purified adopting a two-step procedure used for recoverin purification. We have overexpressed NCS-1 from rat in its myristoylated and nonmyristoylated form in E. coli and purified it from crude lysates using a single-step hydrophobic interaction chromatography. The purified protein was identified by Western blotting and mass spectrometry and assayed for its ability to bind Ca(2+) using a Ca(2+) shift assay, terbium fluorescence, and Stains-all binding. The present protocol provides a rapid, more efficient and simplified, single-step method for purifying NCS-1 for structural and functional studies. This method can also be applied to purify related proteins of the superfamily.

[Problems of prevention of mental disorders in school children]. / Problemy profilaktiki narushenii psikhicheskogo zdorov'ia shkol'nikov.

The paper covers various aspects of schoolchildren's mental adaptation. The study has indicated that mental health is worse in 6th-9th-form pupils than that in junior schoolchildren. There is a relationship of the deviant behavior of schoolchildren and their mental health. The data of an anonymous questionnaire survey are given, which suggest that there is an increase in negative trends towards psychoactive drug abuse. Evidence shows it necessary to apply a comprehensive approach to solving the problems of schoolchildren health care.

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin.

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.

Mapping functional domains of the guanylate cyclase regulator protein, GCAP-2.

Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.

Mapping sites in guanylyl cyclase activating protein-1 required for regulation of photoreceptor membrane guanylyl cyclases.

Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca2+-sensitive manner. It contains four Ca2+-binding motifs, EF-hands, three of which are capable of binding Ca2+. GCAP-1 activates RetGC in low Ca2+ and inhibits it in high Ca2+. In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met157 to Arg182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg178 to Arg182. Another sequence essential for activation is within the N-terminal residues Trp21 to Thr27. The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln33 to the COOH-terminal Gly205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.

Cell-type-dependent activity of the ubiquitous transcription factor USF in cellular proliferation and transcriptional activation.

USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.

Photon-number squeezing in the normal-dispersion regime.

We experimentally demonstrate the generation of amplitude-squeezed light in the normal-dispersion regime and measure by direct detection 1.7+/-0.1 dB (33%) and, with correction for linear losses, 2.5+/-0.2 dB (47%) of noise reduction below the shot-noise level. The dependence of the noise behavior on dispersion is investigated both experimentally and theoretically.