BODIPY Dye Derivative for Irreversible Fluorescent Labeling of Eukaryotic Cells and Their Simultaneous Cytometric Analysis.

In this work, we synthesized a green fluorescent dye derivative, 1,3,5,7-tetramethyl-BODIPY, with a heptyl substituent at the 8-position. The obtained highly hydrophobic compound was able to rapidly and irreversibly bind to eukaryotic cells. Incubation of cells with the dye over different periods of time or at different concentrations allowed us to control the degree of cell labeling and the level of fluorescence. This made it possible to modulate the fluorescence level of different eukaryotic cell cultures and then distinguish them by their level of fluorescence signal in the green channel in cytometric experiments. The labeled cells can be combined and further analyzed in the same test tube under identical conditions using the channels in which the dye does not fluoresce. This approach has been tested on a number of tumor cell cultures containing the HER2 receptor on their surface. The representation of the receptor in these cells was analyzed in one test tube in one run using a HER2-specific ligand based on the hybrid protein DARPin9_29-mCherry, which fluoresces in the red region of the spectrum.

The prevalence of Fabry disease among 1009 unrelated patients with hypertrophic cardiomyopathy: a Russian nationwide screening program using NGS technology.

BACKGROUND: There is a vast number of screening studies described in the literature from the beginning of the twenty-first century to the present day. Many of these studies are related to the estimation of Fabry disease (FD) morbidity among patients from high-risk groups, including adult patients with hypertrophic cardiomyopathy (HCM) and left ventricular hypertrophy (LVH). These studies show diverse detection frequencies (0-12%) depending on the methodology. Our study is the only example of large-scale selective FD screening based on the implementation of next-generation sequencing technology (NGS) as a first-level test to estimate FD morbidity in the Russian population over 18 years of age burdened with HCM. METHODS: The study included 1009 patients (578 males and 431 females), with a median age of 50 years, who were diagnosed with HCM according to current clinical guidelines. In the first stage of screening, all patients underwent molecular genetic testing (NGS method) of target regions. These regions included the coding sequences of 17 genes and mutations that can lead to the development of HCM. Lysosomal globotriaosylsphingosine (lyso-Gb3) concentrations and α-galactosidase A (α-gal A) enzyme activity were measured in the second stage of screening to reveal pathogenic or likely pathogenic variants in the GLA gene. RESULTS: We revealed 8 (0.8%) patients (3 (37.5%) males and 5 (62.5%) females) with an average age of 59 ± 13.3 years who had pathogenic, likely pathogenic variants and variants of uncertain significance (VUS) in the GLA gene (NM_000169.2) as a result of selective screening of 1009 Russian patients with HCM. FD was confirmed via biochemical tests in a male with the pathogenic variant c.902G > A, p.R301Q as well as in two females with likely pathogenic variants c.897C > A, p.D299E and c.1287_1288dup, p.*430Fext*?. These tests showed reduced enzymatic activity and increased substrate concentration. However, a female with the pathogenic variant c.416A > G, p.N139S and with normal enzymatic activity only had increased substrate concentrations. The revealed nucleotide variants and high values of biochemical indicators (lyso-Gb3) in these 4 patients allowed us to estimate the FD diagnosis among 1009 Russian patients with HCM. Mild extracardiac manifestations were observed in these four patients; however, both biochemical values within the reference range in females with the c.971T > G, p.L324W (VUS) variant. α-gal A activity and lyso-Gb3 concentrations were also within the normal range in two males with hemizygous variants, c.546T > C, p.D182D and c.640-794_640-791del (we regarded them as VUS), and in one female with the c.427G > A, p.A143T variant (with conflicting interpretations of pathogenicity). CONCLUSION: The prevalence rate of FD among 1,009 adult Russian patients with HCM was 0.4%. We recommend FD screening among adult patients of both sexes with HCM and an undefined genetic cause via NGS method with subsequent analysis of α-gal A activity and lyso-Gb3 concentration in patients with pathogenic, likely pathogenic variants, and VUS. This strategy identifies patients with an atypical form of FD that is characterized by high residual activity of α-gal A, low concentrations of lyso-Gb3, and minor extracardiac manifestations.

Modeling of surface-induced second-harmonic generation from multilayer structures by the transfer matrix method.

We analytically and numerically investigate surface second-harmonic generation (SHG) from a stack of dielectric layers. We develop a theoretical formalism based on the transfer matrix method for the calculation of the surface-driven second-harmonic radiation from multilayer structures and elaborate it for the case of ultrathin dielectric layers using a power series expansion to derive the effective surface nonlinear tensor for the whole stack. We show that for deeply subwavelength thicknesses of the layers the surface responses from all interfaces can efficiently sum up, leading to largely enhanced efficiency of SHG. As a result, such surface-driven nonlinearity can become comparable to the bulk nonlinearity in noncentrosymmetric semiconductors and can yield high performance for nonlinear nanophotonic applications.

Far-field polarization signatures of surface optical nonlinearity in noncentrosymmetric semiconductors.

We analyse possibilities to quantitatively evaluate the surface second-order optical nonlinearity in noncentrosymmetric materials based on polarization-resolved analysis of far-field radiation patterns of second-harmonic generation. We analytically demonstrate that for plane-wave illumination the contribution to the second-harmonic signal from the surface of a nonlinear medium exhibits different polarization properties and angular dependencies compared to the contribution from the bulk. In view of this, we optimize the illumination geometry in order to enable the most efficient separation and comparison of both nonlinearities. Furthermore, we consider the illumination of an AlGaAs slab by a tightly-focused linearly-polarized Gaussian beam as an alternative measurement geometry. It is found that the reliable separation of the surface nonlinearity contribution as well as a wide range of detectable values can be achieved with this geometry as well.

Repolarization of Ferroelectric Superlattices BaZrO3/BaTiO3.

With the use of the modified Sawyer-Tower scheme and Merz technique, studies were conducted on the repolarization characteristics of ferroelectric (BaZrO3/BaTiO3) superlattices on monocrystalline MgO substrate. Studies of temperature changes in the dielectric hysteresis loops indicated a sufficiently smooth decrease in spontaneous polarization compared with homogeneous barium titanate near the phase transition temperature of the superlattice. Experimental studies of switched currents have shown that the switching processes in the synthesized superlattices are implemented in two stages: activation motion ("creep" mode) and non-activation motion (slip mode). The presence of the activation switching stage and the numerical estimates show that with high probability, the movement of domain boundaries accomplishes the processes of switching in the studied superlattice. The threshold field separating the stated stages decreases with increasing temperature up to the Curie point of the superlattice, similar to the coercive field. Detection of the non-strictly exponential dependence of the switching current on the reverse field strength in the activation stage was modulated by the dependence with the power-law exponent for the applied electric field. Both techniques indicate that the studied superlattices have a small internal displacement field directed from the superlattice to the substrate.

Unusual terahertz waveforms from a resonant medium controlled by diffractive optical elements.

Up to now, full tunability of waveforms was possible only in electronics, up to radio-frequencies. Here we propose a new concept of producing few-cycle terahertz (THz) pulses with widely tunable waveforms. It is based on control of the phase delay between different parts of the THz wavefront using linear diffractive optical elements. Suitable subcycle THz wavefronts can be generated via coherent excitation of nonlinear low-frequency oscillators by few-cycle optical pulses. Using this approach it is possible to shape the electric field rather than the slow pulse envelope, obtaining, for instance, rectangular or triangular waveforms in the THz range. The method is upscalable to the optical range if the attosecond pump pulses are used.

Unipolar subcycle pulse-driven nonresonant excitation of quantum systems.

The interaction of subcycle pulses with quantum systems is considered when the pulse duration becomes much smaller than the timescales of electron oscillations. We show analytically that the interaction process in this case is governed by the electric pulse area. The efficient nonresonant excitation of quantum systems by subcycle pulses with a high degree of unipolarity is demonstrated. The results are confirmed by direct numerical solution of multilevel Bloch equations.

Population density gratings induced by few-cycle optical pulses in a resonant medium.

Creation, erasing and ultrafast control of population density gratings using few-cycle optical pulses coherently interacting with resonant medium is discussed. In contrast to the commonly used schemes, here the pulses do not need to overlap in the medium, interaction between the pulses is mediated by excitation of polarization waves. We investigate the details of the dynamics arising in such ultrashort pulse scheme and develop an analytical theory demonstrating the importance of the phase memory effects in the dynamics.

Generation of unipolar half-cycle pulses via unusual reflection of a single-cycle pulse from an optically thin metallic or dielectric layer.

We propose a strikingly simple method to form approximately unipolar half-cycle optical pulses via reflection of a single-cycle optical pulse from a thin flat metallic or dielectric layer. Unipolar pulses in reflection arise due to specifics of one-dimensional pulse propagation. Namely, we show that the field emitted by the layer is proportional to the velocity of the oscillating charges in the medium, instead of their acceleration. Besides, the oscillation velocity of the charges can be forced to keep a constant sign throughout the pulse duration. That is, reflection of ultrashort pulses from broad-area layers with nanometer-scale thickness can be very different from the common reflection in the case of longer pulses and thicker layers. This suggests a possibility of unusual transformations of few-cycle light pulses in completely linear optical systems.

Synthetic Fluorophores for Visualizing Biomolecules in Living Systems.

The last decade has witnessed significant advance in the imaging of living systems using fluorescent markers. This progress has been primarily associated with the discovery of different spectral variants of fluorescent proteins. However, the fluorescent protein technology has its own limitations and, in some cases, the use of low-molecular-weight fluorophores is preferable. In this review, we describe the arsenal of synthetic fluorescent tools that are currently in researchers' hands and span virtually the entire spectrum, from the UV to visible and, further, to the near-infrared region. An overview of recent advances in site-directed introduction of synthetic fluorophores into target cellular objects is provided. Application of these fluorescent probes to the solution of a wide range of biological problems, in particular, to the determination of local ion concentrations and pH in living systems, is discussed.

[The Development and Application of BODIPY-Labeled Oligonucleotides in Real-Time Polymerase Chain Reaction].

A BODIPY-based (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene, TMB) green fluorescent probe for quantitative real-time polymerase chain reaction (qPCR) was synthesized by azide-alkyne cycloaddition reaction. Comparative studies with analogous fluorescein-based probe were carryed out. We demonstrate that fluorescent probes with TMB fluorophore can be used in qPCR experiments as well.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

[Pulmonary Hypertension in Takayasu Arteritis].

Takayasu arteritis is well-known rare form of large vessel vasculitis. Pulmonary involvement is observed in 50% of cases. However, pulmonary hypertension is a rare complication of pulmonary artery stenosis. In this article we present a rare clinical case of Takayasus arteritis with typical stenosis of subclavian, carotid arteries, and involvement of pulmonary arteries with development of severe pulmonary hypertension. The article also contains discussion of current methods of diagnosis and treatment of pulmonary artery stenosis in Takayasus arteritis with special focus on effectiveness and complications of angioplasty and surgery.

[Intravital and postmortem morphological studies and magnetic resonance imaging of arrhythmogenic right ventricular dysplasia].

The authors conducted histological, immunohistochemical, and morphometric studies of 47 endomyocardial biopsies and 33 hearts with a high morphological criterion for the diagnosis of arrhythmogenic right ventricular dysplasia after F.I. Marcus et al. (2010), as well as intravital and postmortem cardiac magnetic resonance imaging (MRI). The average residual cardiomyocyte area was 39.7 +/- 12.8%, and the average cardiomyocyte diameter was 10.4 +/- 1.5 microm. Muscle fiber atrophy, lipomatosis, and fibrosis in the right ventricle were detected in all cases, myocarditis in 44%, mitral valve prolapse in 42%, and muscle fiber dyscomplexation in the right ventricular in 33%. The most informative signs for the diagnosis of arrhythmogenic right ventricular dysplasia using MRI are right ventricular contraction dyssynchrony and those using a morphological study are a residual cardiomyocyte area and muscle fiber atrophy in the right ventricle. A decrease in or focal absence of gamma-catenin expression in the muscle fibers was observed only in 60% of the patients.

[Age-dependent occupational stress in doctors of various specialties].

A comparative assessment of the extent and structure of the various forms of professional burnout among doctors-organizers as well as therapeutists and surgeons depending on age was given. It is shown that the professional burnout in doctors-organizers conjugates with a high level of emotional tension manifested in avoidance of over-saturated emotional and professional communication outside professional activities, increased irritability and temper, reluctance to exercise empathy towards colleagues and compassion for patients. The comparison of three age groups of physicians to each other in terms of the level and features of the burnout was adduced; the results demonstrate the significant differences between the age periods of 30 and 40 years and over 41 years old. Physicians in the age group of 30-40 years old are inclined to depersonalization at a relatively low level of reduction of professional achievement. It was found that age specificity of formation of the syndrome of professional burnout among surgeons was caused by the increased tendency to development of professional burnout syndrome of young and middle-aged surgeons at low levels of professional burnout among older people.

[3D-structure determination of fluorescent proteins by homology modeling combined with mass spectrometry].

A method for the 3D-structure generation of GFP-like fluorescent proteins is presented. The method is based on a combination of homology modeling for the overall spatial structure determination and mass spectrometry for the chromophore structure identification. The proposed approach can be applied to the spatial structure determination ofnoncrystalizable GFP homologs.

Development of a cryopreservation protocol for testicular interstitial cells with the account of temperature intervals for controlled cooling below -60°Ð¡.

A long course of anticancer therapy may lead to testicular steroidogenesis destruction. Cryopreservation of testicular interstitial cells (TIC) would be a strategy to protect hormonal and fertile potential of pre-pubertal boys treated with chemo - or radiotherapy. The aim of this research was to optimize protocols for freezing of TIC. Essential physical processes associated with the presence of dimethyl sulphoxide (Me(2)SO) in the cryoprotectant solution take place at the temperatures below -60°Ð¡. These processes are the eutectic crystallization at the stage of freezing and the recrystallization before the melting of the eutectic mixture at the stage of heating. Both of the processes affect the viability of the cells subjected to cryopreservation. Temperature intervals when these processes take place were determined by the method of thermoplastic deformation for 10% Me(2)SO selected for cryopreservation of TIC. Rat TIC were cryopreserved using five different protocols which varied in cooling rates within the chosen temperature intervals. Post-thaw cell viability and metabolic activity were evaluated by Trypan Blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining assays. Leydig cell recovery after cryopreservation was measured by 3-beta-hydroxysteroid dehydrogenase reaction. Based on the obtained results, the authors developed a cryopreservation protocol for TIC which makes it possible to achieve great cell viability due to using controlled cooling rates within the temperature intervals below -60°Ð¡.

[Posttranslational reactions resulting in a long-wavelength shift in the spectra of asFP595 protein from Anemonia sulcata].

In most fluorescent proteins characterized by light absorption and emission in the red and the far-red spectral region (550-650 nm), the chromophore pi system is extended at the expense of the additional oxidation of the GFP-like structure and the formation of an acylimine substituent. As distinct from these proteins, the photoactivateable protein asFP595 contains a chromophore with the keto group substituted for an acylimine substituent. In this work, we studied the reactions that result in a bathochromic shift in the spectrum of asFP595. Maturation kinetics analysis has shown the generation of the immature form containing a protonated chromophore (absorption at 420 nm) at the intermediate step, as in the case of other red fluorescent proteins, which then is isobestically converted into the final mature form (568 nm). Mass spectrometric analysis of the chromopeptide isolated from immature asFP595 has demonstrated that the intermediate form contains a GFP-type chromophore. It has also been found that the oxidation of the GFP chromophore is accompanied by the generation of an equimolar amount of hydro gen peroxide. The intermediate products of oxidation have been analyzed by the mutagenesis of the first chromophore-generating amino acid residue. It has been demonstrated that in the case of all mutants studied, chromophore synthesis does not terminate at the stage of the acylimine derivative, but immediately results in the fragmentation of the main chain of the protein and in the formation of the keto form.