Multimodal nonlinear-optical imaging of nucleoli.

Multimodal nonlinear microscopy combining third-harmonic generation (THG) with two- and three-photon-excited fluorescence (2PEF and 3PEF) is shown to provide a powerful resource for high-fidelity imaging of nucleoli and nucleolar proteins. We demonstrate that, with a suitably tailored genetically encoded fluorescent stain, the 2PEF/3PEF readout from specific nucleolar proteins can be reliably detected against the extranucleolar 2PEF/3PEF signal, enabling high-contrast imaging of the key nucleolar ribosome biogenesis components, such as fibrillarin. THG is shown to provide a versatile readout for unstained nucleolus imaging in a vast class of biological systems as different as neurons in brain slices and cultured HeLa cells.

Cell-specific three-photon-fluorescence brain imaging: neurons, astrocytes, and gliovascular interfaces.

We present brain imaging experiments on rat cortical areas, demonstrating that, when combined with a suitable high-brightness, cell-specific genetically encoded fluorescent marker, three-photon-excited fluorescence (3PEF), enables subcellular-resolution, cell-specific 3D brain imaging that is fully compatible and readily integrable with other nonlinear-optical imaging modalities, including two-photon-fluorescence and harmonic-generation microscopy. With laser excitation provided by sub-100-fs, 1.25-µm laser pulses, cell-specific 3PEF from astrocytes and their processes detected in parallel with a three-photon-resonance-enhanced third harmonic from blood vessels is shown to enable a high-contrast 3D imaging of gliovascular interfaces.

Stain-free subcellular-resolution astrocyte imaging using third-harmonic generation.

We demonstrate stain-free, high-contrast, subcellular-resolution imaging of astroglial cells using epi-detected third-harmonic generation (THG). The astrocyte-imaging capability of THG is verified by colocalizing THG images with fluorescence images of astrocytes expressing a genetically encodable fluorescent reporter. We show that THG imaging with an optimized point-spread function can reliably detect significant subcellular features of astrocytes, including cell nuclei, as well as the soma shape and boundaries.

SypHer3s: a genetically encoded fluorescent ratiometric probe with enhanced brightness and an improved dynamic range.

We designed a genetically encoded ratiometric fluorescent probe, SypHer3s, with enhanced brightness and optimized pKa, which responds to pH changes in different cellular compartments. SypHer3s was successfully utilized for imaging the pH dynamics in mitochondria of living neurons and in quantitative pH measurement in zebrafish embryos.

A highly conductive electrolyte for molten oxide fuel cells.

A gas-tight and ductile solid/liquid δ-Bi2O3-0.2 wt% B2O3 electrolyte for molten oxide fuel cells (MOFCs) is developed. The MOFCs are a new class of intermediate temperature fuel cells. The composite, consisting of solid (δ-Bi2O3) and liquid (molten Bi2O3 + B2O3) oxygen ion-conducting phases, demonstrates the promising application as an MOFC electrolyte with the highest oxygen ionic conductivity.

Fiber-optic electron-spin-resonance thermometry of single laser-activated neurons.

Optically detected electron spin resonance in fiber-coupled nitrogen-vacancy (NV) centers of diamond is used to demonstrate a fiber-optic quantum thermometry of individual thermogenetically activated neurons. Laser-induced temperature variations read out from single neurons with the NV-diamond fiber sensor are shown to strongly correlate with the fluorescence of calcium-ion sensors, serving as online indicators of the inward Ca2+ current across the cell membrane of neurons expressing transient receptor potential (TRP) cation channels. Local laser heating above the TRP-channel activation threshold is shown to reproducibly evoke robust action potentials, visualized by calcium-ion-sensor-aided fluorescence imaging and detected as prominent characteristic waveforms in the time-resolved response of fluorescence Ca2+ sensors.

[Main Cellular Redox Couples].

Most of the living cells maintain the continuous flow of electrons, which provides them by energy. Many of the compounds are presented in a cell at the same time in the oxidized and reduced states, forming the active redox couples. Some of the redox couples, such as NAD+/NADH, NADP+/NADPH, oxidized/reduced glutathione (GSSG/GSH), are universal, as they participate in adjusting of many cellular reactions. Ratios of the oxidized and reduced forms of these compounds are important cellular redox parameters. Modern research approaches allow setting the new functions of the main redox couples in the complex organization of cellular processes. The following information is about the main cellular redox couples and their participation in various biological processes.

Fiber-optic control and thermometry of single-cell thermosensation logic.

Thermal activation of transient receptor potential (TRP) cation channels is one of the most striking examples of temperature-controlled processes in cell biology. As the evidence indicating the fundamental role of such processes in thermosensation builds at a fast pace, adequately accurate tools that would allow heat receptor logic behind thermosensation to be examined on a single-cell level are in great demand. Here, we demonstrate a specifically designed fiber-optic probe that enables thermal activation with simultaneous online thermometry of individual cells expressing genetically encoded TRP channels. This probe integrates a fiber-optic tract for the delivery of laser light with a two-wire microwave transmission line. A diamond microcrystal fixed on the fiber tip is heated by laser radiation transmitted through the fiber, providing a local heating of a cell culture, enabling a well-controlled TRP-assisted thermal activation of cells. Online local temperature measurements are performed by using the temperature-dependent frequency shift of optically detected magnetic resonance, induced by coupling the microwave field, delivered by the microwave transmission line, to nitrogen--vacancy centers in the diamond microcrystal. Activation of TRP channels is verified by using genetically encoded fluorescence indicators, visualizing an increase in the calcium flow through activated TRP channels.

[Genetically Encoded Fluorescent Redox Sensors].

Redox processes play a key role in cells of all.organisms. These processes imply directed flows of electrons via so-called redox pairs: substances that exist in both reduced and oxidized states simultaneously within the cell. Examples of redox pairs are NAD+/NADH, NADP+/NADPH, GSSG/2GSH. Until recently, studies of redox processes in the living cells were challenged by the lack of suitable methods. Genetically encoded fluorescent biosensors provide a new way to study biological processes including redox ones. Biosensors allow real-time detection of messengers, metabolites and enzymatic activities in living systems of different complexity from cultured cells to transgenic animals. In this review, we describe the main types of known redox biosensors with examples of their use.

[Compartmentalization of ROS-mediated signal transduction].

The localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion ofthe same components. ROS-generating complexes have been suggested to follow this principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model ofredox signaling compartmentalization. Nevertheless, evidences of local ROS production and restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH-oxidase isoforms, their adapter proteins and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant system which prevents uncontrolled ROS diffusion from the sites of generation to the adjacent subcellular compartments; the current data of the specific localization NADPH-oxidases activity and its influence on intracellular processes; the recent evidences of the ROS diffusion restriction.

[Early invasive and non-invasive approach to treatment of acute non-ST-segment elevation coronary syndrome].

AIM: The aim of the study is to assess the efficiency of early invasive treatment versus conservative treatment for patients with non-ST acute coronary syndrome (ACS), and to study immediate results of percutaneous coronary intervention (PCI) in terms of the risk of adverse events. MATERIAL AND METHODS: The investigation includes 112 patients with non-ST ACS having risk of adverse outcome determined by the GRACE system. More than half of the patients (77/68.8%) underwent diagnostic coronary angiography within 72 hours of admission, and the decision was made on a subsequent treatment strategy (early invasive or conservative approach). In-hospital outcomes (cardiac deaths, nonfatal MI, reMI), cumulative frequency of these events have been studied according to the risk assessment scale GRACE and treatment strategy. RESULTS: Low risk was determined in 33.9% of patients, intermediate--in 32.1%, and high--in 33.9%. On the whole, frequency of myocardial revascularization was assessed as 56.3%. Fifty-one (45.5%) patients received early invasive treatment, 12 (10.7%)--coronary artery bypass grafting (CABG). A conservative approach was used in 49 cases (43.8%). An early invasive treatment was given to 20 patients (52.6%) from each of the low- and high-risk groups, 11 (30.6%)--from the intermediate risk group. In-hospital cardiac deaths were observed only in the high-risk group, and this number was higher in case of conservative treatment versus early invasive treatment: 3 (30%) versus 1 (50%). Non-fatal MIs in the form of re(MI) were also diagnosed only in the high-risk group following conservative treatment: 2 (20.0%). Nevertheless, no reliable difference in the frequency of each complication was found (p = 0.095; p = 0.1). Significant differences in the high-risk group were proven while comparing the impact of early invasive and conservative approaches on the cumulative frequency of in-hospital death and (re)MI cases (50.0% versus 5.0%; p = 0.009). No benefits of an early invasive approach were identified for low-risk and intermediate risk groups. CONCLUSION: Early invasive approach compared with a conservative approach in high risk patients with non-ST acute coronary syndrome can improve clinical outcomes with a decrease in the total frequency of in-hospital death, reMI.

[Signaling function of phagocytic NADPH oxidase: activation of MAP kinase cascades in phagocytosis].

Until recently, the production of reactive oxygen species by NADPH oxidase has been considered only in the context of the oxidative damage to pathogens inside the phagosome. However, homologues of phagocytic NADPH oxidase have been found in almost all cell types, where they produce hydrogen peroxide and thereby regulate the initial intracellular stages of MAP kinase cascades. In the present work, the activation of two MAP kinase cascades, p38 and Erk1/2, during phagocytosis has been studied. It was found that phagocytosis activates both cascades. The activation of Erkl/2 is dependent, and the activation of p38 is not dependent, on the activity of NADPH oxidase. Thus, it can be stated that the activation of MAP kinases in phagocytes during phagocytosis occurs by a mechanism similar to that operating in nonphagocytizing cells, indicating the universality of the function of NADPH oxidases in different cell types.

[NADPH oxidase controls EGF-induced proliferation via the ERK1/2-independent mechanism].

Using HyPer, a ratiometric GFP-based biosensor, the dynamics of H2O2 in living cells has been studied. It was found that activation of the receptor of the epidermal growth factor (EGF) in epithelial cells leads to sustained generation of intracellular H2O2, which is blocked by apocynin, an inhibitor of the assembly of plasma membrane NADPH oxidase. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase should be involved in the process. However, apocynin failed to alter the kinetics of the EGF-stimulated ERK1/2 activation. It was concluded that NADPH oxidase and intracellular H2O2 are the important downstream targets of the EGF receptor, which mediate the proliferation response by mechanisms distinct from the activation of the classical ERK1/2 MAP-kinase pathway.

[Blood lipid spectrum in hypertensive patients and normotensive subjects living in the Khanty-Mansiysk region].

AIM: To evaluate basic parameters of lipid metabolism in patients with arterial hypertension (AH) stage I and II and normotensive subjects living in the far North of the Russian Federation (Khanty-Mansiysk Autonomic Region, Nyagan). MATERIAL AND METHODS: Lipid spectrum was studied in 70 males and females with AH stage I and II, 280 normotensive males and females matched for sex, age, time of residence in the North. RESULTS: Sex-related differences by concentrations of blood lipids become less pronounced with progression of AH while a concentration of atherogenic lipid fractions in AH patients rises. High prevalence of dyslipidemia, primarily hypercholesterolemia, in hypertensive and normotensive subjects is a prognostic factor of coronary atherosclerosis. Paired correlations indirectly confirm the effect of climatic factors on blood lipids. CONCLUSION: It is necessary to correct lipid spectrum and risk factors in hypertensive and normotensive subjects as a prophylactic measure.

[Study of the mechanism of Ras-dva small GTPase intracellular localization].

An analysis of amino acid sequences of small GTPases of the Ras-dva family allowed us to determine the C-terminal prenylation motif, which could be responsible for the membrane localization of these proteins. We demonstrated using in vivo EGFP tracing that the Ras-dva small GTPases from Xenopus laevis embryo cells and NIH-3T3 fibroblasts are localized on both plasma membranes and endomembranes (the endoplasmic reticulum, the Golgi apparatus, and vesicles). At the same time, the replacement of the Cys residue, the SH group of which must be theoretically farnesylated, in the C-terminal prenylation motif of the Ras-dva small GTPase by the Ser residue prevented the membrane localization of the protein. These results indicate that the C-terminal prenylation site is critical for the membrane localization of small Ras-dva GTPases.

Reactive oxygen and nitrogen species: friends or foes?

Chemical and physiological functions of molecular oxygen and reactive oxygen species (ROS) and existing equilibrium between pools of pro-oxidants and anti-oxidants providing steady state ROS level vital for normal mitochondrial and cell functioning are reviewed. The presence of intracellular oxygen and ROS sensors is postulated and few candidates for this role are suggested. Possible involvement of ROS in the process of fragmentation of mitochondrial reticulum made of long mitochondrial filaments serving in the cell as "electric cables", as well as the role of ROS in apoptosis and programmed mitochondrial destruction (mitoptosis) are reviewed. The critical role of ROS in destructive processes under ischemia/reoxygenation and ischemic preconditioning is discussed. Mitochondrial permeability transition gets special consideration as a possible component of the apoptotic cascade, resulting in excessive "ROS-induced ROS release".

Proteinaceous complexes from mitochondrial contact sites.

A Triton X-100 extract from rat brain mitochondria was obtained using low detergent/protein ratio. From this extract a proteinaceous complex was purified; its molecular weight was as high as 880 kD. The complex contained both hexokinase and creatine kinase activity. When incorporated into phospholipid bilayer membranes, the complex formed a channel whose activity was different than the channel activity of purified porin isolated either by adsorption chromatography or by dissociation from protein complexes. A ligand of the mitochondrial benzodiazepine receptor (Ro5-4864) in submicromolar concentrations had an apparent influence on the kinetic behavior of enzymatic coupling of hexokinase and creatine kinase. It is suggested that the 880-kD complex is formed by mitochondrial contact sites. The role of the isolated protein complex in the formation of nonspecific permeability in mitochondria is discussed.

[Acute eye damage from ultraviolet radiation]. / Ostroe porazhenie glaz ul'trafioletovoi radiatsiei.

The eyes of rabbits were UV-irradiated in a range close to the natural spectrum at a dose of 23.2 kJ/m2 (biologically effective dose 1.3 kJ/m2). The condition of optic media was estimated, as well as the parameters of electroretinogram (ERG) induced by single light stimulus of varying intensity and paired light stimuli with different intervals between them. As a result a distinct photokeratoconjunctivitis developed, the amplitude of the ERG a-wave decreased by 30-50% and that of b-wave by 35%, while the latencies of the both waves and the critical frequency of flashes remained unchanged. The cornea transparency did not decrease markedly even during the period of maximum photokeratoconjunctivitis and the changes in the ERG parameters were mostly due to the retina damage by the indirect effect of UV irradiation.

[The electroretinogram parameters and the state of the cornea in the rabbit following acute exposure of the eye to UV-radiation of different intensities]. / Parametry élektroretinogrammy i sostoianie rogovitsy glaza krolika posle ostrogo vozdeistviia na glaz UF-izlucheniia razlichnoi intensivnosti.

The purpose of this study was to investigate wave amplitudes and latencies of electroretinograms (ERG) and to examine the eye cornea of rabbits a month after their eye UV-B-irradiation with a biologically effective dose of 1.3 kJ/m2 at three intensities of 1.62, 0.567 or 0.157 W/m2. It was demonstrated that ERG a- and b-waves decreased significantly 7-20 days after irradiation while latencies remained unchanged. It was also found that during this time period ERG wave amplitudes were dependent on the intensity: at higher intensities amplitudes of both waves diminished to a greater extent. After irradiation with a peak intensity photokeratoconjunctivitis was more severe and prolonged; however, the effect of irradiation with two other intensities was very similar. In view of the fact that the highest intensity of UV-B-radiation was comparable with that beyond the Earth's atmosphere while the lowest intensity was close to that near the Earth's surface, the above findings can be used to solve space biology problems and to assess potential effects of the ozone layer destruction on the eye.