Apoptotic Bodies of Cardiomyocytes and Fibroblasts - Regulators of Directed Differentiation of Heart Stem Cells.

We studied the effects of apoptotic bodies of cardiomyocytes (ApBc) and fibroblasts (ApBf) on myocardial regeneration and contractility in rats and the dynamics of RNA concentrations in cardiomyocytes and fibroblasts at different stages of apoptosis. ApBc increase the contractility of rat myocardium, while ApBf reduce it. ApBc stimulate the development of clones of cardiomyocyte precursors in the myocardium, while ApBf stimulate the formation of endothelial precursor clones. In doxorubicin cardiomyopathy, ApBc, similar to the reference drug (ACE inhibitor) improve animal survival, while ApBf produce no such effect. RNA concentrations in cardiomyocytes and fibroblasts before apoptosis and at the beginning of cell death significantly differed, while in apoptotic bodies of these cells, it was practically the same. It has been hypothesized that RNA complex present in ApBc and ApBf represents an "epigenetic code" of directed differentiation of cardiac stem cells.

Allosteric regulation of the ribosomal A site revealed by molecular dynamics simulations.

The A site of the ribosome, which determines binding and orientation of a new amino acid residue for the peptidyl transferase reaction, was found to occupy two different conformational states upon the molecular dynamics (MD) simulations study of the 70S E. coli ribosome. One of the states, defined as "inactive", appeared in trajectories with E-tRNA, mutations A2531U and UU2492-3C, which are known to decrease the A site affinity to the tRNA. This conformational transition was found to be allosterically connected with conformational alterations in different sites of the macromolecular complex, including the E site of the ribosome and intersubunit bridge B7a located near the E site. The MD simulations of the ribosomes with the A2531U and UU2492-3C mutations known to decrease the A-tRNA retention in the ribosome, demonstrated partial switching of the 16S and 23S rRNA conformation towards its characteristic one in the P/P, E/E state.

Sensors for Proteolytic Activity Visualization and Their Application in Animal Models of Human Diseases.

Various sensors designed for optical and photo(opto)acoustic imaging in living systems are becoming essential components of basic and applied biomedical research. Some of them including those developed for determining enzyme activity in vivo are becoming commercially available. These sensors can be used for various fluorescent signal detection methods: from whole body tomography to endoscopy with miniature cameras. Sensor molecules including enzyme-cleavable macromolecules carrying multiple quenched near-infrared fluorophores are able to deliver their payload in vivo and have long circulation time in bloodstream enabling detection of enzyme activity for extended periods of time at low doses of these sensors. In the future, more effective "activated" probes are expected to become available with optimized sensitivity to enzymatic activity, spectral characteristics suitable for intraoperative imaging of surgical field, biocompatibility and lack of immunogenicity and toxicity. New in vivo optical imaging methods such as the fluorescence lifetime and photo(opto)acoustic imaging will contribute to early diagnosis of human diseases. The use of sensors for in vivo optical imaging will include more extensive preclinical applications of experimental therapies. At the same time, the ongoing development and improvement of optical signal detectors as well as the availability of biologically inert and highly specific fluorescent probes will further contribute to the introduction of fluorescence imaging into the clinic.

Photonic surface waves on metamaterial interfaces.

A surface wave (SW) in optics is a light wave, which is supported at an interface of two dissimilar media and propagates along the interface with its field amplitude exponentially decaying away from the boundary. Research on surface waves has been flourishing in the last few decades due to their unique properties of surface sensitivity and field localization. These features have resulted in applications in nano-guiding, sensing, light-trapping and imaging based on near-field techniques, contributing to the establishment of nanophotonics as a field of research. Up to now, a wide variety of surface waves has been investigated in numerous material and structure settings. This article reviews the recent progress and development in the physics of SWs localized at metamaterial interfaces, as well as bulk media in order to provide broader perspectives on optical surface waves in general. For each type of surface wave, we discuss the material and structural platforms. We mainly focus on experimental realizations in the visible and near-infrared wavelength ranges. We also address existing and potential application of SWs in chemical and biological sensing, and experimental excitation and characterization methods.

Structural Insight into Interaction between C20 Phenylalanyl Derivative of Tylosin and Ribosomal Tunnel.

Macrolides are clinically important antibiotics that inhibit protein biosynthesis on ribosomes by binding to ribosomal tunnel. Tylosin belongs to the group of 16-membered macrolides. It is a potent inhibitor of translation whose activity is largely due to reversible covalent binding of its aldehyde group with the base of A2062 in 23S ribosomal RNA. It is known that the conversion of the aldehyde group of tylosin to methyl or carbinol groups dramatically reduces its inhibitory activity. However, earlier we obtained several derivatives of tylosin having comparable activity in spite of the fact that the aldehyde group of tylosin in these compounds was substituted with an amino acid or a peptide residue. Details of the interaction of these compounds with the ribosome that underlies their high inhibitory activity were not known. In the present work, the structure of the complex of tylosin derivative containing in position 20 the residue of ethyl ester of 2-imino(oxy)acetylphenylalanine with the tunnel of the E. coli ribosome was identified by means of molecular dynamics simulations, which could explain high biological activity of this compound.

The Ribosome as an Allosterically Regulated Molecular Machine.

The ribosome as a complex molecular machine undergoes significant conformational rearrangements during the synthesis of polypeptide chains of proteins. In this review, information obtained using various experimental methods on the internal consistency of such rearrangements is discussed. It is demonstrated that allosteric regulation involves all the main stages of the operation of the ribosome and connects functional elements remote by tens and even hundreds of angstroms. Data obtained using Förster resonance energy transfer (FRET) show that translocation is controlled in general by internal mechanisms of the ribosome, and not by the position of the ligands. Chemical probing data revealed the relationship of such remote sites as the decoding, peptidyl transferase, and GTPase centers of the ribosome. Nevertheless, despite the large amount of experimental data accumulated to date, many details and mechanisms of these phenomena are still not understood. Analysis of these data demonstrates that the development of new approaches is necessary for deciphering the mechanisms of allosteric regulation of the operation of the ribosome.

2-Guanidino-quinazolines as a novel class of translation inhibitors.

A variety of structurally unrelated organic compounds has been reported to have antibacterial activity. Among these, certain small-molecule translation inhibitors have attracted a great deal of attention, due to their relatively high selectivity against prokaryotes, and an appropriate therapeutic index with minor "off target" effects. However, ribosomes are being considered as poorly druggable biological targets, thereby making some routine computational-based approaches to rational drug design and its development rather ineffective. Taking this into account, diversity-oriented biological screening can reasonably be considered as the most advantageous strategy. Thus, using a high-throughput screening (HTS) platform, we applied a unique biological assay for in vitro evaluation of thousands of organic molecules, especially targeted against bacterial ribosomes and translation. As a result, we have identified a series of structurally diverse small-molecule compounds that induce a reporter strain sensitive to translation and DNA biosynthesis inhibitors. In a cell free system, several molecules were found to strongly inhibit protein biosynthesis. Among them, compounds bearing a 2-guanidino-quinazoline core demonstrated the most promising antibacterial activity. With regard to the preliminary structure-activity relationship (SAR) study, we revealed that relatively small substituents at positions 4, 6 and 8 of the quinazoline ring significantly enhance the target activity whereas modification of the guanidine group leads to decrease or loss of antibacterial potency. This novel class of translation inhibitors can properly be regarded as a promising starting point for the development of novel antibacterial therapeutic or screening tools.

New Fluorescent Macrolide Derivatives for Studying Interactions of Antibiotics and Their Analogs with the Ribosomal Exit Tunnel.

Novel fluorescent derivatives of macrolide antibiotics related to tylosin bearing rhodamine, fluorescein, Alexa Fluor 488, BODIPY FL, and nitrobenzoxadiazole (NBD) residues were synthesized. The formation of complexes of these compounds with 70S E. coli ribosomes was studied by measuring the fluorescence polarization depending on the ribosome amount at constant concentration of the fluorescent substance. With the synthesized fluorescent tylosin derivatives, the dissociation constants for ribosome complexes with several known antibiotics and macrolide analogs previously obtained were determined. It was found that the fluorescent tylosin derivatives containing BODIPY FL and NBD groups could be used to screen the binding of novel antibiotics to bacterial ribosomes in the macrolide-binding site.

Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramphenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3-10 µM, which is 100-fold lower than the corresponding values for chloramphenicol amine-ribosome complex. Interaction of the chloramphenicol peptide analogs with ribosomes was simulated by molecular docking, and the most probable contacts of "stop peptide" motifs with the elements of nascent peptide exit tunnel were identified.

[Application of reporter strains for new antibiotic screening].

Screening for new antibiotics remains an important area of biology and medical science. Indispensable for this type of research is early identification of antibiotic mechanism of action. Preferentially, it should be studied quickly and cost-effectively, on the stage of primary screening. In this review we describe an application of reporter strains for rapid classification of antibiotics by its target, without prior purification of an active compound and determination of chemical structure.

Dark-field imaging as a noninvasive method for characterization of whispering gallery modes in microdisk cavities.

Whispering gallery mode microdisk cavities fabricated by direct laser writing are studied using dark-field imaging and spectroscopy in the visible spectral range. Dark-field imaging allows us to directly visualize the spatial intensity distribution of whispering gallery modes. We extract their azimuthal and radial mode indices from dark-field images, and find the axial mode number from the dispersion relation. The scattering spectrum obtained in the confocal arrangement provides information on the density of optical states in the resonator. The proposed technique is a simple noninvasive way to characterize the optical properties of microdisk cavities.

[Current possibilities of clinical applications of breast tumors typing expression].

Whole-genome expression analysis methods significantly clarified contemporary breast cancer classification. Besides today clinical practice lacks the use of expression methods due to complexity of conduction, analysis and lack of clinical application. Further studies of breast cancer expression characteristics and clinical trials with stratification based of phonotypical features may improve the results of existing anticancer agents. Creation of limited clinically applicable test system, which incorporates all the specific breast cancer subtypes is currently needed.

Investigation of Ribosomes Using Molecular Dynamics Simulation Methods.

The ribosome as a complex molecular machine undergoes significant conformational changes while synthesizing a protein molecule. Molecular dynamics simulations have been used as complementary approaches to X-ray crystallography and cryoelectron microscopy, as well as biochemical methods, to answer many questions that modern structural methods leave unsolved. In this review, we demonstrate that all-atom modeling of ribosome molecular dynamics is particularly useful in describing the process of tRNA translocation, atomic details of behavior of nascent peptides, antibiotics, and other small molecules in the ribosomal tunnel, and the putative mechanism of allosteric signal transmission to functional sites of the ribosome.

[Common features of antibacterial compounds: an analysis of 104 compounds library].

Antibacterial compounds are one of the essential classes of clinically important drugs. High throughput screening allowed revealing potential antibiotics active towards any molecular target in bacterial cell. We used a library of 9820 organic compounds with highly diversified structures to screen for antibacterial activity. As the result of automated screening, 103 compounds were found to possess antibacterial activity against Escherichia coli. The properties of these compounds were compared with those of initial library. Non-linear Kohonen mapping was used to analyze the differences between non-active molecules from initial library, identified antibacterial hits and compounds with reported antibacterial activity. It was found that identified antibacterial compounds are located in the separated area of chemical space. It can be therefore suggested that these molecules belong to novel classes of antibacterial compounds and could be studied further.

Regulation of Flagellar Gene Expression in Bacteria.

The flagellum of a bacterium is a supramolecular structure of extreme complexity comprising simultaneously both a unique system of protein transport and a molecular machine that enables the bacterial cell movement. The cascade of expression of genes encoding flagellar components is closely coordinated with the steps of molecular machine assembly, constituting an amazing regulatory system. Data on structure, assembly, and regulation of flagellar gene expression are summarized in this review. The regulatory mechanisms and correlation of the process of regulation of gene expression and flagellum assembly known from the literature are described.

Modeling Interactions of Erythromycin Derivatives with Ribosomes.

Using a method of static simulation, a series of erythromycin A analogs was designed with aldehyde functions introduced instead of one of the methyl substituents in the 3'-N-position of the antibiotic that was potentially capable of forming a covalent bond with an amino group of one of the nucleotide residues of the 23S rRNA in the ribosomal exit tunnel. Similar interaction is observed for antibiotics of the tylosin series, which bind tightly to the large ribosomal subunit and demonstrate high antibacterial activity. Binding of novel erythromycin derivatives with the bacterial ribosome was investigated with the method of fluorescence polarization. It was found that the erythromycin analog containing a 1-methyl-3-oxopropyl group in the 3'-N-position demonstrates the best binding. Based on the ability to inhibit protein biosynthesis, it is on the same level as erythromycin, and it is significantly better than desmethyl-erythromycin. Molecular dynamic modeling of complexes of the derivatives with ribosomes was conducted to explain the observed effects.

Molecular Dynamics Investigation of a Mechanism of Allosteric Signal Transmission in Ribosomes.

The ribosome is a molecular machine that synthesizes all cellular proteins via translation of genetic information encoded in polynucleotide chain of messenger RNA. Transition between different stages of the ribosome working cycle is strictly coordinated by changes in structure and mutual position both of subunits of the ribosome and its ligands. Therein, information regarding structural transformations is transmitted between functional centers of the ribosome through specific signals. Usually, functional centers of ribosomes are located at a distance reaching up to several tens of angstroms, and it is believed that such signals are transduced allosterically. In our study, we attempted to answer the question of how allosteric signal can be transmitted from one of the so-called sensory elements of ribosomal tunnel (RT) to the peptidyl transferase center (PTC). A segment of RT wall from the E. coli ribosome composed of nucleotide residues A2058, A2059, m(2)A2503, G2061, A2062, and C2063 of its 23S rRNA was examined by molecular dynamics simulations. It was found that a potential signal transduction pathway A2058-C2063 acted as a dynamic ensemble of interdependent conformational states, wherein cascade-like changes can occur. It was assumed that structural rearrangement in the A2058-C2063 RT segment results in reversible inactivation of PTC due to a strong stacking contact between functionally important U2585 residue of the PTC and nucleotide residue C2063. A potential role for the observed conformational transition in the A2058-C2063 segment for regulating ribosome activity is discussed.

Mode selection in InAs quantum dot microdisk lasers using focused ion beam technique.

Optically pumped InAs quantum dot microdisk lasers with grooves etched on their surface by a focused ion beam are studied. It is shown that the radial grooves, depending on their length, suppress the lasing of specific radial modes of the microdisk. Total suppression of all radial modes, except for the fundamental radial one, is also demonstrated. The comparison of laser spectra measured at 78 K before and after ion beam etching for a microdisk of 8 µm in diameter shows a sixfold increase of mode spacing, from 2.5 to 15.5 nm, without a significant decrease of the dominant mode quality factor. Numerical simulations are in good agreement with experimental results.

MR imaging of myeloperoxidase activity in a model of the inflamed aneurysm wall.

BACKGROUND AND PURPOSE: Although myeloperoxidase activity in vivo can be visualized by using noninvasive imaging, successful clinical translation requires further optimization of the imaging approach. We report a motion-sensitized driven-equilibrium MR imaging approach for the detection of a myeloperoxidase activity-specific gadolinium-containing imaging agent in experimental aneurysm models, which compensates for irregular blood flow, enabling vascular wall imaging in the aneurysm. MATERIALS AND METHODS: A phantom was built from rotational angiography of a rabbit elastase aneurysm model and was connected to a cardiac pulse duplicator mimicking rabbit-specific flow conditions. A T1-weighted turbo spin-echo-based motion-sensitized driven-equilibrium pulse sequence was optimized in vitro, including the addition of fat suppression and the selection of the velocity-encoding gradient parameter. The optimized sequence was applied in vivo to rabbit aneurysm models with and without inflammation in the aneurysmal wall. Under each condition, the aneurysms were imaged before and after intravenous administration of the imaging agent. The signal-to-noise ratio of each MR imaging section through the aneurysm was calculated. RESULTS: The motion-sensitized driven-equilibrium sequence was optimized to reduce flow signal, enabling detection of the myeloperoxidase imaging agent in the phantom. The optimized imaging protocol in the rabbit model of saccular aneurysms revealed a significant increase in the change of SNR from pre- to post-contrast MR imaging in the inflamed aneurysms compared with naïve aneurysms and the adjacent carotid artery (P < .0001). CONCLUSIONS: A diagnostic MR imaging protocol was optimized for molecular imaging of a myeloperoxidase-specific molecular imaging agent in an animal model of inflamed brain aneurysms.

What do we know about ribosomal RNA methylation in Escherichia coli?

A ribosome is a ribonucleoprotein that performs the synthesis of proteins. Ribosomal RNA of all organisms includes a number of modified nucleotides, such as base or ribose methylated and pseudouridines. Methylated nucleotides are highly conserved in bacteria and some even universally. In this review we discuss available data on a set of modification sites in the most studied bacteria, Escherichia coli. While most rRNA modification enzymes are known for this organism, the function of the modified nucleotides is rarely identified.