RESUMO
The aim of this work was to assess the relationship of rs2230806 SNP of ABCA1 with lipid profile in patients with severe dyslipidemia. The study included 363 patients (42.8% of males), the average age was 48.7 years, 35.5% of patients received hypolipidemic drugs (mainly statins). Quantitative determination of total cholesterol (ТС) and triglycerides (TG) in fasting serum was carried out by a unified enzymatic method, and high density lipoproteins (HDL) - by a direct homogeneous method. Genotype according to the rs2230806 position in the ABCA1 gene was determined by polymerase chain reaction (PCR) «in real time¼ using adjacent samples and melting reaction products after PCR. The frequencies of alleles and genotypes of variant rs2230806 of ABCA1 gene in patients with dyslipidemia did not differ from those in the control group of healthy individuals (athletes). The levels of plasma lipids - TC, TG and HDL cholesterol, on average, in patients with dyslipidemia were 7.8±3,4, 3,4±6,5 and 1.29±0.4 mmol/l, respectively. Compared to different genotypes, the plasma lipid concentrations did not differ significantly, but the analysis of different inheritance models of the allelic variant studied showed a significant association with the level of TG in the additive model, in which each minor allele (a) further enhanced the effect on the level of plasma TG at 1.02 mmol/l (p=0.044). The results of this study demonstrate the effect of a common variant rs2230806 of the ABCA1 gene on the plasma TG level in patients with severe dyslipidemia.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Dislipidemias/genética , Lipídeos/sangue , Alelos , HDL-Colesterol/sangue , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Triglicerídeos/sangueRESUMO
The total of 54 patients with chronic periodontitis of different severity was tested using real-time PCR (Dentoflor kit). The group included 38 patients with chronic gastritis. For the first time, a higher prevalence of Treponema denticola in periodontium of males in comparison with females was demonstrated. The patients with chronic gastritis had more human genome DNA at their periodontium than healthy individuals. Non-parametric statistical analysis demonstrated high association of periodontium colonization with. T. forsythensis and T. denticola (but not Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia) with the severity of the chronic periodontitis.
Assuntos
Bactérias/genética , Periodontite Crônica/microbiologia , Duodenopatias/microbiologia , Microbioma Gastrointestinal/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Gastropatias/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Real-time quantitative PCR (Dentofl or kit) was used to detect DNA of periodontal pathogens in specimens from 92 patients with chronic periodontitis and from a control sample of 12 normal subjects. A bimodal distribution of patients by periodontium colonization with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythensis, and T. denticola was demonstrated. A new approach to interpretation of the results of quantitative evaluation of periodontal pathogens, including the notion of pathological colonization level, led to classification of all cases with chronic generalized periodontitis into 3 groups: associated with A. actinomycetemcomitans, with T. forsythensis/T. denticola complex, and cases of uncertain genesis.
Assuntos
Aggregatibacter actinomycetemcomitans/isolamento & purificação , Periodontite Crônica/microbiologia , Periodonto/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Tannerella forsythia/isolamento & purificação , Treponema denticola/isolamento & purificação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Pessoa de Meia-Idade , Periodonto/patologia , Reação em Cadeia da Polimerase em Tempo Real , Adulto JovemRESUMO
The study revealed positive correlation between bleeding on probing and teeth loss risk with periodontal hypercolonization by Porphyromonas gingivalis, Prevotella intermedia and Treponema denticola. Pathological tooth mobility was associated with hypercolonization by P. intermedia and Tannerella forsythensis. Expression of IL8, TNF-α, MMP8 and MMP9 genes was also assessed in patient groups divided according to the depth of periodontal pockets and-the severity of chronic periodontitis revealing IL8 as positive diagnostic marker.
Assuntos
Periodontite/diagnóstico , Periodontite/genética , Transcriptoma , Adulto , Doença Crônica , Feminino , Marcadores Genéticos , Hemorragia/etiologia , Hemorragia/genética , Humanos , Interleucina-8/genética , Masculino , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Bolsa Periodontal/etiologia , Bolsa Periodontal/genética , Periodontite/complicações , Periodontite/microbiologia , Porphyromonas gingivalis/isolamento & purificação , Prevotella intermedia/isolamento & purificação , Fatores de Risco , Perda de Dente/etiologia , Perda de Dente/genética , Mobilidade Dentária/etiologia , Mobilidade Dentária/genética , Treponema denticola/isolamento & purificação , Fator de Necrose Tumoral alfa/genética , Adulto JovemRESUMO
By using qPCR system, women as well as men were found to have an equal periodontal pathogen colonization. However, the women are subjected to have a higher risk of chronic periodontitis onsets. Women with the chronic periodontitis usually expose an evident hypercolonization with a single pathogen. P. gingivalis is the most prevalent causative agent of the chronic periodontitis in women but not in men. In health as well as in the chronic periodontitis a complex of periodontal pathogens forms such as P. gingivalis, P. intermedia, T. forsythensis and T. denticola. T. forsythensis demonstrates the highest correlation with the chronic periodontitis onset in men. Our data allow us to prove T. forsythensis playing the key role in the forming of periodontal pathogen complex.
Assuntos
Bactérias/classificação , Periodontite Crônica/microbiologia , Microbiota , Bolsa Periodontal/microbiologia , Adulto , Bactérias/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Adulto JovemRESUMO
Allele and genotype frequencies of the -174G/C polymorphism (rs1800795) in the regulatory region of the IL6 gene, which encode anti-inflammatory cytokine interleukin 6, were determined in seven populations representing five ethnic groups from the European part of Russia (440 individuals), as well as in small cohorts that represent populations from 24 countries of Africa and Eurasia (365 individuals). The maps of the geographic distribution of the -174G/C allele frequencies were constructed based on personal (22 populations) and the literature data (66 populations), and the data from dbSNP database obtained by the HapMap project (10 populations). The frequency of the -174G allele varied from 45 to 100% and was characterized by nonrandom geographic distribution. These data could reflect the adaptive load of the alleles examined, which was different in different regions of the world. It is suggested that the level of pathogen prevalence is one of the environmental factors that determine different adaptive values of the IL6*--174G/C alleles. This suggestion is supported by a positive correlation between the -174G allele frequency and level of pathogen prevalence calculated based on historical data (R = 0.768; p < 0.0001).
Assuntos
Frequência do Gene/genética , Interleucina-6/genética , Polimorfismo de Nucleotídeo Único/genética , Sequências Reguladoras de Ácido Nucleico/genética , Meio Ambiente , Projeto HapMap , Humanos , Federação Russa , População Branca/genéticaRESUMO
The study included 179 patients aged 18-45 years (56 with aggressive periodontal disease and 123 with no clinical signs of periodontitis). Gene polymorphism was analyzed by means of real-time PCR and kissing probes in order to reveal marker panel for aggressive periodontal disease predisposition. Significant difference was found for gene MMP9 in rs17576 position for A allele (55.2% in aggressive periodontal disease patients and 69.5% in control group) and in rs3918242 position for C allele (67.7 and 81.7%, respectively).
Assuntos
Periodontite Agressiva/epidemiologia , Periodontite Agressiva/genética , Predisposição Genética para Doença , Adolescente , Adulto , Alelos , Feminino , Marcadores Genéticos , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Polimorfismo Genético , Reação em Cadeia da Polimerase em Tempo Real , Risco , Adulto JovemRESUMO
The representation patterns of 15 cytokines RNA in blood plasma and blood cells of patients with breast cancer and apparently healthy women were investigated. Relative levels of RNA IL-8 and IL-18 in plasma of breast cancer patients are significantly increased compared with control group. At the same time no obvious differences were found in relative concentrations of these transcripts in blood cells of patients and control groups. Relative concentration of IL-8 RNA was higher in blood plasma of locally advanced compared with early breast cancer patients.
Assuntos
Neoplasias da Mama/diagnóstico , Interleucina-18/biossíntese , Interleucina-8/biossíntese , RNA/sangue , Adulto , Idoso , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Transcrição GênicaRESUMO
Mouth human populated set of microorganisms that are in dynamic equilibrium and forming microbiocaenosis. In a situation where this balance is disturbed, there is a "activation" of pathogens, including those that lead to the development of inflammatory periodontal diseases. Quantifying the relation parodontopatogenov in this material may be an important diagnostic tool, but data on the profile of individual microbiota subbiotopov mouth so far very little. In this study, we quantified the six pathogenic representatives of the periodontal pocket microbiota in health and periodontitis. Found that when disease development relationship pathogenic representatives of periodontal pocket microbiota varies considerably.
Assuntos
Periodontite Agressiva/diagnóstico , Periodontite Agressiva/microbiologia , Bactérias/isolamento & purificação , Boca/microbiologia , Adulto , Bactérias/classificação , Feminino , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
The introduction of a broad medical practice PCR "real time" is just beginning and dentistry is no exception. Modern molecular genetic methods provide numerous opportunities for diagnosis, assessment and prediction in patients with inflammatory periodontal diseases. Early and accurate diagnosis can allow in the future reduce the incidence of periodontitis and the progression of its course.
Assuntos
Aggregatibacter actinomycetemcomitans/isolamento & purificação , Metagenoma , Bolsa Periodontal/diagnóstico , Bolsa Periodontal/microbiologia , Reação em Cadeia da Polimerase/métodos , Desoxirribonucleotídeos/genética , Humanos , Fatores de TempoRESUMO
The level of representation of extracellular RNA 14 cytokines in blood plasma in a group of apparently healthy subjects was analyzed. The level of representation of the transcripts of these cytokines in extracellular medium is characterized by specific profile different from the profile of expression of the genes in blood cells.
Assuntos
Citocinas/genética , Perfilação da Expressão Gênica , RNA Mensageiro/sangue , Feminino , Humanos , MasculinoRESUMO
We aimed to develop a pipeline for the bioinformatic analysis and interpretation of NGS data and detection of a wide range of single-nucleotide somatic mutations within tumor DNA. Initially, the NGS reads were submitted to a quality control check by the Cutadapt program. Low-quality 3¢-nucleotides were removed. After that the reads were mapped to the reference genome hg19 (GRCh37.p13) by BWA. The SAMtools program was used for exclusion of duplicates. MuTect was used for SNV calling. The functional effect of SNVs was evaluated using the algorithm, including annotation and evaluation of SNV pathogenicity by SnpEff and analysis of such databases as COSMIC, dbNSFP, Clinvar, and OMIM. The effect of SNV on the protein function was estimated by SIFT and PolyPhen2. Mutation frequencies were obtained from 1000 Genomes and ExAC projects, as well as from our own databases with frequency data. In order to evaluate the pipeline we used 18 breast cancer tumor biopsies. The MYbaits Onconome KL v1.5 Panel ("MYcroarray") was used for targeted enrichment. NGS was performed on the Illumina HiSeq 2500 platform. As a result, we identified alterations in BRCA1, BRCA2, ATM, CDH1, CHEK2, TP53 genes that affected the sequence of encoded proteins. Our pipeline can be used for effective search and annotation of tumor SNVs. In this study, for the first time, we have tested this pipeline for NGS data analysis of samples from patients of the Russian population. However, further confirmation of efficiency and accuracy of the pipeline is required on NGS data from larger datasets as well as data from several types of solid tumors.
Assuntos
Biologia Computacional , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias/genética , Análise de Dados , Humanos , Mutação , Federação RussaRESUMO
Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.
Assuntos
Clonagem Molecular/métodos , Reações Falso-Positivas , Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Telencéfalo/metabolismo , Animais , DNA Complementar/análise , DNA Complementar/genética , Camundongos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
The digestion of the milk sugar (lactose) is observed every normal child but not in every adult. The decreased lactase synthesis in some adults results in problems with digestion of the whole milk (primary hypolactasy). An association of lactase activity in adults with carrying of the allele T within the polymorphism C/T-13910 located upstream of the lactase gene and 100% association of hypolactasy with the genotype C/C has recently been shown for a Finnish sample. In the present work we determined the LCT* C/T_13910 genotypes and allele frequencies in populations from Russia. The genotype C/C frequencies varied from 36.6% for Russians to 88.2% for Chukchi and were close to the published medical and epidemiological data on hypolactasy frequencies in respective populations. Genotyping was performed by three different methods to identify the optimal one. Our results have shown that the studied locus is the key determinant for the primary hypolactasy development in various human populations. Consequently, the DNA diagnostics of the C/C genotype carrying is a promising predictive test to detect the primary hypolactasy long before its clinical development. Practical application of this type of diagnostics would be a step towards the individual-oriented medicine.
Assuntos
Frequência do Gene , Testes Genéticos/métodos , Lactase/genética , Intolerância à Lactose/diagnóstico , Marcadores Genéticos , Genótipo , Humanos , Intolerância à Lactose/genética , Fenótipo , Polimorfismo Genético , População/genética , Prognóstico , Federação RussaRESUMO
The existing diversity of the methods for detecting single nucleotide polymorphisms is so great that may perplex an unsophisticated researcher who chooses the appropriate molecular genetic toolkit. In this work, we tried to systematize and briefly describe the state-of-the-art methods for detecting oligonucleotide polymorphisms that are based on allele-specific PCR and hybridization with oligonucleotide probe as well as to characterize the methods considered with respect to their accuracy, cost, and simplicity.
Assuntos
Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Alelos , Sondas de Oligonucleotídeos/genéticaRESUMO
The registration of the accumulation of polymerase chain reaction (PCR) products in the course of amplification (real-time PCR) requires specific equipment, i.e., detecting amplifiers capable of recording the level of fluorescence in the reaction tube during amplicon formation. By the time the reaction is completed, researchers obtain DNA accumulation graphs. The review discusses the most promising algorithms of analysis of real-time PCR curves and possible errors, whether caused by the software used or the operators' mistakes. The data included will assist researchers in understanding the features of the method to obtain more reliable results.
Assuntos
Algoritmos , Reação em Cadeia da Polimerase , Software , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase/tendências , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
The accuracy of a real-time polymerase-chain-reaction assay for genetically modified sources in food products was determined using two official test systems (kits) of primers and samples. These kits were recommended by the Federal Center of State Sanitary and Epidemiological Surveillance (Russian Ministry of Health) and the European Commission. We used the following three models of thermocyclers: iCycler iQ (BioRad, United States), Rotor-Gene 3000 (Corbett Research, Australia), and DT-322 (DNA-Technology, Russia). Studies of samples that contained 1% genetically modified sources showed that the error of a quantitative assay for genetically modified sources in food products corresponds to 20-30% and does not depend on the kit type and the thermocycler model used.
Assuntos
Primers do DNA/química , Farinha/análise , Alimentos Geneticamente Modificados , Reação em Cadeia da Polimerase , Zea mays , Primers do DNA/genética , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase/instrumentação , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Zea mays/genéticaRESUMO
The main physicochemical characteristics of the major isoform of the laccase secreted by the fungu, Trametes hirsuta 072 were studied. The enzyme belongs to the group of high redox potential laccases (E(T1) = 790 +/- 5), and it oxidizes with high efficiency various substrates of phenolic nature. The gene of this isoform was cloned, and its nucleotide sequence was determined. The length of the complete gene is 2134 bp. It comprises 11 exons and 10 introns. Analysis of the amino acid sequence of T. hirsuta 072 laccase demonstrated a high homology (to 96.9%) to the other laccases secreted by fungi of the genus Trametes.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Lacase/química , Lacase/genética , Polyporales/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Proteínas Fúngicas/isolamento & purificação , Genes Fúngicos , Lacase/isolamento & purificação , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
A new cryI-related sequence designated cryIM was cloned using an immunoscreening technique from ssp. wuhanensis of Bacillus thuringiensis (BT), previously reported to produce multiple Cry proteins [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034]. Analysis of the cryIM nucleotide sequence revealed an ORF, BTII-type promoter-like sequence, peculiar for such genes, a translation initiation element and a putative transcription terminator. Nevertheless, its product was not previously found in the crystals of the host strain [Chestukhina et al. (1994) Can. J. Microbiol. 240, 1026-1034] which shows its weak or absent natural expression. The amino acid sequence of 1151 residues encoded by the continuous reading frame of cryIM is not identical but is essentially similar to the other delta-endotoxins of the CryI class. An IS231-like sequence was found 400 bp downstream of the cryIM stop codon and a fragment of the cryIAb gene was located 3 kb upstream of its initiator codon in the same orientation. Artificial expression of the cloned gene in E. coli under the control of the lacZ promoter allowed us to obtain its hypothetical protein product.