A wealth of genome and transcriptome data obtained using new generation sequencing (NGS) technologies for whole organisms could not answer many questions in oncology, immunology, physiology, neurobiology, zoology and other fields of science and medicine. Since the cell is the basis for the living of all unicellular and multicellular organisms, it is necessary to study the biological processes at its level. This understanding gave impetus to the development of a new direction – the creation of technologies that allow working with individual cells (single-cell technology). The rapid development of not only instruments, but also various advanced protocols for working with single cells is due to the relevance of these studies in many fields of science and medicine. Studying the features of various stages of ontogenesis, identifying patterns of cell differentiation and subsequent tissue development, conducting genomic and transcriptome analyses in various areas of medicine (especially in demand in immunology and oncology), identifying cell types and states, patterns of biochemical and physiological processes using single cell technologies, allows the comprehensive research to be conducted at a new level. The first RNA-sequencing technologies of individual cell transcriptomes (scRNA-seq) captured no more than one hundred cells at a time, which was insufficient due to the detection of high cell heterogeneity, existence of the minor cell types (which were not detected by morphology) and complex regulatory pathways. The unique techniques for isolating, capturing and sequencing transcripts of tens of thousands of cells at a time are evolving now. However, new technologies have certain differences both at the sample preparation stage and during the bioinformatics analysis. In the paper we consider the most effective methods of multiple parallel scRNA-seq using the example of 10XGenomics, as well as the specifics of such an experiment, further bioinformatics analysis of the data, future outlook and applications of new high-performance technologies.
The effect of extremely low frequency electric and magnetic fields (ELF-EMF) on plants and animals including humans is quite a contentious issue. Little is known about ELF-EMF effect on hydrobionts, too. We studied the effect of rectangular voltage pulses of various amplitude, duration, and duty cycle, passed through seawater, on Trichoplax organisms as a possible test laboratory model. Three Placozoa strains, such as Trichoplax adhaerens (H1), Trichoplax sp. (H2), and Hoilungia hongkongensis (H13), were used in experiments. They were picked at the stationary growth phase. Arduino Uno electronics platform was used to generate a sequence of rectangular pulses of given duration and duty cycle with a frequency up to 2 kHz. Average voltage up to 500 mV was regulated by voltage divider circuit. Amlodipine, an inhibitor of calcium channel activity, was used to check the specificity of electrical pulse effect on voltage-dependent calcium channels in Trichoplax. Experimental animals were investigated under a stereo microscope and stimulated by current-carrying electrodes placed close to a Trichoplax body. Variations in behavior and morphological characteristics of Trichoplax body were studied. Stimulating and suppressing effects were identified. Experimental observations were recorded using photo and video techniques. Motion trajectories of individuals were tracked. Increasing voltage pulses with fixed frequency of 20 Hz caused H2 haplotype animals to leave “electrode zone” within several minutes at a voltage of 25 mV. They lost mobility in proportion to voltage increase and were immobilized at a voltage of 500 mV. Therefore, a voltage of 50 mV was used in further experiments. In experiments with two electrodes located on one side instead of both sides of Trichoplax, an animal had more chance to move in various directions. Direction of motion was used as a characteristic feature. Trichoplax were observed to migrate to areas with low density of electric field lines, which are far from electrodes or behind them. Animals from old culture were less sensitive to electrical stimulus. H2 strain was the most reactive one; it demonstrated stronger physiological responses at frequencies of 2 Hz and 2 kHz with a voltage of 50 mV than H1 strain and especially than H13 strain. Motion patterns and animal morphology depended on the duration of rectangular stimulation pulses, their number, amplitude, and frequency. Effects observed varied over a wide range: from direct or stochastic migration of animals to the anode or the cathode or away from it to their immobility, an increase of optical density around and in the middle of Trichoplax plate, and finally to Trichoplax folding and detach from the substrate. Additional experiments on Trichoplax sp. H2 with pulse duration of 35 ms and pulse delay of 1 ms to 10 s showed that the fraction of immobilized animals increased up to 80 % with minimum delay. Nevertheless, in the presence of amlodipine with a concentration of 25 nM, almost all Trichoplax remained fast-moving for several minutes despite exposure to voltage pulses. When using amlodipine with a concentration of 250 nM, experimental animals showed a total discoordination of motion and could not leave an “electrode trap”. Further, Trichoplax plate became rigid, which was expressed in animal shape invariability during motion. Finally, amlodipine with a concentration of 50 μM caused a rapid folding of animal plate-like body into a pan in the ventral-dorsal direction and subsequent dissociation of Trichoplax plate into individual cells. In general, the electrical exposure applied demonstrated a cumulative but a reversible physiological effect, which, as expected, is associated with activity of voltage-dependent calcium channels. Amlodipine at high concentration (50 μM) caused Trichoplax disintegration; at moderate concentration (250 nM), it disrupted the propagation of activation waves that led to discoordination of animal motion; at low concentration (25 nM), it prevented an electric shock.
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