The use of triplet-sensitized photochromism phenomenon for the study of dynamic interaction of membrane proteins

Possibility of registration of protein interactions in the membranes was demonstrated. The membrane preparation of Na+, K+ ATPase was used in the investigations. The Na+, K+ ATPase was bound with 4-acetoamido-4'-isothiocyanatostilbene-2,2' disullfonic acid (SITS) and erythrosinisothiocyanate (ERITC). The label/Na+,K+ATPase (M/M) ratio was equal to 1:1 for SITS and changed from 1:1 to 1:5 for ERITC. The cis-trans isomerization of SITS was initiated by triplet-triplet energy transfer from light excited ERITC to SITS. The kinetics of isomerization was recorded by the SITS fluorescence measurements. The rate constant of triplet-triplet energy transfer (kT) from ERITC to cis isomer of SITS, (3 divided by 7) X 10(3) M-1 s-1 was determined at 25 degrees C. The kT value of the energy transfer between loose molecules of erythrosine and SITS in buffer solution equaled to 7 X 10(7) M-1 s-1. This drop of kT in the membrane at 10(4) reflected the decrease in the frequency of label collisions caused by the increase in the media viscosity and steric hindrances.     

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.     

Experimental study of the dynamics of neutron emission from the GOL-3 multimirror trap

In experiments on the plasma heating and confinement in the GOL-3 multimirror trap, a deuterium plasma with a density of ～10¹⁵ cm^?3 and an ion temperature of 1–2 keV is confined for more than 1 ms. The plasma is heated by a relativistic electron beam. The ion temperature, which was measured by independent methods, reached 1.5–2 keV after the beginning of the beam injection. Since such a fast ion heating cannot be explained by the classical energy transfer from electrons to ions through binary collisions, a theoretical model of collective energy transfer was proposed. In order to verify this model, a new diagnostics was designed to study the dynamics of neutron emission from an individual mirror cell of the multimirror trap during electron beam injection. Intense neutron bursts predicted by this model were detected experimentally. Periodic neutron flux modulation caused by the macroscopic plasma flow along the solenoid was observed. The revealed mechanism of fast ion heating can be used to achieve fusion temperatures in the multimirror trap.     

Diagnostic system for studying generation of subterahertz radiation during beam-plasma interaction in the GOL-3 facility

The design principles and construction of the subterahertz radiometric spectral systems developed for the GOL-3 facility are described. The spectral systems are designed according to the quasi-optical scheme and use multilayer filters based on frequency-selective surfaces. The design and manufacturing technology of such elements are discussed. The results of measuring subterahertz radiation of plasma at the frequency close to the double plasma frequency are presented.     

Creation of a long magnetized plasma column in a metal chamber

A method for creation of a long magnetized column of dense hydrogen plasma in a metal chamber by means of a high-current linear discharge is considered. It is the main method for the formation of preliminary plasma in the GOL-3 multimirror trap, in which a plasma column with a length of up to 12 m and diameter of 8 cm, suitable for conducting experiments on the injection of a relativistic electron beam, was obtained. Conditions for stable discharge operation in the density range of 3 × 10¹⁹–10²² m^?3 are determined, including a discharge with a uniform longitudinal plasma density profile and incomplete initial ionization of hydrogen. It is demonstrated that the system is capable of operating in a magnetic field with a variable configuration and strength of up to 6 T in the solenoidal section and up to 12 T in the end mirrors. It is shown that an important role in the development of a discharge is played by fast electrons with energies corresponding to the initial applied voltage (about 25 kV), which provide primary gas ionization. The properties of low-temperature plasma in such a discharge are discussed.     

Photochemical amplification for horseradish peroxidase-mediated immunosorbent assay.

A new method for lowering the detection limit for a horseradish peroxidase (HRP) label in an enzyme-linked immunosorbent assay (ELISA) is proposed. The method is based on the use of a photochemical reaction of o-phenylenediamine (o-PD) autosensitized oxidation as an enhancement step in ELISA. The assay consists of two successive steps. The first step is a conventional HRP-mediated ELISA, using high-purity o-PD as a substrate. At this step, an o-PD oxidation product, 2,3-diaminophenazine (DAP), is formed in the dark. At the second step, the sample is illuminated at 400-500 nm for several minutes. Under illumination the concentration of DAP is greatly increased, depending on the duration and intensity of irradiation. Providing that the irradiation conditions are standardized, the final DAP concentration is proportional to the concentration of DAP formed by HRP. An ELISA for human carcinoembryonic antigen has demonstrated that the photochemical amplification method allows the detection limit of an assayed antigen to be lowered and the consumption of antibodies to be reduced. At the second step of this assay, the DAP concentration has been increased 50-fold under 4 min of irradiation.     

Differential rotation of plasma in the GOL-3 multiple-mirror trap during injection of a relativistic electron beam

Results of spectral and magnetic diagnostics of plasma differential rotation in the GOL-3 multiplemirror trap are presented. It is shown that the maximum frequency of plasma rotation about the longitudinal axis reaches 0.5 MHz during the injection of a relativistic electron beam into the plasma. The data of two diagnostics agree if there is a region with a higher rotation frequency near the boundary of the electron beam. Plasma differential rotation can be an additional factor stabilizing interchange modes in the GOL-3 facility.