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1.
Adv Exp Med Biol ; 1241: 125-138, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32383119

RESUMO

Chronic stress appears to accelerate biological aging, and oxidative damage is an important potential mediator of this process. Many chronic diseases are accompanied by an increase in overall oxidation of genomic DNA. In course of exposure to daily environmental insults, DNA accumulates oxidative damage, which is, in part, repaired, while the cells with the most damaged DNA die either by necrosis or by apoptosis. The oxidized DNA released from the dying cells contributes to the pool of cell-free/extracellular DNA present in plasma and other biological fluids. This cell-free DNA contains a great deal of 8-oxodG bases. The ratio of 8-oxo-dG and unmodified guanine may serve as a cumulative biomarker of stress encountered by a human body within a previous 24 h-period. This true end-point biomarker may outperform other short-lived molecules that reflect only the most current state of oxidant stress. Patient-specific baselines for oxidative damage may be established by measuring of 8-oxo-dG in circulating DNA. Longitudinal profiling of oxiDNA may aid in reliable quantification of the effects of various self-administered nutraceutical and lifestyle based health interventions. Development of wearable electrochemical sensor patches that will quantify oxiDNA in near real-time is warranted to produce life- and health-modifying event awareness feedback.


Assuntos
8-Hidroxi-2'-Desoxiguanosina/sangue , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/química , Dano ao DNA , DNA/sangue , DNA/química , Nível de Saúde , Estresse Oxidativo , 8-Hidroxi-2'-Desoxiguanosina/química , 8-Hidroxi-2'-Desoxiguanosina/metabolismo , Ácidos Nucleicos Livres/metabolismo , DNA/metabolismo , Estilo de Vida Saudável , Humanos
2.
J Cancer Res Clin Oncol ; 146(8): 1971-1978, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32447484

RESUMO

PURPOSE: Interleukin-10 (IL-10) is an immunoregulatory cytokine and its cervical and serum concentrations have been associated with a poor prognosis of cervical cancer. The rs1800872 polymorphism (c.-592C>A) in the promotor region of the IL-10 gene affects the production and expression of IL-10 and thus is able to determine the immune response profile in the cervix. Therefore, the aim of this work is to state the association between IL-10 c.-592C>A polymorphism and cervical cancer. METHODS: Genomic DNA was extracted from patient's peripheral blood and tumor biopsy. Socio-demographic, sexual behavior and reproductive characteristics data were collected using a questionnaire. RESULTS: Co-dominant model in logistic binary regression adjusted for confounders, showed that patients presenting with C/A genotype had 2.15 times more chances for developing cervical cancer (OR 2.15; CI95% 1.02-4.56). The dominant model, C/A + A/A, was also independently associated with 2.71 times more chances for cervical cancer development when compared to control patients (OR 2.71; CI95% 1.05-4.47). CONCLUSION: Our study analyses show the association between cervical cancer and IL-10 c.-592C>A polymorphism, demonstrating that the allele A presence was independently associated with higher risks of cervical cancer development.


Assuntos
Interleucina-10/genética , Neoplasias do Colo do Útero/genética , Adenocarcinoma/sangue , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adulto , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/imunologia , Estudos de Casos e Controles , DNA/sangue , DNA/genética , Feminino , Genótipo , Humanos , Interleucina-10/biossíntese , Interleucina-10/imunologia , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/imunologia
3.
Ecotoxicol Environ Saf ; 197: 110643, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32315786

RESUMO

Meteorological conditions during pregnancy can affect birth outcome, which has been linked to the H19/H19-differentially methylated region (DMR). However, the detailed mechanisms underlying this association are unclear. This was investigated in the present study to provide epidemiological evidence for elucidating the pathogenesis of adverse birth outcomes. A total of 550 mother-newborn pairs were recruited in Zhengzhou, China from January 2010 to January 2012. Meteorological data including temperature (T), relative humidity (RH), and sunshine duration (SSD) were obtained from the China Meteorological Data Sharing Service System. Bisulfite sequencing PCR was performed to determine the methylation levels of H19/H19-DMR using genomic DNA extracted from maternal peripheral and umbilical cord blood. The results showed that H19-DMR methylation status in cord blood was positively associated with that in maternal blood. Neonatal H19-DMR methylation was negatively associated with T and RH during the first trimester and positively associated with these variables during the third trimester. There was a positive correlation between neonatal H19-DMR methylation and SSD during the second trimester and a negative correlation during the third trimester. Similar associations were observed between maternal H19-DMR methylation and prenatal meteorological conditions. We also observed significant interaction effects of maternal H19/H19-DMR methylation and most prenatal meteorological factors on neonatal methylation, and found that changes in the methylation status of maternal H19-DMR were responsible for the effects of prenatal meteorological conditions on neonatal methylation. In summary, neonatal H19-DMR methylation was significantly associated with prenatal meteorological conditions, which was modified and mediated by maternal H19-DMR methylation changes. These findings provide insights into the relationship between meteorological factors during pregnancy and adverse birth outcomes or disease susceptibility in offspring, and can serve as a reference for environmental policy-making.


Assuntos
Metilação de DNA , Sangue Fetal/química , Exposição Materna/efeitos adversos , Conceitos Meteorológicos , RNA Longo não Codificante/genética , Adulto , China , DNA/sangue , Feminino , Impressão Genômica , Humanos , Recém-Nascido , Gravidez , Regiões Promotoras Genéticas , RNA Longo não Codificante/sangue , Adulto Jovem
4.
PLoS One ; 15(4): e0230482, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32310973

RESUMO

Acute transfusion reactions can manifest in many forms including acute hemolytic transfusion reaction, allergic reaction and transfusion-related acute lung injury. We previously developed an acute hemolytic transfusion reaction rat model mediated by transfusion of incompatible human erythrocytes against which rats have preexisting antibodies resulting in classical complement pathway mediated intravascular hemolysis. In this study, the acute hemolytic transfusion reaction model was adapted to yield an acute lung injury phenotype. Adolescent male Wistar rats were primed in the presence or absence of lipopolysaccharide followed by transfusion of incompatible erythrocytes. Blood was collected at various time points during the course of the experiment to determine complement C5a levels and free DNA in isolated plasma. At 4 hours, blood and lung tissue were recovered and assayed for complete blood count and histological acute lung injury, respectively. Compared to sham animals or animals receiving increasing amounts of incompatible erythrocytes (equivalent to a 15-45% transfusion) in the absence of lipopolysaccharide, lungs of animals receiving lipopolysaccharide and a 30% erythrocyte transfusion showed dramatic alveolar wall thickening due to neutrophil infiltration. C5a levels were significantly elevated in these animals indicating that complement activation contributes to lung damage. Additionally, these animals demonstrated a significant increase of free DNA in the blood over time suggestive of neutrophil extracellular trap formation previously associated with transfusion-related acute lung injury in humans and mice. This novel 'two-hit' model utilizing incompatible erythrocyte transfusion in the presence of lipopolysaccharide yields a robust acute lung injury phenotype.


Assuntos
Lesão Pulmonar Aguda , Modelos Animais de Doenças , Transfusão de Eritrócitos , Lipopolissacarídeos/metabolismo , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/patologia , Animais , Incompatibilidade de Grupos Sanguíneos/metabolismo , Complemento C5a/metabolismo , DNA/sangue , Eritrócitos/metabolismo , Armadilhas Extracelulares/metabolismo , Humanos , Masculino , Infiltração de Neutrófilos , Ratos , Ratos Wistar , Reação Transfusional/patologia
5.
Magy Onkol ; 64(1): 70-72, 2020 Mar 17.
Artigo em Húngaro | MEDLINE | ID: mdl-32181765

RESUMO

During colorectal cancer (CRC) development, in addition to genetic alterations, several epigenetic changes, including DNA methylation in the promoter regions accumulate in tumor cells. Cell-free DNA (cfDNA) in the circulatory system can originate also from tumor tissue; therefore the evaluation of methylated cfDNA in the plasma can be a promising method for early cancer screening. In my Ph.D., I have investigated the rate of cfDNA's release and stability using animal models. I aimed to compile an epigenetic marker panel, which contains genes with altered DNA methylation patterns in the healthy-colorectal adenoma-cancer sequence. I have found that the methylation level of SFRP1, SFRP2, SDC2, and PRIMA1 gene promoters has already increased in adenoma stages in both tissue and plasma samples. Immunohistochemistry analyses indicated decreasing protein expression in parallel with elevated methylation. According to our results, cfDNA amount and the methylation have been influenced by DNA isolation and blood collection methods.


Assuntos
Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/química , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Metilação de DNA , DNA/química , Animais , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , DNA/sangue , DNA/genética , Humanos , Regiões Promotoras Genéticas/genética
6.
Sanid. mil ; 76(1): 8-12, ene.-mar. 2020. ilus, tab, graf
Artigo em Espanhol | IBECS | ID: ibc-193134

RESUMO

INTRODUCCIÓN: la abiotrofia cerebelar equina es una enfermedad neurodegenerativa de etiología genética autosómica y recesiva. Se presenta con más frecuencia en el Pura Raza Árabe. La enfermedad está causada por una mutación puntual que produce una degeneración progresiva de las células de Purkinje. OBJETIVOS: puesta a punto de una técnica sencilla, empleando el análisis de curvas de fusión de alta resolución (HRM), que permita identificar los individuos portadores de la mutación causante de la abiotrofia cerebelosa en los ancestros de un animal enfermo. MATERIAL Y MÉTODOS: extracción de ADN de 93 muestras de sangre provenientes de animales emparentados. Diseño y selección de una pareja de cebadores para la amplificación de una secuencia de 89 pares de bases que contiene la mutación. Amplificación de la secuencia y análisis de las curvas de fusión. RESULTADOS: el análisis genealógico confirmó el carácter autosómico recesivo de la enfermedad. La amplificación no generó fragmentos inespecíficos. La técnica de análisis HRM permitió diferenciar de forma inequívoca los genotipos homocigotos sano y enfermo, y también el genotipo heterocigoto de los portadores sanos. Los resultados coinciden con los obtenidos mediante la técnica de análisis de fragmentos publicada en 2011. CONCLUSIÓN: la técnica desarrollada permite detectar caballos portadores de la abiotrofia cerebelosa de forma sencilla


INTRODUCTION: the equine cerebellar abiotrophy is an autosomal recessive neurodegenerative disease. It has been more frequently described in the Arabian horse. The causative mutation produces a progressive degeneration of Purkinje cells. OBJECTIVES: developing a simple technique based on the high-resolution melt analysis (HRM) in order to identify the carriers of the mutation among a group of ancestors of an affected horse. MATERIAL AND METHODS: DNA extraction of 93 blood samples from a group of related animals. Primers designed to amplify an 89 base pair sequence that contains the mutation. Amplification of the sequence and melting curves analysis. RESULTS: the genealogical analysis confirms the autosomal recessive nature of disease. The amplification did not generate nonspecific fragments. The HRM analysis allowed the differentiation of healthy and affected homozygous genotypes, and also the differentiation of the carrier, heterozygous genotype. Our results concur with those obtained using the fragment analysis technique published in 2011. CONCLUSION: high resolution melting analysis is a simple technique that allows the detection of cerebellar abiotrophy carriers in horses


Assuntos
Animais , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/veterinária , Doenças dos Cavalos/genética , Genótipo , Mutação , DNA/sangue , DNA/genética
7.
Nutr. hosp ; 37(1): 21-27, ene.-feb. 2020. tab
Artigo em Inglês | IBECS | ID: ibc-187570

RESUMO

Objective: to verify the association of serum concentrations of 25-hydroxyvitamin D and glycemic levels with the genetic variants rs1544410 and rs2228570 of the VDR gene in adolescents from the Northeast region of Brazil. Materials and methods: a cross-sectional epidemiological study with 208 adolescents from public schools in the city of João Pessoa (Paraíba, Brazil) between 15 and 19 years of age. Blood samples were collected for DNA extraction and analysis of polymorphisms rs1544410 and rs2228570, as well as biochemical analyses (25-hydroxyvitamin D, parathyroid hormone, calcium and glycemia). Results: the mean age was 17.7 (± 1.14) years. Half of adolescents had sufficient serum levels of 25-hydroxyvitamin D and the other half had insufficient/deficient vitamin. The most frequent genotypic distribution was bb and Ff and of lesser frequency BB and ff. There was a significant relationship between the genotypes of rs1544410 and glycemia values (p = 0.049) in the relationships between the genotypes BBxbb (p = 0.012) and Bbxbb (p = 0.037); (p = 0.036, OR = 2.15, 95 % CI = 1.05-4.41), and in the BB+Bb group analysis when compared to the bb (p = 0.025, OR = 1.89, 95 % CI = 1.08-3.29) presented higher risk of glycemia above the median. On the other hand, when Bb+bb was analyzed in relation to BB, adolescents had a greater chance of blood glucose below the median (p = 0.025, OR = 0.66, CI = 0.47-0.95). Conclusion: this study showed a significant relation of glycemia with the distribution of rs1544410 polymorphism genotypes


Objetivo: verificar la asociación de las concentraciones séricas de 25-hidroxivitamina D y los niveles de glucemia con las variantes genéticas rs1544410 y rs2228570 del gen VDR en adolescentes de la región noreste de Brasil. Materiales y métodos: se realizó un estudio epidemiológico transversal con 208 adolescentes de escuelas públicas en la ciudad de João Pessoa (Paraíba, Brasil) de entre 15 y 19 años de edad. Se recogieron muestras de sangre para la extracción de ADN y el análisis de los polimorfismos rs1544410 y rs2228570, así como para análisis bioquímicos (25-hidroxivitamina D, hormona paratiroidea, calcio y glucemia). Resultados: la edad media fue de 17,7 (± 1,14) años. La mitad de los adolescentes tenía niveles séricos suficientes de 25-hidroxivitamina D y la otra mitad, vitamina insuficiente/deficiente. La distribución genotípica más frecuente fue bb y Ff y la de menor frecuencia, BB y ff. Hubo una relación significativa entre los genotipos de rs1544410 y los valores de glucemia (p = 0,049) en las relaciones entre los genotipos BBxbb (p = 0,012) y Bbxbb (p = 0,037); (p = 0,036, OR = 2,15, IC 95 % = 1,05-4.41), y el análisis del grupo BB + Bb en comparación con el bb (p = 0,025, OR = 1,89, IC 95 % = 1,08-3,29) mostró un mayor el riesgo de glucemia, por encima de la mediana. Por otro lado, cuando se analizó Bb+bb en relación con la BB, los adolescentes tuvieron una mayor probabilidad de que la glucosa en sangre estuviera por debajo de la mediana (p = 0,025, OR = 0,66, IC = 0,47-0,95). Conclusión: este estudio mostró una relación significativa entre la glucemia y la distribución de genotipos de polimorfismo rs1544410


Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto Jovem , Vitamina D/administração & dosagem , Índice Glicêmico/fisiologia , Calcifediol/uso terapêutico , Brasil , Calcifediol/sangue , Estudos Transversais , DNA/sangue , Hormônio Paratireóideo/sangue , Cálcio/sangue , Glicemia/análise , Biomarcadores/análise , Modelos Logísticos
8.
Cancer Sci ; 111(1): 266-278, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746520

RESUMO

According to cancer genome sequences, more than 90% of cases of pancreatic ductal adenocarcinoma (PDAC) harbor active KRAS mutations. Digital PCR (dPCR) enables accurate detection and quantification of rare mutations. We assessed the dynamics of circulating tumor DNA (ct-DNA) in patients with advanced PDAC undergoing chemotherapy using dPCR. KRAS G12/13 mutation was assayed by dPCR in 47 paired tissue- and ct-DNA samples. The 21 patients were subjected to quantitative ct-DNA monitoring at 4 to 8-week intervals during chemotherapy. KRAS mutation was detected in 45 of those 47 patients using tissue DNA. In the KRAS mutation-negative cases, next-generation sequencing revealed KRAS Q61K and NRAS Q61R mutations. KRAS mutation was detected in 23/45 cases using ct-DNA (liver or lung metastasis, 18/19; mutation allele frequency [MAF], 0.1%-31.7%; peritoneal metastasis, 3/9 [0.1%], locally advanced, 2/17 [0.1%-0.2%]). In the ct-DNA monitoring, the MAF value changed in concordance with the disease state. In the 6 locally advanced cases, KRAS mutation appeared concurrently with liver metastasis. Among the 6 cases with liver metastasis, KRAS mutation disappeared during the duration of stable disease or a partial response, and reappeared at the time of progressive disease. The median progression-free survival was longer in cases in which KRAS mutation disappeared after an initial course of chemotherapy than in those in which it was continuously detected (248.5 vs 50 days, P < .001). Therefore, ct-DNA monitoring enables continuous assessment of disease state and could have prognostic utility during chemotherapy.


Assuntos
DNA Tumoral Circulante/genética , DNA/sangue , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/genética , Adulto , Idoso , Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Estudos de Avaliação como Assunto , Feminino , Frequência do Gene/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Prognóstico , Intervalo Livre de Progressão
9.
Anal Chim Acta ; 1093: 106-114, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31735203

RESUMO

Single nucleotide polymorphism (SNP) was associated with many human diseases, therefore, SNP detection was important for early diagnosis and clinical prognosis. Herein, a simple and accurate method for visual detection SNP sites (A/A, G/G, A/G) in CYP1A1 gene related to cancers based on colloidal gold nucleic acid strip biosensor and primer-specific polymerase chain reaction (PCR) was established. This method could directly distinguish SNP sites on strip biosensor by introducing twice PCR amplifications. The second PCR (primer-specific PCR) was performed using specific product of the first PCR as template, thus this twice PCR could reduce non-specific amplification greatly and obtain target product. In addition, single-strand or double-strand DNA (ssDNA or dsDNA) was accurately produced by introducing mismatched base at the 3' end of forward primers in primer-specific PCR. The designed strip biosensor could only combine with the ssDNA, thus visual detection of SNP could be achieved within 10 min by color difference of a pair of strips. 61 human blood samples by this method were identical with those of pyrosequencing. This method had the advantages of rapid, visual and low-cost and was expected to be applied in medical diagnosis.


Assuntos
Técnicas Biossensoriais/métodos , Citocromo P-450 CYP1A1/genética , DNA/sangue , Coloide de Ouro/química , Polimorfismo de Nucleotídeo Único , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Colorimetria/métodos , DNA/genética , Sondas de DNA/química , Ouro/química , Humanos , Nanopartículas Metálicas/química , Reação em Cadeia da Polimerase/métodos
10.
Mikrochim Acta ; 187(1): 7, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31797063

RESUMO

A composite was fabricated from deep eutectic solvent and MnO2 nanosheets (DES/MnO2) and is shown to be a viable oxidase mimic. The property, morphology and composition of DES/MnO2 was characterized. DES/MnO2 displays oxidase-like activity and can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to form a blue product (oxTMB) with an absorption maximum at 652 nm. Due to the presence of the DES, the polyanionic and negatively charged DNA is easily adsorbed on the surface of the composite by hydrogen bonding and electrostatic interactions. This leads to the inhibition of the oxidase-mimicking activity of DES/MnO2. This finding was used to design a colorimetric method for the determination of DNA. The assay work in the 10-100 µg mL-1 DNA concentration range and has a detection limit of 0.37 µg mL-1. The inhibiting mechanism was further studied by zeta potential measurements, dynamic light scattering and transmission electron microscopy. The selectivity study shows the DES/MnO2-TMB system to be highly selective for DNA when compared with many proteins, carbohydrates, salts and amino acid. RNA, on the other hand, interferes. The real sample analysis result illustrates that the new method can be used for the detection of DNA in bovine whole blood. Graphical abstractA novel oxidase mimic based on deep eutectic solvent-functionalized MnO2 nanosheets was synthesized, which can directly catalyze oxidation of 3,3',5,5'-tetramethylbenzidine (TMB, colorless) to oxTMB (blue). A sensitive and convenient colorimetric strategy for visual detection of DNA was established through DES/MnO2-TMB sensing system.


Assuntos
Materiais Biomiméticos/química , Colorimetria/métodos , DNA/análise , Compostos de Manganês/química , Nanoestruturas/química , Óxidos/química , Oxirredutases/metabolismo , Solventes/química , Animais , Bovinos , DNA/sangue , DNA/química
11.
Lipids Health Dis ; 18(1): 217, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31829184

RESUMO

BACKGROUND: Coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide. Previous studies have shown that complement component 3 (C3) is associated with atherosclerosis and cardiovascular risk factors. METHODS: We conducted this study to evaluate the associations between tagSNPs in the C3 gene locus and the CAD susceptibility and lipid levels in the Chinese population. A hospital-based case-control study, including 1017 subjects (580 CAD patients and 437 non-CAD controls), was conducted. TagSNPs in the C3 gene were searched and genotyped by using the polymerase chain reaction-ligase detection reaction method. RESULTS: The C3 levels were positively associated with the low-density lipoprotein cholesterol (LDL-C) levels (r = 0.269, P = 0.001). Compared with those in controls, the serum C3 levels in CAD patients were significantly higher (Control: 0.94 + 0.14 g/l; CAD: 1.10 + 0.19 g/l, P < 0.001). No significant differences in genotype or allele frequencies were observed between CAD patients and controls. The minor T allele of rs2287848 was associated with low apolipoprotein A1 (ApoA1) levels in controls (Bonferroni corrected P, Pc = 0.032). Linkage disequilibrium and haplotype analysis established two haplotype blocks (Block1: rs344555-rs2277984, Block 2: rs2287848-rs11672613) and six haplotypes. No significant associations between haplotypes and the risk of CAD were observed (all Pc > 0.05). CONCLUSIONS: The results revealed that C3 gene polymorphisms were associated with the lipid levels, but not CAD susceptibility in the Chinese population.


Assuntos
Complemento C3/genética , Doença da Artéria Coronariana/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único/genética , Idoso , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , China , Complemento C3/análise , Doença da Artéria Coronariana/sangue , DNA/sangue , Feminino , Genótipo , Haplótipos , Humanos , Desequilíbrio de Ligação , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade
12.
BMC Res Notes ; 12(1): 821, 2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31864401

RESUMO

OBJECTIVES: Genomic DNA (gDNA) is the optimal source of DNA for methylation analysis. This study compared methylation patterns in gDNA derived from blood with cell-line derived DNA (clDNA) from the same individuals. The clDNA had been generated via an Epstein-Barr virus transformation of the participant's lymphocytes. This analysis sought to determine whether clDNA has the potential to be utilised in lieu of finite/unavailable gDNA in methylation analyses using Illumina Infinium MethylationEPIC BeadChip arrays that assess 862,927 CpG sites. RESULTS: DNA samples were divided into two groups with eight gDNA and eight matched clDNA samples compared in each group (n = 16 individuals with 32 samples in total). Methylation patterns for gDNA samples generated for both groups were compared to the clDNA equivalent samples using Partek® Genomics Suite® to assess whether the significantly different CpG sites were consistent between both groups. In total, 28,632 CpG sites with significantly different levels of methylation (p < ×10-8) were common to both groups while 828,072 CpG sites assessed by the MethylationEPIC array were not significantly different in either group. This indicates that there is potential for clDNA to be used as a replacement for finite gDNA samples when absolutely necessary in DNA methylation studies.


Assuntos
Ilhas de CpG/genética , Metilação de DNA/genética , Linhagem Celular , DNA/sangue , DNA/química , DNA/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Epigênese Genética , Feminino , Ontologia Genética , Genômica , Herpesvirus Humano 4 , Humanos , Linfócitos/química , Linfócitos/virologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos
13.
Mikrochim Acta ; 186(12): 760, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712919

RESUMO

A fluorometric method is described for nucleic acid signal amplification through target-induced catalytic hairpin assembly with DNA-templated copper nanoparticles (Cu NPs). The toehold-mediated self-assembly of three metastable hairpins is triggered in presence of target DNA. This leads to the formation of a three-way junction structure with protruding mononucleotides at the 3' terminus. The target DNA is released from the formed branched structure and triggers another assembly cycle. As a result, plenty of branched DNA becomes available for the synthesis of Cu NPs which have fluorescence excitation/emission maxima at 340/590 nm. At the same time, the branched structure protects the Cu NPs from digestion by exonuclease III. The unreacted hairpins are digested by exonuclease III, and this warrants a lower background signal. The method can detect ssDNA (24 nt) at low concentration (44 pM) and is selective over single-nucleotide polymorphism. On addition of an aptamer, the strategy can also be applied to the quantitation of thrombin at levels as low as 0.9 nM. Graphical abstractSchematic representation of target-induced catalytic hairpin assembly to form branched DNA template for the in situ synthesis of fluorescent Cu nanoparticles.


Assuntos
DNA/sangue , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Espectrometria de Fluorescência/métodos , Trombina/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Cobre/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Exodesoxirribonucleases/química , Corantes Fluorescentes/síntese química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico
14.
Mikrochim Acta ; 186(12): 843, 2019 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-31768709

RESUMO

Voltammetric detection of the K-ras gene fragment was accomplished through the combined application of (a) a switchable DNA nanostructure, (b) the use of hairpin probe and exonuclease III (Exo III)-assisted signal amplification, (c) a split G-quadruplex, and (d) by exploiting the redox activity of DNAzyme. Three assistant oligonucleotides were designed to construct a DNA tweezer on a gold electrode. It is in "open state" in the absence of K-ras DNA. Then, a hairpin probe was introduced, whose stem-loop structure can be opened through hybridization with the K-ras DNA. Exo III is added which hydrolyzes the complementary region of the hairpin sequence to release a single-stranded rest fragment. The ssDNA hybridizes with the DNA tweezer on the electrode which thereby is switched to the "closed state". This leads to the formation of G-quadruplex due to the shortened distance of the split G-quadruplex-forming sequences in the tweezer. The voltammetric signal of the G-quadruplex-hemin complex, with a peak near -0.3 V vs. Ag/AgCl, is used as the signal output. Under the optimal conditions, the current response in differential pulse voltammetry (DPV) increases linearly with the concentration of K-ras DNA in the range of 0.01-1000 pM, and the detection limit is 2.4 fM. The assay can clearly discriminate K-ras DNA from a single-base mutation. The method has excellent selectivity and was applied to the determination of K-ras DNA in (spiked) serum samples. Graphical abstractSchematic representation of a method for the determination of the K-ras gene fragment through a combination of switchable DNA tweezer, split G-quadruplex, and exonuclease III (ExoIII)-assisted target recycling signal amplification.


Assuntos
Técnicas Biossensoriais/métodos , DNA/sangue , Técnicas Eletroquímicas/métodos , Genes ras , Nanoestruturas/química , Oligodesoxirribonucleotídeos/química , Sequência de Bases , DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Quadruplex G , Ouro/química , Hemina/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Mutação , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética
15.
PLoS One ; 14(11): e0224677, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31703075

RESUMO

Chicken mitochondrial DNA is a circular molecule comprising ~16.8 kb. In this study, we used next-generation sequencing to investigate mitochondrial heteroplasmy in the whole chicken mitochondrial genome. Based on heteroplasmic detection thresholds at the 0.5% level, 178 cases of heteroplasmy were identified in the chicken mitochondrial genome, where 83% were due to nucleotide transitions. D-loop regionwas hot spot region for mtDNA heteroplasmy in the chicken since 130 cases of heteroplasmy were located in these regions. Heteroplasmy varied among intraindividual tissues with allele-specific, position-specific, and tissue-specific features. Skeletal muscle had the highest abundance of heteroplasmy. Cases of heteroplasmy at mt.G8682A and mt.G16121A were validated by PCR-restriction fragment length polymorphism analysis, which showed that both had low ratios of heteroplasmy occurrence in five natural breeds. Polymorphic sites were easy to distinguish. Based on NGS data for crureus tissues, mitochondrial mutation/heteroplasmy exhibited clear maternal inheritance features at the whole mitochondrial genomic level. Further investigations of the heterogeneity of the mt.A5694T and mt.T5718G transitions between generations using pyrosequencing based on pedigree information indicated that the degree of heteroplasmy and the occurrence ratio of heteroplasmy decreased greatly from the F0 to F1 generations in the mt.A5694T and mt.T5718G site. Thus, the intergenerational transmission of heteroplasmy in chicken mtDNA exhibited a rapid shift toward homoplasmy within a single generation. Our findings indicate that heteroplasmy is a widespread phenomenon in chicken mitochondrial genome, in which most sites exhibit low heteroplasmy and the allele frequency at heteroplasmic sites changes significantly during transmission events. It suggests that heteroplasmy may be under negative selection to some degree in the chicken.


Assuntos
Galinhas/genética , Genoma Mitocondrial , Animais , DNA/sangue , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Mutação/genética , Polimorfismo Genético
16.
Clin Lab ; 65(11)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710449

RESUMO

BACKGROUND: Genomic studies facilitate comprehension of the pathophysiology, diagnosis, and treatment of chronic diseases. Such studies require sufficient and good quality DNA isolated from a large number of blood samples. This study attempts to obtain a high-quality genomic DNA isolated from a large number of blood samples using a simple and cheap method. METHODS: The EasyPure® Genomic DNA Kit (Transgen Biotech) was modified to increase the amount of DNA recovery: a few steps and two additional column elutions were added to the original manufacturer´s procedure. RESULTS: The amount of DNA isolated from frozen blood samples increased by an average of 56%. Its 260/280 ratio and electrophoretic mobility properties make it suitable for genomic studies. CONCLUSIONS: A relatively low-cost commercial column and a simple modification of the manufacturer´s protocol, provided a simple and cheap procedure to isolate high-quality DNA from a large number of blood samples suitable for genomic studies.


Assuntos
Células Sanguíneas/química , Coleta de Amostras Sanguíneas/economia , DNA/isolamento & purificação , Técnicas Genéticas/economia , Análise Custo-Benefício , DNA/sangue , Humanos
17.
Mikrochim Acta ; 186(11): 716, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654133

RESUMO

A fluorometric method is described for the determination of DNA. It involves the use of graphene oxide (GO), exonuclease III (Exo III), and two specially designed fluorophore-labeled hairpin probes (HP1 and HP2). Different from other GO-based DNA assays, the method takes advantage of the distinct binding abilities of GO with hairpin DNA probes and single nucleotides. GO serves as a strong quencher for fluorescent labels to ensure a very low background signal. Two reaction cycles mediated by Exo III are employed to enhance the signals. The combination of GO-induced quenching and Exo III-mediated dual regeneration of analytes leads to a detection limit as low as 1 pM for the model analyte human hemochromatosis protein (HFE) gene. The method is also applicable for the determination of HFE gene spiked into fetal bovine serum. Graphical abstract Schematic representation of a GO-based, Exo III-assisted method for dual-signal amplified detection of DNA, for which human haemochromatosis protein (HFE) gene is designed as the model target. The assay involves graphene oxide (GO), exonuclease (Exo III), and two specially designed, fluorophore-labelled hairpin probes (HP1 and HP2).


Assuntos
DNA/sangue , Exodesoxirribonucleases/química , Grafite/química , Espectrometria de Fluorescência/métodos , Animais , Bovinos , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico
18.
Anal Chim Acta ; 1088: 144-149, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31623710

RESUMO

Sensitive detection of DNA is conducive to enhance the accuracy of diseases diagnosis and risk prediction. In this work, we report the use of activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP) as a novel on-chip amplification strategy for the fluorescence detection of DNA. More specifically, the target DNA was captured by the on-chip immobilized hairpin DNA probes. Upon hybridization, exposed 3'-N3 of the hairpin was used to attach AGET ATRP initiators onto the silicon surface by click chemistry. Then, numerous fluorescent labeling linked to the end of the probes via the formation of long chain polymers of fluorescein o-acrylate, which in turn amplified the fluorescence signal for DNA detection. Under optimal conditions, it showed a good linear range from 100 fM to 1 µM in DNA detection, with the limit of detection as low as 4.3 fM. Moreover, this strategy showed good detection performance in complex real serum samples, the fluorescence intensity of 0.1 nM tDNA in 1% fetal bovine serum samples was 97.6% of that in Tris-EDTA buffer. Based on its high sensitivity, reduced cost and simplicity, the proposed signal amplification strategy displays translational potential in clinical application.


Assuntos
Acrilatos/química , Técnicas Biossensoriais/métodos , Sondas de DNA/química , DNA/análise , Fluoresceína/química , Sequências Repetidas Invertidas , Limite de Detecção , Sequência de Bases , DNA/sangue , DNA/química , DNA/genética , Sondas de DNA/genética , Humanos , Polímeros/química , Espectrometria de Fluorescência
19.
Analyst ; 144(22): 6671-6680, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31596277

RESUMO

Androgen-receptor splice variant 7 (AR-V7) is a highly promising liquid biopsy predictive biomarker showing primary or acquired resistance to novel androgen receptor signaling inhibitors in metastatic castration resistant prostate cancer (mCRPC). We present for the first time the expression pattern of AR-FL, AR-V7, and AR-567es at a quantitative level in circulating tumor cells (CTCs) and paired plasma-derived extracellular vesicles in mCRPC. We first developed and analytically validated a novel multiplex RT-qPCR assay for AR full length (AR-FL), AR-V7, AR-567es and AR-total. We then quantified the expression levels of AR-splice variants, CK-19 (epithelial marker) and B2M (reference gene) in EpCAM+ CTCs, and paired plasma-derived extracellular vesicles isolated from peripheral blood (20 mL) of 62 mCRPC patients and 10 healthy donors. CTCs were enumerated using the FDA-cleared CellSearch® system. In CTCs AR-FL was detected in 64/69 (92.3%), AR-V7 in 34/69 (49.3%), AR-567es in 16/69 (23.2%) and AR-total in 62/69 (89.9%). In 52 out of 69 samples, paired plasma-derived extracellular vesicles were analyzed. AR-FL was detected in 40/52 (76.9%), AR-V7 in 4/52 (7.7%), AR-567 in 2/52 (3.8%) and AR total in 39/52 (75.0%). In all cases AR splice variants were expressed in higher levels in CTCs than in paired extracellular vesicles, while AR-V7 was detected in higher percentages than in AR-567es. Using CellSearch®, CTCs were detected in 52/69 (75.4%) mCRPC patient samples; 27/52 (51.9%) of these samples were CTC+/AR-V7+ and 14/52 (26.9%) were CTC+/AR-567es+, while 7/17 (41.2%) were CTC-/AR-V7+ and 2/17 (11.8%) were CTC-/AR-567es+. Our results reveal for the first time a remarkable heterogeneity in the expression levels of AR-FL, AR-V7 and AR-567es in EpCAM+ CTCs and paired extracellular vesicles between individual mCRPC patients. The clinical significance of this finding will be further investigated in a large patient cohort with respect to therapy response.


Assuntos
Biomarcadores Tumorais/sangue , DNA/sangue , Vesículas Extracelulares/química , Células Neoplásicas Circulantes/química , Neoplasias de Próstata Resistentes à Castração/diagnóstico , Receptores Androgênicos/genética , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex/métodos , Neoplasias de Próstata Resistentes à Castração/sangue , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reprodutibilidade dos Testes , Microglobulina beta-2/genética
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