A novel preliminary metabolomic panel for IHD diagnostics and pathogenesis.

Cardiovascular disease (CVD) represents one of the main causes of mortality worldwide and nearly a half of it is related to ischemic heart disease (IHD). The article represents a comprehensive study on the diagnostics of IHD through the targeted metabolomic profiling and machine learning techniques. A total of 112 subjects were enrolled in the study, consisting of 76 IHD patients and 36 non-CVD subjects. Metabolomic profiling was conducted, involving the quantitative analysis of 87 endogenous metabolites in plasma. A novel regression method of age-adjustment correction of metabolomics data was developed. We identified 36 significantly changed metabolites which included increased cystathionine and dimethylglycine and the decreased ADMA and arginine. Tryptophan catabolism pathways showed significant alterations with increased levels of serotonin, intermediates of the kynurenine pathway and decreased intermediates of indole pathway. Amino acid profiles indicated elevated branched-chain amino acids and increased amino acid ratios. Short-chain acylcarnitines were reduced, while long-chain acylcarnitines were elevated. Based on these metabolites data, machine learning algorithms: logistic regression, support vector machine, decision trees, random forest, and gradient boosting, were used for IHD diagnostic models. Random forest demonstrated the highest accuracy with an AUC of 0.98. The metabolites Norepinephrine; Xanthurenic acid; Anthranilic acid; Serotonin; C6-DC; C14-OH; C16; C16-OH; GSG; Phenylalanine and Methionine were found to be significant and may serve as a novel preliminary panel for IHD diagnostics. Further studies are needed to confirm these findings.

Changes in the Expression of Pluripotency Factor Oct-4 and Intensity of Apoptosis in the Uterus during Spontaneous and Immune-Dependent Abortions in Mice.

We studied the expression of pluripotency factor Oct-4 and the intensity of apoptosis in the uterus during spontaneous and immune abortions in mice. Increased expression of factor Bax and reduced protein Bcl-2 synthesis in cells of the decidual membrane and decreased Oct-4 expression in the myometrium and perimetrium were detected. Thus, both spontaneous and immune-dependent abortions impair the apoptosis processes in the decidua and the formation of a pool of Oct-4+ cells in the uterus. In immune-dependent abortions, the intensity of apoptosis of decidual cells was lower than in spontaneous abortion. Low expression of the transcription factor Oct-4 in the myometrium and perimetrium characterizes pregnancy failure irrespective of its mechanisms.

[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.

Morphofunctional Changes in Colon after Cold Stress in Male C57BL/6 Mice Susceptible and Tolerant to Hypoxia.

There are individual differences in the tolerance to hypoxia and stress. Stress can contribute to the development of various diseases, including inflammatory bowel disease. It was found that inflammatory bowel diseases in animals susceptible to hypoxia runs more severe course than in tolerant animals. We studied morphofunctional changes in the colon under conditions of modeled cold stress in male C57BL/6 mice susceptible and tolerant to hypoxia. The animals were daily subjected to cold stress (20 min at -20°C) for 2 weeks. Cold stress was followed by an increase in the volume fraction of goblet cells in the colon and production of mucins by these cells in mice tolerant to hypoxia and an increase in cell content in the lamina propria of the colon mucous membrane in animals susceptible to hypoxia. The number of serotoninproducing endocrine cells increased in both groups, but these changes were more pronounced in mice susceptible to hypoxia.

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.

Infradian Rhythm in Proliferative Activity of a Culture of Embryonic Fibroblast-Like Cells from C57BL/6 Mice.

We studied daily dynamics of proliferative activity of embryonic fibroblast-like cells in culture during the logarithmic growth phase. Daily increase in cell count in the culture showed a 4-day rhythm that persisted over 3 weeks of culturing. In cultures from different animals, the phase of this rhythm was synchronous and did not depend on the moment of cell isolation. It can be hypothesized that the 4-day biorhythm of proliferative activity of embryonic fibroblast-like cells in culture is determined by external environmental factors, probably of electromagnetic nature.

[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.

[Medical subject headings for the scientific groups evolution analysis on the example of academician A.I. Archakov's scientific school]. / Meditsinskie predmetnye rubriki dlia analiza évoliutsii nauchnykh grupp na primere nauchnoi shkoly akademika A.I. Archakova.

This paper proposes a method of comparative analysis of scientific trajectories based on bibliographic profiles. The bibliographic profile ("meshprint") is a list of MeSH terms (key terms used to index articles in the PubMed), indicating the relative frequency of occurrence of each term in the scientist's articles. Comparison of personalized bibliographic profiles can be represented in the form of a semantic network, where the nodes are the names of scientists, and the relationships are proportional to the calculated measures of similarity of bibliographic profiles. The proposed method was used to analyze the semantic network of scientists united by the academic school of the academician A.I. Archakov. The results of the work allowed us to show the relationship between the scientific trajectories of one scientific school and to correlate the results with world trends.

Methods of Computational Interactomics for Investigating Interactions of Human Proteoforms.

Human genome contains ca. 20,000 protein-coding genes that could be translated into millions of unique protein species (proteoforms). Proteoforms coded by a single gene often have different functions, which implies different protein partners. By interacting with each other, proteoforms create a network reflecting the dynamics of cellular processes in an organism. Perturbations of protein-protein interactions change the network topology, which often triggers pathological processes. Studying proteoforms is a relatively new research area in proteomics, and this is why there are comparatively few experimental studies on the interaction of proteoforms. Bioinformatics tools can facilitate such studies by providing valuable complementary information to the experimental data and, in particular, expanding the possibilities of the studies of proteoform interactions.

Morphological Characteristic of Melanoma B16 Progression in C57BL/6 Mice with High and Low Resistance to Hypoxia.

The features of B16 melanoma progression in male C57BL/6 mice with initially high and low resistance to hypoxia were studied. To assess the resistance to hypoxia, the mice were placed in a low-pressure chamber at a simulated altitude of 10,000 m. One month after testing, B16 melanoma was inoculated to high- and low-resistant animals. In 19 days after melanoma transplantation, the severity of melanoma progression was assessed by morphological and immunofluorescent methods. The expression of vegf-a and hif-1a in the liver of melanomabearing and control mice was evaluated by real-time PCR. Tumor growth progression was more pronounced in low-resistant mice, which was seen from high weight of the primary tumor node, relative necrosis area, proliferation rates (mitotic index and number of Ki-67+ cells), and expression of vegf-a gene in the liver. In high-resistant to hypoxia animals, the number of caspase-3+ cells dying by apoptosis was higher. The data on more rapid melanoma progression in mice with low resistance to hypoxia should be considered during the search of new prognostic markers and methods for therapy of malignant neoplasms.

Infradian and Ultradian Rhythms of Body Temperature Resumption during Hibernation.

The rhythms of short-term arousal episodes, associated with normalization of low body temperature, were studied in hibernating Erinaceus roumanicus. The episodes of body temperature recovery during hibernation were 1.7 times more incident during the acrophase of 4.058-day rhythm of glucocorticoid hormones, detected previously, than during the batiphase of this rhythm. Ultradian rhythm of arousal episodes conformed to a 4-h biorhythm: the maximum number of body temperature resumption episodes was recorded at 00.00-01.00, 04.00-05.00, 08.00-09.00, 12.00-13.00, 16.00-17.00, and 20.00-21.00. These data indicated that in mammals the mechanisms of infradian and ultradian rhythm maintenance were stable and did not depend on body temperature or were determined by external factors with periods of 4.058 days and 4 h.

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.

[Multiomics study of HepG2 cell line proteome]. / Mul'tiomnaia strategiia issledovaniia proteoma kletochnoi linii gepatotselliuliarnoi kartsinomy HepG2.

Current proteomic studies are generally focused on the most abundant proteoforms encoded by canonical nucleic sequences. Transcriptomic and proteomic data, accumulated in a variety of postgenome sources and coupled with state-of-art analytical technologies, allow to start the identification of aberrant (non-canonical) proteoforms. The main sources of aberrant proteoforms are alternative splicing, single nucleotide polymorphism, and post-translational modifications. The aim of this work was to estimate the heterogeneity of HepG2 proteome. We suggested multiomics approach, which combines transcriptomic (RNAseq) and proteomic (2DE-MS/MS) methods, as a promising strategy to explore the proteome.

[PCR analysis of the absolute number of copies of human chromosome 18 transcripts in liver and HepG2 cells]. / PTsR-analiz absoliutnogo chisla kopii transkriptov khromosomy 18 cheloveka v kletkakh pecheni i linii HepG2.

Using reverse transcription in conjunction with the quantitative real-time PCR or digital droplet PCR, the transcriptome profiling of human chromosome 18 has been carried out in liver hepatocytes and hepatoblastoma cells (HepG2 cell line) in terms of the absolute number of each transcript per cell. The transcript abundance varies within the range of 0.006 to 9635 and 0.011 to 4819 copies per cell for HepG2 cell line and hepatocytes, respectively. The expression profiles for genes of chromosome 18 in hepatocytes and HepG2 cells were found to significantly correlate: the Spearman's correlation coefficient was equal to 0.81. The distribution of frequency of transcripts over their abundance was bimodal for HepG2 cells and unimodal for liver hepatocytes. Bioinformatic analysis of the differential gene expression has revealed that genes of chromosome 18, overexpressed in HepG2 cells compared to hepatocytes, are associated with cell division and cell adhesion processes. It is assumed that the enhanced expression of those genes in HepG2 cells is related to the proliferation activity of cultured cells. The differences in transcriptome profiles have to be taken into account when modelling liver hepatocytes with cultured HepG2 cells.

[Postgenomic Medicine: Alternative to Biomarkers].

In the article relevance of high-performance postgenomic technologies is tackled. The intrinsic problems of the implementation of genomics, proteomics and metabolomics in routine clinical practice are considered. Further development of postgenomic medicine requires severe change in the current research approaches. Avenue for development of such approaches are illustrated by metabolome research of human blood plasma. The postgenomic biomarkers are pictured as molecular iceberg, greater part of which is inaccessible for detection with measurement methods. Due to diversity of protein forms the spectrum of molecular markers will always evaluate in terms of incompleteness and inconsistence regardless of technological development level. These properties of «big data¼ are typical of data intensive domains. Special computational methods are essential for data intensive analytics and hardly suitable for the evidence-based medicine.

[The Russian part of the human proteome project:first results and prospects].

The article summarizes the achievements of the pilot phase (2010-2014) of the Russian part of the international project "Human Proteome" and identifies the directions for further work on the study of the human chromosome 18 proteome in the framework of the project main phase (2015-2022). The pilot phase of the project was focused on the detection of at least one protein for each chromosome 18 protein-coding gene in three types of the biological material. The application of mass spectrometric detection of proteins by the methods of multiple reactions monitoring (MRM) and gene-centric approach made it possible to detect 95% of master forms of proteins, for 60% of which the quantitative assessment of the protein content was obtained in at least one type of the biological material. The task of the main phase of the project is to measure the proteome size of healthy individuals, taking into account the modified protein forms, providing for both the bioinformatics prediction of the quantity of proteins types and the selective experimental measurement of single proteoforms. Since the ranges of protein concentrations corresponding to the normal physiological state have not been identified, the work of the main phase of the project is focused on the study of clinically healthy individuals. The absence of these data complicates significantly the interpretation of the patients' blood proteomic profiles and prevents creating diagnostic tests. In the long term prospect, implementation of the project envisages development of a diagnostic test system based on multiple reactions monitoring (MRM) for quantitative measurement of the protein forms associated with some diseases. Development of such test systems will allow predicting the extent of risk of different diseases, diagnosing a disease at its early stage and monitoring the effectiveness of the treatment.

[International criteria of the research activity of groups and individual scientists in biology and medicine].

Comparison data on results of efficiency of the research institutes (RI) of RAMS by international and own evaluation criteria of the research activity of above institutions in 2011 are presented. The consistency of international and own evaluation results was observed in 21RI (39%), the institutions-leaders found, and in 13 RI (24%) - less effective institutions. Thus, the use of own evaluation criteria only complicates the unbiased assessment of the Russia's position in the international scientific rating.

[Knowledgebase as a tool for monitoring post-genomic medico-biological research].

On-going molecular biological researches are characterized by exponential increase in the amount of experimental data which dictates the necessity to develop relevant information technologies. The knowledge-based approach appears to be a most promising and flexible tool for analytical processing biomedical information that allows new relationships between study objects to be established. In this article we analyze the work of researchers based at the Institute of Biomedical Chemistry with the materials of the PubMed biomedical library. A list of publication IDs for a half-year period has been compiled to elucidate individual profiles of research activities. Statistical analysis of medical subject headings (MeSH) reveals typical profiles of research of interest for separate divisions of the Institute. The proposed approach may be recommended as a means for improving the efficiency and coordination of biomedical research.