Analysis of the Morphology of Monocytes and Lymphocytes from COVID-19 Patients Using Low-Voltage Scanning Electronic Microscopy.

Severe course of COVID-19 is largely determined by hyperactivation of the immune system, or cytokine storm, in which immune cells (lymphocytes, monocytes, etc.) play a major role. Using low-voltage scanning electron microscopy, we studied the morphology of lymphocytes and monocytes during cytokine storm. Monocytes and lymphocytes were isolated by fluorescence sorting from the blood of healthy volunteers (n=6) and patients with COVID-19 (n=5) during cytokine storm (IL-6>23 ng/ml, smear positive for SARS-CoV-2). For each patient, 11-32 individual cells were analyzed at magnification of 18-32,000 times. Measurements showed that monocyte size was increased during cytokine storm (p=0.0001).

[The Efficiency of Immunoprecipitation of microRNA/Ago2 Complexes from Human Blood Plasma Is Protocol Dependent].

The study of extracellular miRNA is one of the most dynamic areas of modern biomedical research. Oftentimes, there is a need to isolate miRNAs associated with a particular carrier, for example, a ribonucleoprotein complex. The most thoroughly investigated protein component of these complexes is Ago2. Complexes are commonly isolated by immunoprecipitation with specific antibodies. Here we compare three methods for immunoprecipitating Ago2/microRNA complexes from blood plasma. In the first protocol, anti-Ago2-antibodies are added to the plasma following protein A-sepharose. In second protocol, anti-Ago2-antibodies are bound to sepharose from the very beginning, and then mixed with plasma. The third protocol differs from the second in that sepharose is blocked by non-specific antibodies at the final stage. To compare the efficiency of these protocols, the levels of miR-16-5p, miR-21-5p, and miR-144-3p were analyzed after precipitation with anti-Ago2 antibodies and control antibodies. For miR-16-5p all protocols were efficient, for miR-21-5p only the second technique yielded results, while for miR-144-3p none of the protocols resulted in extraction. Thus, we conclude that different protocols for immunoprecipitation of microRNA/Ago2 complexes favor different miRNAs.

EXTRACELLULAR VESICLES FROM BLOOD PLASMA STUDIED BY LOW VOLTAGE SCANNING ELECTRON MICROSCOPY.

Extracellular vesicles are subspherical membranous structures secreted by cells and enriched with different types of biological molecules. The number and the molecular content of these structures depend on pathological conditions and the physiological state of the organism. Extracellular vesicles play an important role in intercellular communication and represent potential disease biomarkers. However, mechanisms of formation, functions and morphological characteristics of extracellular vesicles are still studied insufficiently. Low voltage scanning electron microscopy is a promising method to investigate extracellular vesicles, since it does not require conductive coating and therefore enables a high-resolution visualization of morphological details of nanosized objects. This paper presents the results of low voltage scanning electron microscopy study of morphology and size of objects from blood plasma fractions.

[Ubiquitination of EGF receptors with C-terminal domain deletion and point mutations during endocytosis].

Analysis of ubiquitination of EGF receptor carrying different mutations of C-terminal domain was done. The mutants differed both by the set of major autophosphorylation sites that determine the way of interaction with ubiquitin-ligase c-Cbl, and by the presence of lysine residues which can be possible acceptor sites for ubiquitin. It was found that the receptor lacking tyrosine kinase activity due to lysine for phenylalanine substitution at ATP-binding site of tyrosine kinase (TK) domain (K721) failed to be ubiquitinated as well as the receptor without all binding sites for c-Cbl (CD165), while dynamics and pattern of ubiquitination of other deletion mutants was significantly different. The mutant lacking Grb2 binding sites but able to bind c-Cbl directly (CD123) was minimally ubiquitinated and only at early stages upon EGF endocytosis stimulation. At the same time, the receptor possessing all binding sites for Cbl but lacking C-terminal domain of 63 aminoacid residues (CD63) which contains two autophosphorylation sites (Y1148 and 1173) and 4 lysines, was less ubiquitinated and had more low-ubiquitinated forms comparing to the WT one. However, these lysines are not acceptor sites for ubiquitin since the full-size receptor lacking like CD63 the same major autophosphorylation sites underwent ubiquitination similar to the deletion mutant. Thus, C-terminal region of the EGF receptor, being not a substrate for ubiquitination per se, is involved in its regulation. It was also found that ubiquitination pattern at fast endocytosis differed from those at slow one. In the first case the total level of EGFR decreased dramatically as a result of efficient lysosomal degradation. The level of receptor-associated c-Cbl was practically the same, while the total intracellular c-Cbl dropped. Treatment of cells with proteasomal inhibition MG132 blocked the loss of Cbl only partially. In the second case, total amount of both EGF receptor and c-Cbl did not notably change that suggested recycling pathway for receptors even despite them beeng ubiquitinated.

[Analysis of tyrphostin AG1478 effect on behavior of internalized EGF receptor at different stages of endocytosis].

In the present work the effect of specific inhibitor of the receptor tyrosine kinase, tyrphostin AG1478, has been analyzed for behavior of internalized EGF receptor at different stages upon stimulation of endocytosis. It was found that tyrphostin addition in 30 min after endocytosis stimulation resulted in recycling of a significant portion of 125I-EGF onto cell surface. This portion was decreasing with time. EGF-receptor complexes, being recycled under action of AG1478, however, did not dissociate possibly because of tyrphostin ability to initiate receptor oligomerization in the absence of the ligand which can possibly affect dissociation constants. It was found that only a portion of EGF receptor localized in early endosomes was able to recycle upon TK inhibition. Addition of the inhibitor in 30 and 60 min after endocytosis stimulation resulted in decrease of labeled EGF degradation. At early stages internalized EGF-receptor complexes was blocked mostly in early endosomes, while at late stages their accumulation occured in incompletely matured late endosomes. These data speak in favor of late endocytic stage existence transition through which depends on the receptor TK. Besides, tyrphostin addition in 90 min after endocytosis led not to decrease, but on the contrary, to increase in degradation. That speaks about theexistence of the mechanisms providing a time window during which receptor TK can carry out the functions which are not connected directly with endocytosis.