Electrochemical profiling of poliovirus particles inactivated by chemical method and ionizing radiation.

Electrochemical profiling of formaldehyde-inactivated poliovirus particles demonstrated a relationship between the D-antigen concentration and the intensity of the maximum amplitude currents of the poliovirus samples. The resultant signal was therefore identified as electrochemical oxidation of the surface proteins of the poliovirus. Using registration of electrooxidation of amino acid residues of the capsid proteins, a comparative electrochemical analysis of poliovirus particles inactivated by electrons accelerated with doses of 5 kGy, 10 kGy, 15 kGy, 25 kGy, 30 kGy at room temperature was carried out. An increase in the radiation dose was accompanied by an increase in electrooxidation signals. A significant increase in the signals of electrooxidation of poliovirus capsid proteins was detected upon irradiation at doses of 15-30 kGy. The data obtained suggest that the change in the profile and increase in the electrooxidation signals of poliovirus capsid proteins are associated with an increase in the degree of structural reorganization of surface proteins and insufficient preservation of the D-antigen under these conditions of poliovirus inactivation.

Perspectives for the creation of a new type of vaccine preparations based on pseudovirus particles using polio vaccine as an example.

Traditional antiviral vaccines are currently created by inactivating the virus chemically, most often using formaldehyde or ß-propiolactone. These approaches are not optimal since they negatively affect the safety of the antigenic determinants of the inactivated particles and require additional purification stages. The most promising platforms for creating vaccines are based on pseudoviruses, i.e., viruses that have completely preserved the outer shell (capsid), while losing the ability to reproduce owing to the destruction of the genome. The irradiation of viruses with electron beam is the optimal way to create pseudoviral particles. In this review, with the example of the poliovirus, the main algorithms that can be applied to characterize pseudoviral particles functionally and structurally in the process of creating a vaccine preparation are presented. These algorithms are, namely, the analysis of the degree of genome destruction and coimmunogenicity. The structure of the poliovirus and methods of its inactivation are considered. Methods for assessing residual infectivity and immunogenicity are proposed for the functional characterization of pseudoviruses. Genome integrity analysis approaches, atomic force and electron microscopy, surface plasmon resonance, and bioelectrochemical methods are crucial to structural characterization of the pseudovirus particles.

[Metabolome profiling in the study of aging processes]. / Metabolomnoe profilirovanie v izuchenii protsessov stareniia.

Aging of a living organism is closely related to systemic metabolic changes. But due to the multilevel and network nature of metabolic pathways, it is difficult to understand these connections. Today, this problem is solved using one of the main approaches of metabolomics - untargeted metabolome profiling. The purpose of this publication is to systematize the results of metabolomic studies based on such profiling, both in animal models and in humans.

Increasing the Efficiency of Cytochrome P450 3A4 Electrocatalysis Using Electrode Modification with Spatially Ordered Anodic Aluminum Oxide-Based Nanostructures for Investigation of Metabolic Transformations of Drugs.

A new approach for modifying electrodes using porous membranes based on anodic aluminum oxide with pore diameters of 0.1 and 0.2 µm and a membrane-like substance didodecyldimethylammonium bromide (DDAB) was proposed to study the electrocatalytic efficiency of the system. This approach makes it possible to increase the catalytic efficiency of the cytochrome P450 3A4-dependent N-demethylation of erythromycin by 132% when using a membrane with pore diameter of 0.1 µm and by 32% when using a membrane with 0.2 µm pore size. Electrode modification using porous membranes shifted the potential of electrochemical reduction and catalysis of cytochrome P450 3A4 in positive direction by 0.070-0.050 V, which indicates a thermodynamically more favorable process of electron transfer and enzymatic electrocatalysis.

[Nonimmunogenic staphylokinase in the treatment of acute ischemic stroke (FRIDA trial results)]. / Neimmunogennaya stafilokinaza ­ novyi tromboliticheskii preparat v lechenii ishemicheskogo insul'ta (rezul'taty issledovaniya FRIDA).

AIM OF THE STUDY: To investigate the efficacy and safety of non-immunogenic staphylokinase (NS) compared with alteplase (A) in patients with acute ischemic stroke (AIS) within 4.5 h after symptom onset. MATERIAL AND METHODS: 336 patients with IS within 4.5 h after symptom onset were included in a randomized, open-label, multicenter, parallel-group, non-inferiority comparative trial of NS vs A (168 patients in each group). NS was administered as an intravenous bolus in a dose of 10 mg, regardless of body weight, over 10 s, A was administered as a bolus infusion in a dose of 0.9 mg/kg, maximum 90 mg over 1 hour. The primary efficacy endpoint was a favorable outcome, defined as a modified Rankin scale (mRS) score of 0-1 on day 90. Safety endpoints included all-cause mortality on day 90, symptomatic intracranial haemorrhage, and other serious adverse events (SAEs). RESULTS: At day 90, 84 (50%) patients reached the primary endpoint (mRS 0-1) in the NS group, 68 (41%) patients - in the A group (p=0.10, OR=1.47, 95% CI=0.93-2.32). The difference between groups NS and A was 9.5% (95% CI= -1.7-20.7) and the lower limit of the 95% CI did not cross the margin of non-inferiority (pnon-inferiority<0.0001). There were no significant differences in the frequency of deaths between the groups: on day 90, 17 (10%) patients in the NS group and 24 (14%) in the A group had died (p=0.32). There was a trend towards significant differences in the frequency of symptomatic intracranial haemorrhage: NS group - 5 (3%) patients, A group - 13 (8%) patients (p=0.087, OR=0.37, 95% CI=0.1-1.13). There were significant differences in the number of patients with SAEs: in the NS group - 22 (13%) patients, in the A group - 37 (22%) patients (p=0.044, OR=0.53, 95% CI=0.28-0.98). CONCLUSION: The presented results of the FRIDA trial are the first in the world to use a drug based on NS in patients with IS. It has been shown that a single bolus (within 10 s) administration of NS at a standard dose of 10 mg, regardless of body weight, allows to conduct fast, effective and safe thrombolytic therapy in patients with IS within 4.5 h after symptom onset. In further clinical tials of NS, it is planned to expand the therapeutic window beyond 4.5 h after symptom onset in patients with IS.

[Splice variants of mRNA of cytochrome P450 genes: analysis by the nanopore sequencing method in human liver tissue and HepG2 cell line]. / Splais-varianty mRNK genov tsitokhromov R450: analiz metodom nanoporovogo sekvenirovaniia v tkani pecheni cheloveka i kletkakh linii HepG2.

The analysis of cytochrome P450 transcripts was carried out by the nanopore sequencing in liver tissue samples of three donors and HepG2 line cells. It has been demonstrated that direct mRNA sequencing with a MinION nanopore sequencer (Oxford Nanopore Technologies) allows one to obtained quantitative profiles for transcripts (and their splice variants) of cytochrome P450 superfamily genes encoding isoforms involved in metabolism of the large (~80%) part of drugs. The splice variant profiles substantially differ for donors. The cytochrome P450 gene expression at the transcript level is significantly weaker in cells of the HepG2 line compared with that in the normal liver tissue. This limits the capability of the direct mRNA nanopore sequencing for studying alternative splicing of cytochrome P450 transcripts in HepG2 cells. Both quantitative and qualitative profiles of the cytochrome P450 gene expression at the transcript level are notably differ in human liver tissue and HepG2 cells.

[PCR analysis of the expression of chromosome 18 genes in human liver tissue: interindividual variability]. / PTsR-analiz ékspressii genov khromosomy 18 v tkani pecheni cheloveka: mezhindividual'naia variabel'nost'.

Using human chromosome 18 (Ch18) genes as an example, a PCR analysis of the interindividual variability of gene expression in liver tissue was performed. Although the quantitative profiles of the Ch18 transcriptome, expressed in the number of cDNA copies per single cell, showed a high degree of correlation between donors (Pearson correlation coefficients ranged from 0.963 to 0.966), the expression of the significant number of genes (from 13% to 19%, depending on the method of experimental data normalization) varied by more than 4-fold when comparing donors pairwise. At the same time, the proportion of differentially expressed genes increased with a decrease in the level of their expression. It is shown that the higher quantitative variability of low-abundance transcripts is mainly not technical, but biological. Bioinformatic analysis of the interindividual variability of the differential expression of chromosome 18 genes in human liver tissue did not reveal any statistically significant groups of genes related to certain biological processes that indicated a rather transient nature of the interindividual variability of their expression, probably reflecting the response of cells of an individual to specific external stimuli.

[Ten years of the Russian metabolomics: history of development and achievements]. / Desiat' let rossiiskoi metabolomike: istoriia razvitiia i osnovnye rezul'taty.

Metabolomics is one of the omics sciences, the technologies of which are widely used today in many life sciences. Its application influenced the discovery of new biomarkers of diseases, the description of biochemical processes occurring in many organisms, laid the basis for a new generation of clinical laboratory diagnostics. The purpose of this review is to show how metabolomics is represented in the studies of Russian scientists, to demonstrate the main directions and achievements of the Russian science in this field. The review also highlights the history of metabolomics, existing problems and the place of Russian metabolomics in their solution.

[Mass-spectrometric MRM analysis of FDA-verified proteins in the blood plasma of healthy volunteers]. / Mass-spektrometricheskii MRM analiz FDA-verifitsirovannykh belkov v plazme krovi zdorovykh dobrovol'tsev.

The proteomic composition of a biological sample serves as the most important feature of a biological object, and it allows discriminating normal and pathological conditions. Targeted mass spectrometric analysis, namely, multiple reaction monitoring (MRM) using synthetic isotopically-labeled internal standard (SIS), is the main alternative to the ELISA method for the analysis of diagnostically significant proteins. Based on the MRM results, a prototype test system has been developed; it employs the targeted mass spectrometric method for multiplex, quantitative analysis of FDA-verified proteins in whole blood plasma. Using this approach, it was possible to measure the content of 42 proteins in 31 samples in a concentration range spanning five orders of magnitude. The interindividual variability for 30 of the 42 registered proteins was less than 40%. The largest scatter was observed for haptoglobin (68%), immunoglobulin heavy constant delta IGHD (90%), angiotensin (72%), sex hormone-binding globulin SHBG (100%) and lipoprotein-(a) (136%). The obtained results on the concentration of proteins correlate with published data (Hortin et al., 2008, Clinical Chemistry, 54, 1608) with R2=0.84. The developed prototype test system based on targeted mass spectrometric analysis of proteins can be considered as an alternative to methods using monoclonal antibodies.

[Blood metabolome analysis for creating a digital image of a healthy person]. / Metabolomnyi analiz krovi dlia sozdaniia tsifrovogo obraza zdorovogo cheloveka.

In the frame of the work, data on the implementation of metabolomics tests in medicine have been systematized. Based on the obtained data, a set of protocols was proposed, the sequential realization of which makes it possible to conduct a blood metabolome analysis for medical purposes. Using this analysis and the number of blood samples from healthy volunteers, a prototype of a healthy person's metabolomic image has been developed; it allows visually and digitally to assess the compliance of the human blood metabolome with the norm. At the same time, 99% of the metabolic processes reflected in the blood plasma are estimated. If abnormalities are detected, the metabolomic image allows to get the value of these deviations of metabolic processes in digital terms.

Comparative Analysis of Blood Plasma Proteome in Patients with Renal Cell Carcinoma.

Comparative mass spectrometric analysis of protein composition was carried out in 36 blood plasma specimens from patients with renal cell carcinoma and 20 specimens from donors. Analysis of protein composition of plasma specimens devoid of the major protein fractions showed a 20-50% higher level of protein identifications in patient' specimens. Specimens of the control and experimental series were similar by protein composition, 70-80% identifications in experimental and control series coinciding. High similarity of biological processes with participation of the proteins identified in both series was observed. The greater part of proteins in both series were located extracellularly and were exosomal (specimens from renal cancer patients) or vesicular (specimens from healthy volunteers).

[Quantitative targeted screening of proteins associated with lung adenocarcinoma cancer by the method of selected reaction monitoring]. / Napravlennyi kolichestvennyi skrining markerov adenokartsinomy legkogo metodom monitoringa vybrannykh reaktsii.

In the present study, we applied selected reaction monitoring (SRM) to a group of proteins that were previously reported to be associated with lung cancer (Novikova S.E. et al. (2017) Biomeditsinskaya khimiya, 63, 181-210. [1]). Measurements were performed on 59 plasma samples. These samples included: 23 samples of plasma of patients diagnosed with lung adenocarcinoma (LAC), 11 samples of plasma of patients diagnosed with squamous cell lung carcinoma (SqCC), 25 samples of donors with no previous history of oncological diseases, and one pooled sample from each of the above group. As a result of the SRM measurements 52 proteins were detected at least in one individual plasma sample. Statistical analysis showed that there were two groups confidently differentiated by the concentration value of 8 proteins wherein 5 proteins displayed increased level (P00738, P26639, P21926, P08603, P51149) in LAC group and 3 proteins (P51884, O15162, Q8N2K0) indicated diminishing the concentration level towards the control level. Data on protein concentrations obtained for LAC and SqCC did not distinguish the samples by statistical clustering analysis. These potential biomarkers can be used for further development of methods for early diagnostics of lung cancer.

[Improving of HDL capacity for macrophages cholesterol efflux after plasma incubation with phospholipid nanoparticles]. / Povyshenie sposobnosti lipoproteinov vysokoi plotnosti k vyvedeniiu kholesterina iz makrofagov pri inkubatsii plazmy s ul'tramalymi fosfolipidnymi chastitsami.

In connection with recent data about antiatherogenic importance of not only plasma HDL concentration, but of their cell cholesterol efflux capacity as well, the possibility of its correction by phospholipid (PL) nanoparticles was studied. Blood plasma was incubated with earlier elaborated PL nanoparticles emulsion with the particle diameter up to 30 nm, and HDL cholesterol efflux capacity of apo B-depleted plasma was studied. Using macrophages THP-1 preloaded 3H-cholesterol were used. The addition of incubated plasma supernatants with the elevated PL/apo A-1 ratio to cell media resulted in almost increase in two fold 3H-cholesterol efflux as compared with native HDL. The maximal efflux was observed at the PL/apo A-1 ratio of 1.06 as compared with native apo B-depleted plasma (the PL/apo A-1 ratio of 0.85). Results suggest possible usage of ultrasmall PL nanoparticles for regeneration of impaired antiatherogenic HDL functionality. This approach seems to be predominant compared with the usage of PL emulsions with detergent or apoprotein A1.

[Methods for determining of cytochrome P450 isozymes functional activity]. / Metody opredeleniia funktsional'noi aktivnosti izofermentov tsitokhroma P450.

The review is dedicated to modern methods and technologies for determining of cytochrome P450 isozymes functional activity, such as absorbance and fluorescent spectroscopy, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), Raman, Mossbauer, and X-ray spectroscopy, surface plasmon resonance (SPR), atomic force microscopy (AFM). Methods of molecular genetic analysis were reviewed from personalized medicine point of view. The use of chromate-mass-spectrometric methods for cytochrome P450-dependent catalytic reactions' products was discussed. The review covers modern electrochemical systems based on cytochrome P450 isozymes for their catalytic activity analysis, their use in practice and further development perspectives for experimental pharmacology, biotechnology and translational medicine.

[Use of DNA-aptamers for enrichment of low abundant proteins in cellular extracts for quntitative detection by selected reaction monitoring]. / Primenenie DNK-aptamerov dlia obogashcheniia nizkopredstavlennykh belkov v kletochnykh ékstraktakh dlia kolichestvennoi detektsii metodom monitoringa vybrannykh reaktsii.

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of target protein with aptamers immobilized on a solid phase was studied. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as model target protein. It has been demonstrated that the affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads results in an approximately 10-fold increase of the concentration of target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM is possible without an interference from other peptides present in a tryptic digest.

[Pharmacological targets for dislipidemies correction. Opportunities and prospects of therapeutic usage]. / Farmakologicheskie misheni korrektsii dislipidemii. Vozmozhnosti i perspektivy terapevticheskogo ispol'zovaniia.

Literature data on influence of existing and new groups of drug preparations for dyslipidemias correction are systemized, and molecular mechanisms of their effects are reviewed. The results of experimental and clinical investigations aimed at revealing of new pharmacological targets of dyslipidemias correction were analyzed. The approaches for activation of high density lipoproteins functionality are described. The implementation of alternative preparations with new alternative mechanisms of action may be suggested to improve the effectiveness of traditional treatment in the future.

A New Method for Quantitative Determination of Steroid Metabolites of Cytochrome P450-Dependent Reactions Using Fluorescent Spectroscopy.

A method for determination of hydroxylase activity of cytochrome P450 3A4 (CYP3A4) towards its substrate hydrocortisone using fluorescent analysis of the product was developed. 6ß-hydroxycortisol, formed during CYP3A4-dependent electrocatalysis, has a characteristic fluorescent peak at λ = 427 ± 2 nm after treating with the sulfuric acid : ethanol (3 : 1) mixture and excitation at λ = 365 nm, which is different from the substrate (hydrocortisone) fluorescence (λ = 525 ± 2 nm). The limit of detection of 6ß-hydroxycortisol was 0.32 µM. The developed analytical approach was used to determine the kinetic parameters of CYP3A4-dependent hydrocortisone hydroxylation.

The detection of hepatitis c virus core antigen using afm chips with immobolized aptamers.

In the present study, the possibility of hepatitis C virus core antigen (HCVcoreAg) detection in buffer solution, using atomic force microscope chip (AFM-chip) with immobilized aptamers, has been demonstrated. The target protein was detected in 1mL of solution at concentrations from 10-10М to 10-13М. The registration of aptamer/antigen complexes on the chip surface was carried out by atomic force microscopy (AFM). The further mass-spectrometric (MS) identification of AFM-registered objects on the chip surface allowed reliable identification of HCVcoreAg target protein in the complexes. Aptamers, which were designed for therapeutic purposes, have been shown to be effective in HCVcoreAg detection as probe molecules.

Quantitative target proteomics of chromosome 13 human blood plasma proteins.

Quantitative proteomic analysis of 50 blood plasma samples of healthy volunteers who underwent a comprehensive medical examination and were found eligible for space flights was performed. As a result of directed mass spectrometric analysis, signals for 128 proteins, which accounted for nearly 40% of the total number of chromosome 13 gene products, were detected. The analysis of interindividual variation of concentrations of chromosome 13 proteins showed the presence of a pool comprising 41 proteins with a low variation (CV < 30%), which can potentially be used as biomarkers.

[Protective action of fish muscle extracts against cellular senescence induced by oxidative stress]. / Zashchitnoe deistvie ékstraktov iz myshts ryb ot indutsirovannogo okislitel'nym stressom kletochnogo stareniia.

Muscle extracts of some fish species, i.e. pike (Esox lucius), sterlet (Acipenser ruthenus), pink salmon (Oncorhynchus gorbuscha) and, to a lesser extent, perch (Perca fluviatilis) and Russian sturgeon, (Acipenser gueldenstaedtii) prevent the development of premature senescence of the human embryonic fibroblasts induced by the sublethal concentration of H2O2. Muscle extracts of other fish species tested, i.e. coho salmon (Oncorhynchus kisutch) and zander (Sander lucioperca), have not demonstrated this feature. Cell proliferation increased after the action of the senescence-inhibiting muscle extracts. Possible mechanisms of the action of nature biologically active compounds that interfere with the development of stress-induced cell senescence are discussed.