Potential value of high-throughput single-cell DNA sequencing of Juvenile myelomonocytic leukemia: report of two cases.

Juvenile myelomonocytic leukemia (JMML) is a rare myeloproliferative disease of early childhood that develops due to mutations in the genes of the RAS-signaling pathway. Next-generation high throughput sequencing (NGS) enables identification of various secondary molecular genetic events that can facilitate JMML progression and transformation into secondary acute myeloid leukemia (sAML). The methods of single-cell DNA sequencing (scDNA-seq) enable overcoming limitations of bulk NGS and exploring genetic heterogeneity at the level of individual cells, which can help in a better understanding of the mechanisms leading to JMML progression and provide an opportunity to evaluate the response of leukemia to therapy. In the present work, we applied a two-step droplet microfluidics approach to detect DNA alterations among thousands of single cells and to analyze clonal dynamics in two JMML patients with sAML transformation before and after hematopoietic stem cell transplantation (HSCT). At the time of diagnosis both of our patients harbored only "canonical" mutations in the RAS signaling pathway genes detected by targeted DNA sequencing. Analysis of samples from the time of transformation JMML to sAML revealed additional genetic events that are potential drivers for disease progression in both patients. ScDNA-seq was able to measure of chimerism level and detect a residual tumor clone in the second patient after HSCT (sensitivity of less than 0.1% tumor cells). The data obtained demonstrate the value of scDNA-seq to assess the clonal evolution of JMML to sAML, response to therapy and engraftment monitoring.

Practical Aspects of Using Multifractal Formalism to Assess the Morphology of Biological Tissues.

The aim of the study is to identify practical aspects of using multifractal formalism to assess the morphology of biological tissues. Materials and Methods: The objects of the study were histological images of lung tissues of Wistar rats without pathology and with detected pathological changes, obtained at 50×, 100×, 200× magnifications. Image processing was carried out using the ImageJ/Fiji universal software. The multifractal spectrum of the images, processed to obtain a linear contour, was calculated with the use of FracLac - a module for ImageJ. This module was used to determine the scaling exponent (the function of the Rényi exponent, τ(q)) and the singularity spectrum itself. Results: The singularity spectra for tissues with no pathology have signs of multifractality. The image spectrum of tissue with pathology is shifted to the left relative to the spectrum characteristic of tissue without pathology. A decrease in the spectral height in the presence of pathology indicates a "simplification" of the alveolar pattern, which is presumably associated with the presence of widespread vasculitis, since it causes areas of hemorrhage to appear on the image; this leads to leveling the contour of the alveolar pattern, reducing the surface area of the alveoli and emerging areas inflamed by erythrocytes. At lower magnification, images with pathology lose signs of multifractality. Conclusion: Correct results of assessing multifractal spectra of histological images can be achieved at 200× magnification and preprocessing to obtain linear contours. Significant differences between the morphological structure of lung tissues with and without pathology are observed when comparing the height, width, and position of the spectrum relative to the origin.

Properties and Activity of Peptide Derivatives of ACE2 Cellular Receptor and Their Interaction with SARS-CoV-2 S Protein Receptor-Binding Domain.

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture.

Procoagulant Platelets: Mechanisms of Generation and Action.

During the past decades, it has been increasingly recognized that the major function of accelerating membrane-dependent reactions of blood coagulation is predominantly implemented by a subset of activated platelets. These procoagulant platelets (also called collagen- and thrombin-activated or COAT, coated, necrotic, although there could be subtle differences between these definitions) are uniquely characterized by both procoagulant activity and, at the same time, inactivated integrins and profibrinolytic properties. The mechanisms of their generation both in vitro and in situ have been increasingly becoming clear, suggesting unique and multidirectional roles in hemostasis and thrombosis. In this mini-review, we shall highlight the existing concepts and challenges in this field.

Study of Hematological Parameters and Morphometric Indices of Erythrocytes in Rats Exposed to Calcium Oxide Nanoparticles.

In female Wistar rats, we studied the effects of daily 20-day administration of intragastric suspension of nanosized calcium oxide on hematological indices (the counts of erythrocytes, leukocytes, platelets, hemoglobin, hematocrit, basophils, lymphocytes, monocytes, stab/segmented neutrophils, and the eosinophil-to-lymphocyte ratio) and morphometric parameters of erythrocytes (equivalent Feret's diameter, sphericity, aspect ratio, and convexity). The study revealed significantly (up to 3.1 times) decreased levels of hemoglobin, platelets, hematocrit, averaged content of hemoglobin in erythrocyte, and platelet volume relatively to the control values. The established morphometric parameters of blood erythrocytes attested to predominance of spherocytes, which can be regarded as systemic response of the body to calcium oxide nanoparticles fraught with possible deterioration of perfusion in microvasculature and hypoxia in the tissues.

Programmed Cell Death and Functional Activity of Platelets in Case of Oncohematologic Diseases.

Programmed cell death of non-nucleated blood cells - platelets - could be associated with pathophysiology of oncologic and oncohematologic diseases. It contributes to both bleedings (caused by the thrombocytopenia, which is induced by elimination of the platelets) and thrombosis (caused by the processes of blood coagulation on the surface of phosphatidylserine exposing platelets). Here we characterized functional responses of platelets from the patients with various oncological disorders undergoing chemotherapy and compared them to the platelets from the healthy donors and platelets pre-incubated with apoptosis inducer ABT-737. Some patients exhibited diminished capability of platelets to aggregate. Immunophenotyping of these platelets revealed their pre-activation in comparison to the platelets from the healthy donors. Calcium signaling analysis revealed that in the patient-derived platelets, as well as in the apoptotic platelets, intracellular calcium levels were increased in resting cells. However, moderate level of this increase together with weak expression of phosphatidylserine allows us to assume that apoptotic processes in the circulating platelets from the patients are limited.

New insights into ligand binding by plant lipid transfer proteins: A case study of the lentil Lc-LTP2.

Lipid transfer proteins (LTPs) are an important class of plant proteins containing an internal cavity and binding hydrophobic ligands. Although LTP structures and functions are well studied, mechanisms of ligand binding remain unclear. Earlier, we discovered the lentil lipid transfer protein Lc-LTP2 capable of binding and transfer various ligands. We have shown that the "bottom" entrance of the Lc-LTP2 cavity takes part in attachment to the micelle surface and in lipids uptake. Here, we studied the role of Arg45 and Tyr80, located at the "bottom" entrance, in Lc-LTP2 ligand binding. We obtained recombinant mutant analogs of Lc-LTP2 (R45A, Y80A, R45A/Y80A), investigated their ability to bind fatty acids and lysolipids, as well as performed molecular modeling of the protein-ligand complexes. We showed that replacement of one or both residues led to a change of the internal hydrophobic cavity dimensions. As a result, lipids may change their orientation into the protein cavity, and thereby binding ability of mutant analogs may be affected as well. In the present work, we revealed an important role of Arg45 and Tyr80 in stabilization of the Lc-LTP2 complexes with both fatty acids and lysolipids with different ligand orientation.

Morphometry of Sperm Head in Rats Treated with an Antifungal Medication.

The parameters of sperm head in Wistar male rats orally treated with antifungal agent for 48 days during spermatogenesis were studied by the method of image analysis. The degree of roundness (roughness) of sperm head was calculated. Significant differences in morphometric parameters of sperm head, such as length, width, head angle, and roundness were revealed between the treatment and control group. The index of deformation of sperm head in the treatment group rats was 4.93 arb. units. These data indicated microcephaly accompanied by the enlargement of the head, transition of an acute angle to a right angle, and acquiring of a round form. Potential gonadotoxicity was confirmed by the analysis of the functional activity of spermatozoids of male rats (increased count of spermatozoa with head pathology), fertilization ability (enhanced fertility index), and genotoxicity (increased number of chromosomal aberrations polychromatophilic erythrocytes of murine bone marrow). These changes can be responsible for reduced fertility.

[Diagnostics of nasopharyngeal carcinoma with Epstein-Barr virus (Herpesviridae, Lymphocryptovirus, HHV-4) serological and molecular markers in cases of undetected primary tumor location.]

INTRODUCTION: The reasons of late diagnosis of nasopharyngeal carcinoma (NPC) are the long asymptomatic course of the pathological process, the anatomical structure of the nasopharynx, often small, visually and endoscopically undetectable tumor and other factors. It is proved that the Epstein-Barr virus (EBV) is an etiological agent in the most common undifferentiated non-keratinizing histological type of NPC (uNPC). OBJECTIVES: The aim of the work was to assess the significance of diagnostic markers of EBV (titers of humoral antibodies to the virus and the concentration of viral DNA in plasma) for the diagnosis of uNPC in a group of patients with metastatic lesions of the cervical lymph nodes without an identified localization of the primary tumor focus. MATERIAL AND METHODS: The material for the study was blood plasma of 83 patients with metastatic lesions of the cervical lymph nodes and not established localization of the primary tumor. Plasma samples were tested for the anti-EBV IgG and IgA antibody content and titers and the concentration of viral DNA. RESULTS AND DISCUSSION: The data obtained indicate that the parallel testing of blood plasma for EBV-specific antibodies and viral load is a useful tool for preliminary screening of uNPC patients. The final diagnosis is confirmed by the data of subsequent morphological and instrumental studies. Several examples also show that the concentration of viral DNA in the blood plasma of patients with uNPC reflects the effect of the therapy and the prognosis of the disease: remission, stabilization of the tumor process, relapse or metastasis. CONCLUSION: Although the titers of virus-specific antibodies are found to reflect clinical manifestations of the disease less accurately than the plasma concentrations of viral DNA, serological markers are extremely important for the preliminary diagnostics of uNPC in cases of undetected primary tumor location. They are also useful for primary screening of this neoplasm among individuals at risk.

EPSTEIN-BARR VIRUS AND NASOPHARYNGEAL CARCINOMA: VIRAL MARKERS FOR DIAGNOSTICS AND ASSESSMENT OF CLINICAL STATUS OF PATIENTS.

The etiological role of the Epstein-Barr virus (EBV) in the development of an undifferentiated histological variant of nasopharyngeal carcinoma (uNPC) found for the first time in regions with a high incidence of this pathology, the Southern provinces of China and the countries of Southeast Asia, and later in the rest of the world, has served as a basis for the widespread use of EBV serological markers for the diagnosis of this form of tumor. In recent years, the use of a test based on the quantitative determination of the EBV DNA concentration in the blood plasma of uNPC patients for early detection and monitoring of the disease has become widespread in endemic regions. In non-endemic regions, such studies virtually have not been carried out, and moreover, the comparative evaluation of the significance of two viral markers, serological and EBV DNA load in the bloodstream of uNPC patients, for diagnostics and evaluation of the therapeutic effect was not investigated. The aim of this study was to compare the clinical value of two serological markers and plasma EBV DNA load in uNPC patients from non-endemic region (Russia). The obtained results indicate that IgA antibodies to the viral capsid antigen (IgA/VCA) and plasma EBV DNA concentration can be successfully used for the diagnosis of uNPC, while IgG/VCA antibodies have no practical significance as an uNPC marker. In addition, it was found that plasma EBV DNA load is more sensitive marker of uNPC than IgA/VCA titers because DNA copy numbers reflect more accurately the effect of the therapy and the clinical state of patients at the stages of remission or relapse. It was shown for the first time that in the non-endemic region the simultaneous evaluation of IgA/VCA antibody levels and the plasma EBV DNA loads are the most effective markers for the diagnostics of uNPC. However, we believe, that it is more practical to use IgA/VCA antibody levels for uNPC screening, and plasma EBV DNA copies - for monitoring of the disease.

Epstein-Barr virus biomarkers for nasopharyngeal carcinoma in non-endemic regions.

The Epstein-Barr virus (EBV) plays a key role in the development of undifferentiated nasopharyngeal carcinoma (uNPC). In uNPC endemic regions EBV-specific antibodies and plasma EBV DNA load are used as markers for the early detection of uNPC and monitoring of the disease. In non-endemic regions, such studies were practically not conducted. The aim of this study was to compare the clinical significance of EBV serological markers and plasma EBV DNA levels for uNPC patients in a non-endemic region, Russia. The results obtained indicate that both viral capsid antigen/immunoglobulin A (VCA/IgA) antibodies and plasma EBV DNA copies can effectively be used for nasopharyngeal carcinoma (NPC) diagnosis. Besides, plasma EBV DNA load was found to be a more sensitive marker of uNPC than VCA/IgA antibody titres, as it reflected the effect of the therapy in stages of remission and relapse of the disease more precisely. Our study, for the first time, demonstrates that the simultaneous use of plasma EBV DNA loads and VCA/IgA antibody levels are indispensable markers for uNPC in non-endemic regions: a serological marker can be more effectively used for NPC screening, but EBV DNA copies are better for monitoring the disease. However, both markers turned out to be practically unsuitable for assessing the clinical status of patients. Serological markers did not correlate with any signs of the tumour process estimated by tumour, node and metastasis (TNM) classification and the plasma EBV DNA loads correlated only with the size of the pathologically altered lymph nodes (N). Additional study is required to confirm these findings.

Functional characteristics and clinical effectiveness of platelet concentrates treated with riboflavin and ultraviolet light in plasma and in platelet additive solution.

BACKGROUND AND OBJECTIVES: Pathogen reduction technologies may affect platelet quality during storage. We studied functional characteristics and clinical effectiveness of platelet concentrates (PCs) treated with Mirasol in plasma and in platelet-additive solution SSP+. MATERIALS AND METHODS: Mirasol-treated, gamma-irradiated and untreated apheresis PCs were examined on days 0, 1, 3 and 5 of storage. Phosphatidylserine, P-selectin and active glycoprotein IIb/IIIa were analysed using flow cytometry before and after platelet stimulation. Platelet count increments, the numbers of inefficient transfusions and post-transfusion reactions were analysed to estimate clinical effectiveness. RESULTS: A significant increase in all platelet activation markers occurred during storage in all PC groups. Activation markers in Mirasol-treated samples were already significantly higher compared with the control ones on the day of harvesting, and continued to grow during the storage. Mirasol treatment increased the number of platelets with a mitochondrial membrane potential loss. On the 3rd day of storage, 50% of Mirasol-treated platelets did not respond to activation; on the 5th day, none did. This agreed well with a decrease (approximately twofold) in the effectiveness of Mirasol-treated PC transfusions. Transfusions of PCs stored in SSP+ were accompanied by fewer inefficient transfusions and post-transfusion reactions than of PCs stored in plasma. CONCLUSION: Treatment with Mirasol decreased platelet function, particularly profoundly on the 5th day of storage, and led to a decrease in the effectiveness of transfusions. SSP+ did not affect laboratory parameters significantly compared with plasma, but decreased the percentage of transfusion complications.

Diagnostic value of the Epstein-Barr virus serological markers in patients with nasopharyngeal carcinoma in cases of undetectable primary tumor location.

The goal of this work was to describe a method for diagnosis of the non-keratinizing nasopharyngeal carcinoma (nNPC) in cases of the undetectable primary tumor location. The method is based on evaluation of IgG and IgA antibody levels to the capsid (VCA) and early antigens (EA) of the Epstein-Barr virus (EBV). The diagnosis of nNPC is established by a so-called decision rule. The latter was created by mathematical processing of the method of multifactor analysis of the results of anti-EBV antibody testing of 72 patients with clinically and morphologically confirmed nNPC and 72 patients with other head and neck benign tumors (OHNT) not associated with EBV, which were tested as a control group. The diagnostic value of the decision rule which was tested in the group of 77 patients with confirmed nNPC and 231 patients of a control group was high. The numbers of false negative and false positive cases were equal to 5.2% (4/77) and 6.5% (17/231), respectively. Among 32 patients with undetectable primary tumors the decision rule was able to identify 11 cases of nNPC. This diagnosis later was confirmed by morphological and instrumental methods of study. Only in two cases, false negative result was obtained (2/32; 6.3%) indicating that the serological diagnostics of nNPC with the decision rule is highly specific but not exact. Thus, the data obtained allowed us to conclude that the serological testing of EBV specific antibody evaluated by the decision rule can be recommended as an important test supplementing the standard methods of pdNPC diagnostics including cases with undetected primary tumor location.

Molecular epidemiology of tuberculosis in the Tula area, Central Russia, before the introduction of the Directly Observed Therapy Strategy.

Tuberculosis remains a major public health concern in Russia and worldwide. Given the great geographical, ethnic, and socio-economic heterogeneities between Russian regions, epidemiological data cannot be generalized from a regional to a country-wide level. We present data on the epidemiology of tuberculosis in Central Russia. We report a high level of resistance to major antitubercular drugs in both new and previously treated patients in the region. The level of drug resistance in new cases was almost twice as high as the estimated average national level. The Mycobacterium tuberculosis strains that circulated in the region were predominantly represented by LAM-RUS and Beijing genotypes. These two lineages were strongly associated with drug resistance and clustering. Using molecular epidemiology techniques, we showed a high interpenetration by M. tuberculosis strains between the prison and civilian populations. A limited number of identical strains were responsible for the majority of drug-resistant tuberculosis cases in both settings.

[Interaction of the photosensitizer 13,15-N-(3'-hydroxypropyl)cycloimide of chlorin p(6) with normal and cancerous blood cells].

The interaction of 13,15-N-(3'-hydroxypropyl)cycloimide of chlorin p(6) (CIC) with normal blood cells and human K562 and HL60 myeloid leukemia cells was studied. CIC was found to be bound by the erythrocyte membrane but did not penetrate into the cytoplasm. It is characterized by a diffuse distribution in the cytoplasm of normal leukocytes, whereas its diffuse distribution in K562 and HL60 cells is accompanied by perinuclear accumulation and binding to the plasma membrane. The average cytoplasmic concentration corresponding to the CIC accumulation in leukemic cells at saturation is 2.2 to 2.6 times higher than that in normal leukocytes. CIC is more intensely accumulated in granulocytes than in lymphocytes. The kinetics of the cellular uptake and efflux was characterized. The normal leukocytes and erythrocytes were found to be 1.5 times and 3 to 4 times less sensitive, respectively, to the photodynamic action of CIC than the K562 and HL60 cells.

Studying of Membrane Localization of Recombinant Potassium Channels in E.coli.

The effective expression of recombinant membrane proteins in E.coli depends upon the targeting and insertion of proteins into the cellular membrane, as well as on those proteins adopting the correct spatial structure. A significant technological problem involves the design of approaches for detecting the location of target proteins within a host cell. Using a hybrid potassium channel KcsA-Kv1.3 as a model, we developed a technological scheme which is suitable for the study of membrane localization in E.coli cells of recombinant proteins containing voltage-gated eukaryotic potassium channels as the functional active site. The scheme involves both biochemical and fluorescent methods for detecting target proteins in the cytoplasmic membrane of E.coli, as well as the study of the ligand-binding activity of membrane-embedded proteins.

[Predominant genotypes of Mycobacterium tuberculosis strains from prisoners in Ozerky].

The correlation of the Beijing/W and LAM strains with multiple drug resistance was studied. The transmissibility of the multidrug-resistant strains of these families was demonstrated.

[Effect of catabolic plasmids on physiological parameters and efficiency of oil destruction by bacteria of the genus Pseudomonas].

The ability of microbial degraders of polycyclic aromatic hydrocarbons to grow at 24 degrees C in liquid mineral medium supplemented with oil as the sole source of carbon and energy was studied. Growth characteristics (CFU) and the level of oil destruction by plasmid-bearing and plasmid-free strains were determined after seven days of cultivation. The presence of catabolic plasmids in the degrader strains, including rhizosphere pseudomonads, was shown to increase cell growth and enhance the level of oil degradation. Strain Pseudomonas chlororaphis BS 1391 bearing plasmid pBS216 was found to be the most effective oil degrader.

[Molecular-genetic method of identification of drug-resistant Mycobacterium tuberculosis].

Above two million people annually die worldwide of tuberculosis. There has been a serious uptrend in the tuberculosis morbidity observed during the recent decade in Russia. The growing prevalence of drug-resistant strains of pathogens is highly dangerous. A timely diagnosis reduces significantly the risk of epidemics. The use of molecular methods cuts the time of tuberculosis diagnosis, including its resistant forms, from several weeks to several days. The presented survey contains a discussion of modem methods used for identification of Mycobacterium tuberculosis resistance to the main anti-TB drugs.

[Production of DNA-hydrolyzing antibody BV04-01 Fab fragment in methylotrophic yeast Pichia pastoris]. / Produktsiia Fab-fragmenta DNK-gidrolizuiushchego antitela BV04-01 v metilotrofnykh drozhzhakh Pichia pastoris.

Efficient system for producing the recombinant Fab of DNA-hydrolyzing antibody BV04-01 in metylotrophic yeast Pichia pastoris was developed. Addition of peptides encompassing the Jun-Fos leucine zipper at the C-termini of the antibody chains facilitated the in vivo assembling of the Fab. The yield of secreted functionally active BV04-01 Fab was about 3 mg/L. Catalytic efficiency of supercoiled DNA hydrolysis by the Fab obtained was 1.8 x 10(6) M(-1) min(-1).