Isolation of formononetin-7-O-β-D-glucopyranoside from the grass of Ononis arvensis L. and the assessment of its effect on induced platelet activation

Introduction. Analysis of the clinical and laboratory picture of the SARS-CoV-2 infection suggests the presence of microcirculation and oxygen transport disorders, hemolysis of erythrocytes, intra-alveolar fibrin formation and microthrombus formation in the patient’s pathogenesis. Accordingly, the search for potential anticoagulants, erythrocyte antiplatelet agents, membrane stabilizing drugs and mild thrombolytic drugs can prevent the development of life-threatening complications and reduce the mortality of COVID-19 patients.Aim. Isolation of formononetin-7-O-β-D-glucopyranoside from the grass of Ononis arvensis L. and identification of the molecular mechanisms of its effect on platelet activation in vitro, induced by TRAP-6 (Thrombin receptor activated peptide) and ADP (adenosine diphosphate).Materials and methods. Terrestrial parts of Ononis arvensis L. were collected in the SPCPU nursery of medicinal plants (Leningrad region, Vsevolozhsky district, Priozerskoe highway, 38 km). Isolation of formononetin-7-O-β-D-glucopyranoside was carried out by preparative high performance liquid chromatography on a Smartline device (Knauer, Germany) equipped with a spectrophotometric detector. The structure of formononetin-7-O-β-D-glucopyranoside was confirmed by one-dimensional and two-dimensional NMR spectroscopy (Bruker Avance III, 400 MHz, Germany), as well as high-resolution mass spectrometry (HR-ESI-MS) (Bruker Micromass Q-TOF, Germany). The study of the effect of formononetin- 7-O-β-D-glucopyranoside on induced platelet activation was carried out on human platelets isolated from the blood of healthy volunteers. To research the effect of formononetin-7-О-β-D-glucopyranoside on platelet aggregation flow cytofluorometry with Cyto-FLEX (Beckman-Coulter, USA) was used.Results and discussion. According to the method of fractionation and purification of the total extract of O. arvensis developed in previous studies, formononetin-7-O-β-D-glucopyranoside was isolated in an individual form for subsequent biological studies with a total yield of 30 % in comparison with its content in the original extract. In samples with formononetin-7-O-β-D-glucopyranoside and ADP, there is a pronounced inhibition of platelet activation – the percentage of active platelets ranges from 6.3–6.6 % at doses of formononetin-7-O-β-D-glucopyranoside 1 μM, 3 μM and 30 μM. The inhibitory effect of formononetin-7-O-β-D-glucopyranoside is not dose-dependent (p ≤ 0.05). In samples with formononetin-7-O-β-D-glucopyranoside and TRAP, there is also a pronounced inhibition of platelet activation. The percentage of active platelets is 8 % at 1 μM formononetin-7-O-β-D-glucopyranoside doses, 15 % at 3 μM doses, and 16 % at 30 μM doses.Conclusion. Administration of formononetin-7-O-β-D-glucopyranoside at doses of 1 μM, 3 μM, 30 μM strongly inhibits platelet activation induced by ADP and TRAP-6. For ADP, there is no dose-dependent effect, while for TRAP there is a weak dose-dependent effect, the greatest inhibition efficiency is achieved with the minimum investigated dose of 1 μM. In all cases, the results obtained are statistically significant.

INSIGHT INTO ANTIMICROBIC EVALUATION TECHNIQUES AND THEIR ROLE IN BETTER ASSESSMENT OF ANTIMICROBIC AGENTS: A REVIEW

At present, researchers have a keen interest in creating advanced antimicrobic agents to overcome microbial resistance from various new sources either from plants or microbes. For this, robust attention has been given to the advancement in the rapid antimicrobic assessment strategies and methods to develop antimicrobic agents. Some of the antimicrobic techniques such as well diffusion using broth and agar and disc diffusion are more prominent in assaying antimicrobial testing while techniques such as bioluminescence, flow cytofluorometry methods are less used due to specific equipment’s, high calibration and evaluation processes. Thus, the information of precise antimicrobial techniques is must to the new researchers for antimicrobic testing. In this review article, various antimicrobic techniques with their advantages and limitations are being reported which are currently being carried out for antimicrobial testing.

Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates

Most glycosyltransferases show remarkable gross and fine substrate specificity, which is reflected in the old one enzyme-one linkage paradigm. While human Gb3/CD77 synthase is a glycosyltransferase that synthesizes the Galα1→4Gal moiety mainly on glycosphingolipids, its pigeon homolog prefers glycoproteins as acceptors. In this study, we characterized two Gb3/CD77 synthase paralogs found in pigeons (Columba livia). We evaluated their specificities in transfected human teratocarcinoma 2102Ep cells by flow cytofluorometry, Western blotting, high-performance thin-layer chromatography, mass spectrometry and metabolic labelling with 14C-galactose. We found that the previously described pigeon Gb3/CD77 synthase (called P) can use predominately glycoproteins as acceptors, while its paralog (called M), which we serendipitously discovered while conducting this study, efficiently synthesizes Galα1→4Gal caps on both glycoproteins and glycosphingolipids. These two paralogs may underlie the difference in expression profiles of Galα1→4Gal-terminated glycoconjugates between neoavians and mammals.

Activity of Negative Regulation of the PD-1/PD-L1/PD-L2 T-Cell Response System in Patients with Pneumonia and Influenza A (H1N1)

Systemic inflammation is an integral pathophysiological component of many critical illnesses. The systemic inflammatory response is based on a cascade of interactions leading to hypercytokinemia and, as a consequence, multiple organ failure, which is one of the main causes of mortality in intensive care units.Aim of the study. To evaluate the activity of the negative regulation system of T-cell response by determining the plasma levels of PD-1, PD-L1 and PD-L2 molecules in pneumonia patients with influenza A (H1N1).Materials and methods. 85 patients with pneumonia and underlying influenza A (H1N1) were examined. Among them there were 30 patients with severe pneumonia, and 55 patients with non-severe pneumonia. Plasma levels of PD-1, PD-L1, PD-L2 molecules was determined by flow cytofluorometry method.Results. In patients with severe pneumonia and underlying influenza A (H1N1), the plasma level of PD-1 receptor increased 4.6-fold, while the concentration of its ligands PD-L1 and PD-L2 increased 10.6 and 2.2-fold, respectively.Conclusion. Significant increase in levels of PD-1 and its ligands PD-L1 and PD-L2 in patients with pneumonia and underlying influenza A (H1N1) indicates the involvement of negative regulation system of T-cell response in the cascade of immunological reactions and is associated with the severe disease. Possible correction of immune reactions realized through PD-1/PD-L1/PD-L2 complex in critically ill patients is a promising research avenue.

MO438STUDY OF IMMUNOLOGICAL STATUS IN PATIENT OF CKD IN PREDIALYSIS STAGES

Background and Aims It is known that inflammation is an important factor associated with the variability of the clinical picture in patients with chronic diseases. However, the question remains not completely clear: are immunological changes predictors of less stable control of the course of CKD. The aim of this study was a comprehensive assessment of the immunological status in patients with pre-dialysis stages of CKD. Method 80 patients (46 men and 36 women) with CKD in the outcome of nephropathies of diabetic and nondiabetic etiology, admitted to the Republican Medical Scientific and Practical Center for Therapy and Medical Rehabilitation, were examined. Chronic pyelonephritis without exacerbation was diagnosed in 31 patients, chronic glomerulonephritis - in 29, diabetic nephropathy - in 25 polycystic kidney disease - in 6 patients. Glomerular filtration was calculated based on serum creatinine concentration using the CKD-EPI formula (2011). A complex of immunological studies was performed. Immunotyping of lymphocytes was performed by flow cytofluorometry with the characterization of the main markers of immunocompetent cells. Results In patients with CKD, violations were observed in almost all links of immunological reactivity, first of all, a significant decrease in indicators of natural resistance (CD16 +, PS, PI) (p <0.05). The results of immunological monitoring showed a predominant violation of cellular defense mechanisms (CD3 +, CD4 +, CD8 +) (p <0.05), a multiple increase in inflammatory cytokines Il-4, TNF-α, IL-6 (p <0.05), in the absence of activation of humoral link and inadequacy of elimination mechanisms (CEC, p <0.05). Conclusion Our study showed that the immunological parameters were characterized by insufficient activation of the first line of defense of natural resistance, as well as specific mechanisms - an imbalance of the cellular and humoral links of immunity, hyperactivation of cytokine regulation in the absence of effector functions.

Preoperative administration of corticosteroids in thoracic anesthesiology

Annotation. One lung ventilation (OLV) is one of the most difficult intraoperative methods of respiratory support for anesthesiologists. OLV should provide the most comfortable surgical field, maintaining proper gas exchange and minimizing damage to both lungs. This anesthetic procedure has a significant inflammatory response, so using perioperative corticosteroid therapy to suppress inflammatory mediators is recommended as an approach to improving prognosis. Therefore, the aim of this study was to determine how the preoperative administration of methylprednisolone affects the systemic proinflammatory response of cytokines during thoracic surgery. The analysis was performed for 80 patients who underwent surgeries in the thoraco-abdominal department of the Shalimov National Institute of Surgery and Transplantology. Patients were divided into 2 groups (study – 40 patients who were administered methylprednisolone 10 mg / kg intravenously during induction of anesthesia and control – 40 patients without methylprednisolone). Before surgery and in the postoperative period on days 1, 3 and 5, the surface phenotype of peripheral blood lymphocytes and the expression of IL-6 by monocytes were determined by flow cytofluorometry. The author's MedStat package was used for statistical analysis (Lyakh Yu.E., Guryanov V.G., 2004–2012). Postoperative indicators of IL-6 monocyte expression in the blood of patients administered methylprednisolone were significantly lower on the 1st and 3rd postoperative day (p<0.001). Thus, preoperative administration of methylprednisolone reduces the release of pro-inflammatory cytokines and improves the condition of patients after thoracic surgery. We consider it expedient to conduct further research on the administration of methylprednisolone for several days in the postoperative period.

Technology for Determining the Inflow from Near and Far Zones of Fractures During Hydraulic Fracturing by Chemical Tracers in a Production Well

The tracer-based production logging technology can be used to obtain the well production data continuously for several years without the need for risky well interventions and expensive equipment. The paper examines the case of placing polymer-coated tracers dopped proppant in a horizontal well with ten multi-stage frac intervals and using two different tracers dopped proppant codes for two frac ports (the first and the last ones) to identify the performance of the far and near zones of a hydraulic fracture. Upon the completion of the hydraulic fracturing operations, the collected reservoir fluid samples were studied in the laboratory. Chemical tracers contained in the samples were detected by flow cytofluorometry using custom-tailored machine learning-based software. The studies helped identify the productivity of each frac port, calculate the contribution of each port in percentage points, and also evaluate the productivity of the near and far hydraulic fracture zones in the first and the last intervals. The analysis provided data on the exact content of oil and water in the production profile for each frac interval. The results of tracer-based logging in the well in question revealed that the interval productivity is changing in the course of several months of surveillance. The most productive ports and those showing increasing oil flow rate were identified during quantitative analysis. The use of tracer dopped proppant with different codes within one multi-stage frac interval enabled detecting a peak release of chemical tracers from the far fracture zone in the initial periods of well operation followed by a consistent smoothing of the far and near zones’ production profiles. Laboratory analysis of reservoir fluid samples and hydraulic fracturing simulations proved the uniform distribution of proppant across the entire reservoir pay zone and laid the foundation for further research required to better understand the fracture geometry and reduce uncertainties in production optimization operations.

Acute Myeloid Leukemia Patient-Derived Xenograft Models Generated with the Use of Immunodeficient NSG-SGM3 Mice

Background. Up to the present the survival rates of acute myeloid leukemia (AML) patients have remained low. A successful AML management presupposes generating personalized models of the disease. The most promising research activity in this field is creation of AML patient-derived xenograft models using the advanced strain of immunodeficient humanized NSG-SGM3 mice. Aim. To generate AML patient-derived xenograft models using immunodeficient NSG-SGM3 mice. Materials & Methods. The creation of PDX models was based on bone marrow aspirates taken from 4 patients with newly diagnosed AML who were treated at the V.A. Almazov National Medical Research Center. Patient-derived tumor cells were transplanted to NSG-SGM3 mice. Test experiment consisted in injecting AML cells OCI-АМL2 and HL60 in NSG-SGM3 mice. The efficacy of tumor engraftment was evaluated in terms of physical condition of animals and laboratory tests (blood count, blood smear, PCR, and flow cytofluorometry). Results. The engraftment of applied tumor cells derived from AML patients was achieved in half (2 out of 4) of the transplanted tumor samples. In mice with successful transplantation leukocytosis was reported. Blast cells were identified in peripheral blood on Day 30 after transplantation. The mice with injected AML cells OCI-АМL2 and HL60 showed a more aggressive course of disease. Among tested approaches to evaluate tumor engraftment in mouse recipients, the PCR method was marked by highest sensitivity. Conclusion. The use of immunodeficient humanized NSG-SGM3 mice enables successful generation of AML patient-derived xenograft models.

INTERFERON STATUS OF ENDOMETRY IN PATIENTS WITH MISCARRIAGE AND “THIN ENDOMETRY” SYNDROME

The article presents the results of examination of 21 patients with miscarriage and “thin endometry” syndrome compared with 20 patients without reproductive losses and the presence of normal endometrial thickness. Interferon gamma intracellular production by cytotoxic endometrial lymphocytes was investigated by flow cytofluorometry. Based on the study, it was found that in patients with miscarriage and “thin endometry” syndrome, the interferon gamma intracellular production by cytotoxic CD8+ lymphocytes was sharply suppressed by almost 36 times, by CD56+ lymphocytes - by 13 times, by CD16+ lymphocytes - by 4.5 times.

Levels of isoforms of fibronectin and α5/CD49e integrin on lymphocytes and in blood plasma in the conditions of chronic diffuse liver diseases

Chronic diffuse liver diseases are characterized by accumulation of complex inflammatory infiltrate in the liver tissues, blood, and lympha, and activation of the immune system. Leukocytes become involved in the area of inflammation after the activation of receptors of blood adhesia, particularly integrins and their ligands. Plasma lymphocytes quickly activate the function of integrins by changing their conformation, leading to high affinity and underlying the formation of strong stable connection between the components of extracellular matrix. A vitally important role in the process of liver fibrogenesis is performed by a pro-fibrogenicic protein fibronectin which induces the expresson of collagen genes and precedes the deposition of other components of matrix. The studies were conducted in the group of patients suffering from chronic diffuse liver diseases of non-viral etiology aged 28–60 years, n = 36 and in the group of 15 practically healthy volunteer donors aged 25 to 52 years without a history of liver diseases using the methods of flow cytofluorometry, immunoenzymatic analysis, and quantitative real-time polymerase chain reaction. The patients of the group with chronic diffuse liver diseases were observed to have statistically significant decrease in the concentration of plasmatic form of fibronectin measuring 27.6% compared with the control group. We determined increase in the concentration of cellular fibronectin in blood plasma of patients with the diseases on average accounting for 63.8% compared with the norm, and the highest increase in this parameter equaling 77.2% was seen in patients suffering from drug-induced hepatitis. Significant increase in the level of exposure of cellular FN on blood lymphocytes was determined in patients with chronic diffuse liver diseases, measuring 231.8%, whereas the level of plasmatic form of fibronectin in these cells was decreased (statistically unreliable). For α5-integrin subunit, we determined a 390.8% increase in the level of its exposure in blood lymphocytes in the surveyed groups compared with the control. Level of blood lymphocytes that express the cellular fibronectin significantly decreased by 140.1%. Statistical characteristics of diagnostic possibility of the parameters of the level of plasmatic and cellular fibronectin in blood, determined over the analysis of ROC-curves, demonstrated excellent informativeness of these tests. Analysis of the possibility of predicting the presence of pathology using the model of logistic regression revealed zero error of prediction and maximum efficiency of the tests: intensity of exposure of α5-integrin receptor on the surface of lymphocytes, intensity of exposure of plasmatic fibronectin on the surface of lymphocytes, intensity of exposure of cellular fibronectin on the surface of lymphocytes, concentration of plasmatic fibronectin in blood, concentration of cellular fibronectin in blood plasma. These parameters may be proposed for further surveys for developing serologic biomarkers based on the parameters for diagnostics of chronic diffuse liver diseases.