Diamond-based HBAR as a high-pressure sensor.

Features of an application of a High-overtone Bulk Acoustic Resonator (HBAR) as a high-pressure sensor have been considered. In this way, the second version of an integrated measurement system combining a Diamond Anvil Cell (DAC) and an HBAR operating in the microwave frequency band from 1.3 to 3.7 GHz was developed. A specific configuration of HBAR based on a piezoelectric layered structure as "Al/ASN/Mo/(100) diamond" was proposed. Two independent methods of pressure control were used to calibrate the embedded HBAR as a pressure sensor: a stress-induced shift of the diamond Raman line and the shift of the R1 luminescence line of Cr3+ in the ruby matrix. A stable correlation between the frequency shifts of the acoustic overtones in the HBAR, the shift of the diamond Raman line and the shift of the R1 line with a change in pressure applied to the W-gasket with embedded ruby particles was established in the range of 0 … 30 GPa. The sensitivity of an investigated sensor to the pressure variation was found to be equal 1ΔPΔff=4.8∙10-4GPa-1. The maximal value of 30 GPa obtained in a given work can be easily increased after a minor reconfiguration of the DAC. Considering the range of 0 - 5 GPa a proposed built-in DAC acoustoelectronic sensor has the better performance and sensitivity compared with known methods of a pressure measurement.

HspBP1 in Complex with the Peptide of the Innate Immunity Protein Tag7 is Able to Lyse Tumor Cells Carrying TNFR1 Receptor.

The search for new cytotoxic agents capable of lysing tumor cells is an important task in the fight against cancer. Here we have shown that the HspBP1 protein, the chaperone of the heat shock protein Hsp70, is able to form a complex with the previously discovered peptide (17.1) of the innate immunity protein Tag7. Experiments using thermophoresis demonstrated that the affinity of the Tag7 protein peptide 17.1 to the HspBP1 molecule is 100 times higher than that of the full-sized Tag7 molecule. The addition of the 17.1-HspBP1 complex to tumor cells induces apoptosis and necroptosis in them. The results obtained in this work can be used to develop promising antitumor drugs.

Incubation of Lymphocytes with IL-2 Causes the Appearance of Antitumor T Cells Carrying FC, CD25, and LFA-1 on the Surface.

To carry out antitumor activity against cells that have lost surface antigens, human lymphocytes must have a certain repertoire of surface proteins capable of contacting a tumor cell and inducing programmed cell death in it. In this work, we showed that activation of healthy donor cells by IL-2 cytokine within 6 days causes the appearance of FasL, CD25, and LFA-1 proteins on CD8+CD25+ T lymphocytes, and also converts the LFA-1 protein into an active form having a high affinity for its target, ICAM-1 integrin. The appearance of these proteins on the surface of this subpopulation of lymphocytes allows them to induce programmed cell death in HLA-negative tumor cells.

Tag7-Mts1 Complex Activates Chemotaxis of Regulatory T Cells.

One of the basic features of immune system is the ability to sustain balance between activation and suppression of effector lymphocytes. In this process a key role belongs to the subpopulation of cells called regulatory T cells (Treg). Many cancer and autoimmune diseases are caused by malfunctions of Treg, and investigation of this subpopulation is important for development of new therapeutic approaches. In this study, we demonstrate that regulatory T cells can migrate along the concentration gradient of Tag7-Mts1 complex, and also they produce agents that induce blood cells migration.

A new mouse unilateral model of diffuse alveolar damage of the lung.

OBJECTIVE AND DESIGN: The existing biological models of diffuse alveolar damage (DAD) in mice have many shortcomings. To offset these shortcomings, we have proposed a simple, nonsurgical, and reproducible method of unilateral total damage of the left lung in ICR mice. This model is based on the intrabronchial administration of a mixture of bacterial lipopolysaccharide (LPS) from the cell wall of S. enterica and α-galactosylceramide (inducing substances) to the left lung. METHODS: Using computer tomography of the lungs with endobronchial administration of contrast material, we have been able to perform an operative intravital verification of the targeted delivery of the inducer. The model presented is characterized by more serious and homogeneous damage of the affected lung compared to the existing models of focal pneumonia; at the same time, our model is characterized by longer animal survival since the right lung remains intact. RESULTS: The model is also characterized by diffuse alveolar damage of the left lung, animal survival of 100%, abrupt increases in plasma levels of TNFa, INFg, and IL-6, and significant myocardial overload in the right heart. It can be used to assess the efficacy of innovative drugs for the treatment of DAD and ARDS as the clinical manifestations that are developed in patients infected with SARS-CoV-2. Morphological patterns of lungs in the noninfectious ("sterile") model of DAD induced by LPS simultaneously with α-galactosylceramide (presented here) and in the infectious model of DAD induced by SARS-CoV-2 have been compared. CONCLUSION: The DAD model we have proposed can be widely used for studying the efficacy of candidate molecules for the treatment of infectious respiratory diseases, such as viral pneumonias of different etiology, including SARS-CoV-2.

Mechanisms of Action of the PGLYRP1/Tag7 Protein in Innate and Acquired Immunity.

One of the promising fields of modern molecular biology is the search for new proteins that regulate the various stages of the immune response and the investigation of the molecular mechanisms of action of these proteins. Such proteins include the multifunctional protein PGLYRP1/Tag7, belonging to the PGRP-S protein family, whose gene was discovered in mice at the Institute of Gene Biology, Russian Academy of Sciences, in 1996. PGLYRP1/Tag7 is classified as a protein of innate immunity; however, it can also participate in the regulation of acquired immunity mechanisms. In this paper, we consider the involvement of PGLYRP1/Tag7 in the triggering of antimicrobial defense mechanisms and formation of subsets of cytotoxic lymphocytes that kill tumor cells. The paper emphasizes that the multifaceted functional activity of Tag7 in the immune response has to do with its ability to interact with various proteins to form stable protein complexes. Hsp70-associated Tag7 can induce the death of tumor cells carrying the TNFR1 receptor. Tag7, associated with the Mts1 (S100A4) protein, can stimulate the migration of innate and adaptive immune cytotoxic lymphocytes to a lesion site. Involvement of Tag7 in the regulation of immunological processes suggests that it may be considered as a promising agent in cancer therapy. These properties of Tag7 were used to develop autologous vaccines that have passed the first and second phases of clinical trials in patients with end-stage melanoma and renal cancer. The C-terminal peptide of Tag7, isolated by limited proteolysis, was shown to protect the cartilage and bone tissue of the ankle joint in mice with induced autoimmune arthritis and may be a promising drug for suppressing the development of inflammatory processes.

Innate Immune Protein Tag7 Stimulates the Appearance of Cytotoxic NK Cells after Incubation with Lymphocytes.

Tag7 (PGRP-S) is an innate immune protein that is involved in the antibacterial and antitumor defense and stimulates the maturation of cytotoxic lymphocyte subpopulations. It was found that the incubation of lymphocytes with Tag7 for 3 days promotes the appearance of cytotoxic NK cells that are active against a number of tumor cell lines.

Tag7 (PGLYRP1) Can Induce an Emergence of the CD3+CD4+CD25+CD127+ Cells with Antitumor Activity.

We have shown that in the human peripheral blood cells, the innate immunity protein Tag7 can activate a subpopulation of CD3+CD4+CD25+ cells, which have antitumor activity. These cells can induce lysis of HLA-negative tumor cell lines. The Hsp70 stress molecule on the surface of the tumor cells is used as a recognition target, while the Tag7 protein on the lymphocyte membrane acts as a receptor for Hsp70. We have also demonstrated that this subpopulation of the CD4+CD25+ cells is CD127 positive and hence is not the Treg cells. Our data suggest that this subpopulation of cells is identical to the CD4+CD25+ lymphocytes, which are activated in the leukocyte pool by the IL-2 cytokine.

Tag7-Mts1 Complex Induces Lymphocytes Migration via CCR5 and CXCR3 Receptors.

The discovery of new chemokines that induce the migration of lymphocytes to the infection site is important for the targeted search for therapeutic agents in immunotherapy. We recently showed that Tag7 (PGLYRP1), an innate immunity protein, forms a stable complex with the Ca2+ -binding protein Mts1 (S100A4), which is able to induce lymphocyte movement, although the individual Tag7 and Mts1 do not have this activity. The purpose of this study is to identify receptors that induce the migration of lymphocytes along the concentration gradient of the Tag7-Mts1 complex, and the components of this complex capable of interacting with these receptors. The study investigated the migration of human PBMC under the action of the Tag7-Mts1complex. PBMC of healthy donors were isolated using a standard Ficoll-Hypaque gradient centrifugation procedure. It has been established that the movement of PBMC along the concentration gradient of the Tag7-Mts1 complex is induced by the classical chemotactic receptors CCR5 and CXCR3. It has been shown that only Mts1 is able to bind to the extracellular domain of CCR5, however, this binding is not enough to induce cell movement. A comparative analysis of the primary and 3D structures of the three proteins revealed the homology of the amino acid sequence fragments of the Tag7-Mts1 protein complex with different sites of the CCR5 receptor ligand - MIP1α protein. In conclusion, it should be noted that the Tag7-Mts1 complex can be considered as a new ligand of the classical chemotactic receptors CCR5 and CXCR3.

Lymphocytes incubated in the presence of IL-2 lose the capacity for chemotaxis but acquire antitumor activity.

Naïve non-activated lymphocytes are capable of releasing the chemoattractant complex Tag7-Mts1 and can migrate along the gradient of its concentration. After activation of these cells by IL-2, they acquire the abilities to kill tumor cells and to release the cytotoxic Tag7-Hsp70 complex, which is accompanied by a loss of both the Tag7-Mts1-mediated lymphocyte chemotaxis and the ability to release this chemoattractant into the conditioned medium.

Apoptotic tumor cell death under the influence of the cytotoxic complex Tag7-Hsp70 is induced by interaction with the TNFR1 receptor.

The results of comparative analysis of interaction between the protein cytotoxic complex Tag 7-Hsp70 and the Tag 7 component of this complex with TNFR1 receptor in solution and in tumor cells are presented.

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1.

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7-Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4(+) and CD8(+) lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.

Kinetic Parameters and Cytotoxic Activity of Recombinant Methionine γ-Lyase from Clostridium tetani, Clostridium sporogenes, Porphyromonas gingivalis and Citrobacter freundii.

The steady-state kinetic parameters of pyridoxal 5'-phosphate-dependent recombinant methionine γ -lyase from three pathogenic bacteria, Clostridium tetani, Clostridium sporogenes, and Porphyromonas gingivalis, were determined in ß- and γ-elimination reactions. The enzyme from C. sporogenes is characterized by the highest catalytic efficiency in the γ-elimination reaction of L-methionine. It was demonstrated that the enzyme from these three sources exists as a tetramer. The N-terminal poly-histidine fragment of three recombinant enzymes influences their catalytic activity and facilitates the aggregation of monomers to yield dimeric forms under denaturing conditions. The cytotoxicity of methionine γ-lyase from C. sporogenes and C. tetani in comparison with Citrobacter freundii was evaluated using K562, PC-3, LnCap, MCF7, SKOV-3, and L5178y tumor cell lines. K562 (IC50=0.4-1.3 U/ml), PC-3 (IC50=0.1-0.4 U/ml), and MCF7 (IC50=0.04-3.2 U/ml) turned out to be the most sensitive cell lines.

Cell death of L-929 cells induced by cytotoxic complex Tag7-Hsp70 is analogous to the death of the same cells induced by TNF-α.

The identification and studying the molecular bases of functioning of new cytotoxic agents finds an important implication in developing drugs for fighting with tumors. While investigating the cytotoxic action of protein complex Tag7-Hsp70 which was opened in our laboratory previously we found that Tag7-Hsp70 demonstrated the same specificity in regard to different tumor target cells as it was for classical cytokine TNF-α. L-929 cells and Jurkat cells appeared to be good targets representing up to 30% of dead cells within a population and HeLa cells--bad targets representing less than 5% of dead cells after 20 h of incubation with either of the cytotoxic agents. While investigating the action of either TNF-α or Tag7-Hsp70 on L-929 cells we detected two peaks of death: after 3 h and after 20 h. For both cytotoxic agents we observed the first, smaller (13-15%), peak to be eliminated after the addition of caspase inhibitor YVAD-CHO and the second, greater (25-30%), peak to become even bigger in presence of caspase inhibitor. Probably, protein complex Tag7-Hsp70 interacts like TNF-α with a receptor on the surface of tumor cells that results in triggering two alternative mechanisms of programmed cell death: apoptosis and necroptosis.

Effect of exercise on the expression of HSPBP1, PGLYRP1, and HSPA1A genes in human leukocytes.

The effects of 30-min medium-intensity exercise on the expression of genes encoding heat shock protein 70 (HSPA1A) and its cochaperones HSP-70-binding protein 1 (HSPBP1) and Tag7 (PGLYRP1) in human leukocytes were studied. Transcription activities of HSPA1A and PGLYRP1 genes increased immediately after medium-intensity exercise, while activity of HSPBP1 gene remained unchanged. During recovery after exercise, the expression of HSPA1A gene virtually did not change, while the expression of PGLYRP1 gene continued to increase and after 90 min more than 2-fold surpassed the basal level.

Interactions and possible functional characteristics of Tag7-S100A4 protein complex.

Peptidoglycane-recognizing protein Tag7 formed a complex with S100A4 (a representative of S100 protein family), the apparent dissociation constants in the absence and presence of Ca2+ were 2 x l0(-8) M and 10(-9) M, respectively. Analysis of fluorescence spectra of hydrophobic fluorescent probe 2-toluidinyl naphthalene-6-sulfonate in the presence of S100A4 and Tag7 proteins showed that extensive area or several sites are involved into the complex formation between these proteins. The formation of Tag7-S100A4 complex had virtually no effect on the role of S100A4 in the regulation of intracellular Ca2+ metabolism. Removal of not only Tag7, but also S100A4 from neutrophil conditioned medium reduced lysis of E. coli cell, while addition of the Tag7-S100A4 complex to the medium restored antibacterial activity.