RESUMO
Genitourinary cancer (GUC) represents more than one fifth of all human cancers. This makes the development of approaches to its early diagnosis an important task of modern biomedicine. Circulating microRNAs, short (17-25 nucleotides) non-coding RNA molecules found in human biological fluids and performing a regulatory role in the cell, are considered as promising diagnostic and prognostic biomarkers of cancers, including GUC. In this review we have considered the current state of research aimed at assessing microRNAs as biomarkers of such human GUC types as malignant tumors of the bladder, kidney, prostate, testicles, ovaries, and cervix. A special attention has been paid to studies devoted to the identification of microRNAs in urine as a surrogate "liquid biopsy" that may provide the simplest and cheapest approach to mass non-invasive screening of human GUC. The use of microRNA panels instead of single types of microRNA generally leads to higher sensitivity and specificity of the developed diagnostic tests. However, to date, work on the microRNAs assessment as biomarkers of human GUC is still of a research nature, and the further introduction of diagnostic tests based on microRNAs into practice requires successful clinical trials.
Assuntos
Biomarcadores Tumorais , Neoplasias Urogenitais , Humanos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Neoplasias Urogenitais/diagnóstico , Neoplasias Urogenitais/genética , Neoplasias Urogenitais/metabolismo , Feminino , Masculino , MicroRNAs/urina , MicroRNAs/sangue , Prognóstico , Biópsia Líquida/métodos , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismoRESUMO
Using the method of shotgun mass spectrometry, we have evaluated changes in the proteomic profile of HaCat cells in response to the treatment with sodium dodecyl sulfate (anionic surfactant) and Triton-X100 (non-ionic surfactant) in two concentrations (12.5 µg/ml and 25.0 µg/ml). The study revealed induction of orphan CYP2S1 (biotransformation phase I) in response to Triton-X100. We have identified proteins of II (glutathione-S-transferases, GSTs) and III (solute carrier proteins, SLCs) biotransformation phases, as well as antioxidant proteins (peroxiredoxins, PRDXs; catalase, CAT; thioredoxin, TXN). Thus, proteins of all three xenobiotic detoxification phases were detected. The presented results suggest a new prospect of using HaCaT keratinocytes as a model of human epidermis for studying the metabolism of drugs/toxicants in human skin in vitro.
Assuntos
Proteômica , Tensoativos , Humanos , Tensoativos/farmacologia , Queratinócitos , Linhagem Celular , Pele , Octoxinol , Sistema Enzimático do Citocromo P-450RESUMO
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.
Assuntos
Sequenciamento por Nanoporos , Sistema Enzimático do Citocromo P-450/genética , Células Hep G2 , Humanos , Fígado , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Cromossomos Humanos Par 18 , Fígado , Cromossomos Humanos Par 18/genética , Biologia Computacional , Perfilação da Expressão Gênica , Humanos , Reação em Cadeia da Polimerase , TranscriptomaRESUMO
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.
Assuntos
Proteínas Sanguíneas , Proteínas , Proteômica , Proteínas Sanguíneas/análise , Ensaio de Imunoadsorção Enzimática , Voluntários Saudáveis , Humanos , Espectrometria de MassasRESUMO
The effects of medium viscosity on the decay rate of the 4a-hydroperoxyflavin intermediate of the bioluminescent reaction was investigated. It was found that at low concentrations of glycerol or sucrose (viscosity 1.1-1.3 cP) the decay rate rises, whereas a further increase in viscosity to 6.2 cP leads to a decrease in the decay rate following a power function with an exponent of 0.82-0.84. Using molecular dynamics methods, it was shown that the presence of glycerol and sucrose molecules causes a change in the mobility of the amino acid residues in the active center of luciferase, particularly those responsible for binding of flavin. The results obtained are indicative of two opposite effects of viscous media with glycerol and sucrose: (1) destabilization of 4a-hydroperoxyflavin due to a change in the structural and dynamic properties of the protein and (2) stabilization of this intermediate by the decrease in the diffusion rate of its decay products.
Assuntos
Meios de Cultura/metabolismo , Luciferases/química , Luciferases/metabolismo , Photobacterium/enzimologia , Difusão , Cinética , ViscosidadeRESUMO
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.
Assuntos
Metaboloma , Metabolômica , Voluntários Saudáveis , Humanos , PlasmaRESUMO
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.
Assuntos
Algoritmos , Bibliometria , Medical Subject Headings , Editoração/tendências , PubMedRESUMO
Using random (combinatorial) DNA-libraries with various degrees of diversity, it was shown that their amplification by polymerase chain reaction in real time resulted in appearance of a maximum on amplification curves. The relative decrease of fluorescence after passing the maximum was directly proportional to the logarithm of the number of oligonucleotide sequence variants in the random DNA-library provided that this number was within in the interval from 1 to 104 and remained practically unaltered when the number of variants was in the interval from 105 to 108. The obtained dependence was used in the course of SELEX to evaluate changes in the diversity of random DNA-libraries from round to round in selection of DNA-aptamers to the recombinant SMAD4 protein. As a result, oligonucleotides containing sequences able to form a site of SMAD4-DNA interactions known as SBE (SMAD-binding element) have been selected thus indicating that the SMAD4-SBE interaction dominates the aptamer selection.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA/química , Biblioteca Gênica , Técnica de Seleção de Aptâmeros , Reação em Cadeia da PolimeraseRESUMO
The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Reação em Cadeia da Polimerase em Tempo Real , Biblioteca Gênica , Cinética , Técnica de Seleção de AptâmerosRESUMO
Proteome profiling of human testicular biopsies was performed using tandem mass spectrometry with electrospray ionization. Protein identification results were compared for the Mascot commercial search engine, the SearchGUI noncommercial package, and their analog IdentiProt based on the open-source IdentiPy algorithm (http://hg.theorchromo.ru/identipy). A feature of IdentiPy is an automatic optimization of MS/MS search parameters. A set of protein identifications obtained with IdentiPy was consequently greater by one third than the sets with the other search engines. For the first time, an IdentiPy/IdentiProt search was conducted within the Progenesis LC-MS framework, which allows spectrum alignment, and the proteome profile obtained with alignment was compared with that obtained using the ProteoWizard converter. A total of 16 human chromosome 18 proteins were identified, including the myelin basic protein, which is not characteristic of testicular tissue.
Assuntos
Algoritmos , Proteômica , Espectrometria de Massas em Tandem , Testículo/patologia , Biópsia , Bases de Dados de Proteínas , Humanos , Masculino , SoftwareRESUMO
A critical analysis of proteomes provides a basis for understanding the operation of complex biochemical systems. A personalized approach to therapy takes into account biological uniqueness of each patient at genome, transcriptome, and proteome levels, and is a priority area in molecular medicine. The identification of proteoforms, which have dramatic impact on the phenotype of a disease, is a fundamental task of personal molecular profiling. Considerable progress of proteomic approaches presented new avenues for accurate, specific, and high-performance protein analysis. Thus, the identification of new efficient bio-markers can be expected based on studies of aberrant proteoforms associated with various diseases.
Assuntos
Medicina Molecular/métodos , Medicina de Precisão/métodos , Proteoma/metabolismo , Proteômica/métodos , Animais , Humanos , Proteoma/genéticaRESUMO
OMERO service was used to annotate the cell line HaCaT microscope images by two independent expert groups. The images were obtained in the course of developing tissue-engineered epithelium which consisted of several layers of the keratinocytes. Evaluation of expert opinions was performed by calculation of specificity, sensitivity and accuracy. The best convergence of opinions (91%) was achieved for the confluence of the cell monolayers. Accuracy 70% was observed in determining the extent of cell differentiation after 10 days of incubation. The paper illustrates the usefulness of OMERO service for dynamic cross-validation of quality in the development and standardization of cell preparations.
Assuntos
Pele , Diferenciação Celular , Queratinócitos , Controle de Qualidade , Engenharia TecidualRESUMO
A modification of the enzymatic method for the preparation of combinatorial random DNA libraries, which combines amplification in isolated microvolumes with the simultaneous incorporation of modified nucleotides and subsequent separation of DNA strands, was developed. Deoxyuridine triphosphate with hydrophobic substituents such as structural analogues of amino acid side chains in the C5 position of the pyrimidine ring was used to introduce modifications into DNA. To prevent competitive amplification, which reduces the representativeness of combinatorial libraries, PCR in inverse emulsion was used. The separation of the strands of PCR products was carried out. There were six single-stranded DNA libraries with complete substitution of deoxythymidine via modified analogues with various functional groups. These DNA libraries are suitable for generating aptamers to protein targets through additional hydrophobic interactions from the introductions of appropriate modifications, and are completely compatible with the SELEX aptamer selection methodology.
Assuntos
Aptâmeros de Nucleotídeos , DNA/isolamento & purificação , Biblioteca Gênica , Reação em Cadeia da Polimerase , Técnica de Seleção de AptâmerosRESUMO
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.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Cromossomos Humanos Par 13/genética , Proteômica , Voluntários Saudáveis , HumanosRESUMO
Oxidative stress is a universal response of the skin cell damage of various origins. Sodium dodecyl sulfate (SDS, sodium lauryl sulfate) is an anionic surfactant commonly used as an emulsifying detergent in household cleaners. Sodium dodecyl sulfate is the reference compound for testing toxicity on cellular skin models. The effect of sodium dodecyl sulfate in sub toxic dose 25 µg/mL during 48 h on the protein profile of human keratinocytes HaCaT was studied by tandem mass spectrometry with electrospray ionization. In total, 1064 proteins were found in immortalized human keratinocytes HaCaT, of which about 80% were identified by two or more peptides. The change of the 217 proteins content was revealed, among them 39 according to Gene Ontology are associated with oxidative stress. It has been found that sodium dodecyl sulfate leads to a decrease in the number of proteins/peptides containing carboxymethylated and/or carboxyethylated lysine. We concluded about the promising of the cells redox-balance analysis at testing chemicals in the doses, which do not lead to a decrease in their viability. Possible involvement of sodium dodecyl sulfate in the development of cutaneous neoplasia is discussed.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Queratinócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteoma/biossíntese , Proteômica , Dodecilsulfato de Sódio/farmacologia , Linhagem Celular Transformada , Humanos , Queratinócitos/citologiaRESUMO
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.
Assuntos
Proteoma , Transcriptoma , Processamento Alternativo , Perfilação da Expressão Gênica , Células Hep G2 , Humanos , Polimorfismo de Nucleotídeo Único , Processamento de Proteína Pós-Traducional , Proteômica , Análise de Sequência de RNA , Espectrometria de Massas em TandemRESUMO
The effects of sodium dodecyl sulfate (25 mg/ml) and Triton X-100 (12.5 mg/ml and 25 mg/ml) on the HaCaT immortalized keratinocytes exposed to these surfactants for 48 h were studied. Using shotgun proteomics, a comparative analysis of the proteomic profiles of control and experimental cells after surfactants exposure was carried out. 260 common proteins were identified in control and experimental cells; 33 proteins were found in cells exposed to all three treatments, but not in control cells. These 33 proteins apparently reflect a nonspecific (universal) response of cells to toxic damage by the surfactants. These proteins are associated with activation of cell proliferation, changes in the functional activity of their ER and mitochondria, increased mRNA stability and activation of protein degradation processes in the cells. The possibility of using these proteins as a nonspecific parameter of cell response to cytotoxic damage is discussed. The mass spectrometry proteomics data ("raw", "mgf" and "xml" files) have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD007789 and PXD007776.
Assuntos
Detergentes/efeitos adversos , Queratinócitos/efeitos dos fármacos , Proteoma/metabolismo , Células Alimentadoras , Humanos , Queratinócitos/metabolismo , Proteômica , Pele , Dodecilsulfato de SódioRESUMO
The human body is an open system that receives a variety of xenobiotics in the course of dietary route or respiration and in the form of the drugs. As a lump sum scores of toxic and potentially toxic substances are detected in a human body that significantly affect health and human lifespan. There are also thousands of diseases, dozens of which latently occur in the body of each person. Traditional diagnosis is not able to screen all the variety of xenobiotics and potential human diseases. For this purpose metabolomic blood test is available which is of non-targeted (review) nature. The test can reveal all the diversity of low molecular weight substances in blood, including tens of thousands of xenobiotics and markers of different diseases. Detection of xenobiotics in the blood, directional detoxification and subsequent monitoring of "body's chemical purity" together with the control of "normality" of all biochemical processes in the organism, using metabolomics blood tests is a necessary and presumably a sufficient condition in the implementation of inherent human genotype longevity. This article describes the purpose, implementation and interpretation of metabolomic blood test facilitating the implementation of this method in the Russian Federation, in order to significantly increase the average life expectancy.
Assuntos
Testes Hematológicos/métodos , Longevidade/fisiologia , Metaboloma , Metabolômica/métodos , Xenobióticos/sangue , Biomarcadores/sangue , Dieta/efeitos adversos , Exposição Ambiental/efeitos adversos , Microbioma Gastrointestinal/fisiologia , Interação Gene-Ambiente , Testes Hematológicos/instrumentação , Humanos , Expectativa de Vida , Estilo de Vida , Metabolômica/instrumentação , Federação Russa , Xenobióticos/administração & dosagemRESUMO
Viability of keratinocytes of HaCaT immortalized line incubated with sodium dodecyl sulfate for 3 min, 1 and 48 h, was studied by light microscopy, MTT test, and neutral red absorption test. The IC50 values were determined for each of the studied lengths of exposure. HaCaT cells exhibited a dose-dependent decrease of viability under the effect of sodium dodecyl sulfate, proportional to the length of exposure. The values measured by different methods (MTT test and neutral red absorption test) varied, the differences were determined by the duration of exposure to sodium dodecyl sulfate. The dispersion of values for 1 and 48 h exposure, obtained by MTT method, was greater than of the values obtained by neutral red absorption test.