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2.
Front Immunol ; 12: 628564, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34211456

RESUMO

Neutrophil extracellular traps (NETs) and mitochondrial DNA (mtDNA) are inflammatory mediators involved in the development of type 1 diabetes (T1D). Pancreas-infiltrating neutrophils can release NETs, contributing to the inflammatory process. Levels of NETs are increased in serum from patients with T1D and mtDNA is increased in adult T1D patients. Our aim was to investigate extracellular DNA (NETs, mtDNA and nuclear DNA) in children with newly diagnosed T1D and in children at high risk of the disease. We also elucidated if extracellular DNA short after diagnosis could predict loss of endogenous insulin production. Samples were analysed for mtDNA and nuclear DNA using droplet digital PCR and NETs were assessed by a NET-remnants ELISA. In addition, in vitro assays for induction and degradation of NETs, as well as analyses of neutrophil elastase, HLA genotypes, levels of c-peptide, IL-1beta, IFN and autoantibodies (GADA, IA-2A, IAA and ZnT8A) were performed. In serum from children 10 days after T1D onset there was an increase in NETs (p=0.007), mtDNA (p<0.001) and nuclear DNA (p<0.001) compared to healthy children. The elevated levels were found only in younger children. In addition, mtDNA increased in consecutive samples short after onset (p=0.017). However, levels of extracellular DNA short after onset did not reflect future loss of endogenous insulin production. T1D serum induced NETs in vitro and did not deviate in the ability to degrade NETs. HLA genotypes and autoantibodies, except for ZnT8A, were not associated with extracellular DNA in T1D children. Serum from children with high risk of T1D showed fluctuating levels of extracellular DNA, sometimes increased compared to healthy children. Therefore, extracellular DNA in serum from autoantibody positive high-risk children does not seem to be a suitable biomarker candidate for prediction of T1D. In conclusion, we found increased levels of extracellular DNA in children with newly diagnosed T1D, which might be explained by an ongoing systemic inflammation.


Assuntos
Núcleo Celular/genética , DNA Mitocondrial/sangue , DNA/sangue , Diabetes Mellitus Tipo 1/sangue , Armadilhas Extracelulares/metabolismo , Adolescente , Fatores Etários , Autoanticorpos/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Feminino , Humanos , Estudos Longitudinais , Masculino , Medição de Risco , Fatores de Risco , Regulação para Cima
3.
Methods Mol Biol ; 2324: 339-360, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34165725

RESUMO

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/sangue , Pseudogenes , Sequência de Bases , Biomarcadores Tumorais/genética , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/genética , Meios de Cultura , Meios de Cultivo Condicionados , DNA/sangue , DNA/genética , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , DNA de Cadeia Simples/sangue , Células Progenitoras Endoteliais/citologia , Sangue Fetal/citologia , Glioblastoma/patologia , Humanos , Mutagênese Insercional , Proteína Homeobox Nanog/genética , Neoplasias/genética , Células-Tronco Neurais/citologia , Prognóstico , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Homologia de Sequência do Ácido Nucleico , Transfecção , Células Tumorais Cultivadas
4.
BMC Cancer ; 21(1): 482, 2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931014

RESUMO

BACKGROUND: This retrospective study aimed to evaluate the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) with stereotactic body radiation therapy (SBRT) and to elucidate potential mechanisms of acquired resistance. METHODS: Patients with advanced NSCLC harboring positive EGFR mutations after initial TKI therapy for at least 8 weeks were eligible for SBRT between August 2016 and August 2019. Eligible patients were treated with thoracic SBRT, and TKI was continued after SBRT until it was considered ineffective. The control group was treated with TKIs monotherapy. Propensity score matching (PSM, ratio of 1:2) was used to account for differences in baseline characteristics. Overall survival (OS), progression-free survival (PFS), treatment safety and resistance mechanisms were evaluated. RESULTS: Three hundred eight patients were included in the study population. Among them, 262 patients received TKIs alone, and 46 patients received TKIs with SBRT. Baseline characteristics were not significantly different between the two cohorts after PSM. The median PFS was 19.4 months in the TKIs +SBRT group compared to 13.7 months in the TKIs group (p = 0.034). An influence on OS has not yet been shown (p = 0.557). Of the 135 patients evaluated after PSM, 28 and 71 patients in the TKIs and TKIs +SBRT cohorts, respectively, had plasma cell-free DNA (cfDNA) next-generation sequencing (NGS) performed at baseline and disease progression. In the TKIs +SBRT cohort, the NGS results showed that T790M mutations were detected in 64.3% (18/28) of patients. Patients in the TKIs cohort exhibited fewer T790M-positive mutations (40.8%, p = 0.035) compared to patients in the TKIs +SBRT cohort. CONCLUSION: Real world data prove that TKIs plus thoracic SBRT significantly extend PFS with tolerable toxicity. The mutation ratio of T790M was increased in the TKIs +SBRT group compared to the TKIs only group. Further randomized studies are warranted.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/terapia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/terapia , Inibidores de Proteínas Quinases/uso terapêutico , Radiocirurgia/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimiorradioterapia/métodos , DNA/sangue , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Intervalo Livre de Progressão , Pontuação de Propensão , Inibidores de Proteínas Quinases/efeitos adversos , Radiocirurgia/efeitos adversos , Estudos Retrospectivos , Adulto Jovem
5.
Gynecol Obstet Invest ; 86(1-2): 177-184, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33895751

RESUMO

OBJECTIVES: Insulin receptor substrate 1 (IRS1) is a crucial factor in the insulin signaling pathway. IRS1 gene polymorphism rs1801278 in mothers has been reported to be associated with gestational diabetes mellitus (GDM). However, it is not clear whether IRS1 gene polymorphism rs1801278 in fetuses is associated with their mothers' GDM morbidity. The purpose of this study is to analyze the association between maternal, fetal, or maternal/fetal IRS1 gene polymorphism rs1801278 and GDM risk. DESIGN: The study was a single-center, prospective cohort study. In total, 213 pairs of GDM mothers/fetuses and 191 pairs of control mothers/fetuses were included in this study. They were recruited after they underwent oral glucose tolerance test during 24-28 weeks of gestation and followed up until delivery. All participants received the conventional interventions (diet and exercise), and no special therapy except routine treatment. METHODS: A total of 213 pairs of GDM mothers/fetuses and 191 pairs of normal blood glucose pregnant mothers/fetuses were ge-notyped using PCR and DNA sequencing from January 2015 to September 2016. Maternal/fetal IRS1 gene polymorphism rs1801278 was analyzed and compared between 2 groups. RESULTS: There were no significant differences in the frequency of individual mothers' or fetuses' IRS1 rs1801278 polymorphisms between 2 groups; if both the mothers and fetuses carried A allele, significantly lower GDM morbidity was observed in the mothers. LIMITATIONS: The sample size was relatively small as a single-center study. CONCLUSIONS: Our study suggested that maternal/fetal rs1801278 polymorphism of IRS1 is a modulating factor in GDM; both mothers/fetuses carrying the A allele of rs1801278 may protect the mothers against the development of GDM.


Assuntos
Diabetes Gestacional/genética , Feto , Proteínas Substratos do Receptor de Insulina/genética , Polimorfismo Genético/genética , Adulto , Alelos , China , DNA/sangue , Feminino , Frequência do Gene , Genótipo , Idade Gestacional , Teste de Tolerância a Glucose , Humanos , Insulina , Gravidez , Cuidado Pré-Natal , Estudos Prospectivos , Análise de Sequência de DNA
6.
Transfusion ; 61(7): 2008-2013, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33929058

RESUMO

BACKGROUND: Minority RBC donors are important to support the transfusion needs of patients with sickle cell disease. Testing of donors for sickle cell trait (SCT) is performed to avoid transfusion of hemoglobin S+ (HbS+) RBCs to specific patient groups and to investigate leukoreduction failures. A screening assay based on hemoglobin solubility is commonly used. The purpose of this study was to validate a DNA approach for HbS screening. METHODS: Hemoglobin solubility screening (Pacific Hemostasis or SICKLEDEX) and PreciseType human erythrocyte antigen (HEA)-HbS (Immucor) targeting c.20A>T in the ß-globin gene were performed according to manufacturer's directions. Resolution of differences in results included gene sequencing and high-performance liquid chromatography (HPLC). RESULTS: Initial validation of HEA-HbS performed by testing 60 known samples, 20 HbS/A, A/A, and S/S, gave expected results. However, in the subsequent parallel testing phase, 4/58 samples HbS+ by solubility assay tested negative by HEA-HbS; the negative results were confirmed by ß-globin gene sequencing. Samples from donors self-identifying as White testing HbS+ by solubility assay (n = 60) were retested by HEA-HbS and HPLC. The HEA-HbS assay was concordant with HPLC which is recognized as the gold standard for hemoglobin variation. CONCLUSION: A DNA-based approach is an alternative to screen donors for SCT, found in approximately 7% of Black and 1.7% of our random donors. HEA-HbS correlated with HPLC results in all samples tested, supporting the use of HEA-HbS as the test of record. The method allows higher throughput screening and testing at the donor center allows association of the screening result with the donor record to avoid repeat testing.


Assuntos
Doadores de Sangue , DNA/genética , Seleção do Doador/métodos , Grupos Étnicos/genética , Traço Falciforme/diagnóstico , Adulto , Cromatografia Líquida de Alta Pressão , DNA/sangue , Feminino , Hemoglobina Falciforme/análise , Hemoglobina Falciforme/química , Humanos , Masculino , Grupos Minoritários , Cidade de Nova Iorque/epidemiologia , Estudos Retrospectivos , Análise de Sequência de DNA , Traço Falciforme/etnologia , Traço Falciforme/genética , Solubilidade , Globinas beta/genética
7.
Nucleic Acids Res ; 49(13): e75, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-33905513

RESUMO

Technological advances in rare DNA mutations detection have revolutionized the diagnosis and monitoring of tumors, but they are still limited by the lack of supersensitive and high-coverage procedures for identifying low-abundance mutations. Here, we describe a single-tube, multiplex PCR-based system, A-Star, that involves a hyperthermophilic Argonaute from Pyrococcus furiosus (PfAgo) for highly efficient detection of rare mutations beneficial from its compatibility with DNA polymerase. This novel technique uses a specific guide design strategy to allow PfAgo selective cleavage with single-nucleotide resolution at 94°C, thus mostly eliminating wild-type DNA in the denaturation step and efficiently amplifying rare mutant DNA during the PCR process. The integrated single-tube system achieved great efficiency for enriching rare mutations compared with a divided system separating the cleavage and amplification. Thus, A-Star enables easy detection and quantification of 0.01% rare mutations with ≥5500-fold increase in efficiency. The feasibility of A-Star was also demonstrated for detecting oncogenic mutations in solid tumor tissues and blood samples. Remarkably, A-Star achieved simultaneous detection of multiple oncogenes through a simple single-tube reaction by orthogonal guide-directed specific cleavage. This study demonstrates a supersensitive and rapid nucleic acid detection system with promising potential for both research and therapeutic applications.


Assuntos
Proteínas Argonauta , Análise Mutacional de DNA/métodos , Reação em Cadeia da Polimerase/métodos , DNA/sangue , Clivagem do DNA , Humanos , Mutação , Neoplasias/sangue , Neoplasias/genética , Oncogenes , Pyrococcus furiosus
8.
Food Chem Toxicol ; 152: 112163, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33836211

RESUMO

Comet assay, applied to in vitro, in vivo and ex vivo systems, is a quick, simple, and sensitive method for the detection of genotoxicity. In general, fresh whole blood or peripheral blood mononuclear cells (PBMCs) are used in the assay for the determination of DNA damage and repair. In this study, the effects of storage conditions on genotoxicity assessed by Comet assay in human whole blood and lymphocyte samples, were evaluated. Whole blood and lymphocyte samples were stored at 4 °C for 1, 2, 3, 4, 5 and 7 days; at -20 °C for 1 month and at -80 °C for 3, 6 and 12 months. 1% DMSO was used as cryoprotectant. No significant differences in DNA damage were demonstrated in all of the storage conditions and durations, and the results were similar according to the median values (p < 0.05). According to Spearman or Pearson correlations, an important correlation was found between the DNA damage of the fresh samples and the samples which were kept at -80 °C for 6 months with temperature and time (p < 0.01 for Pearson and p < 0.05 for Spearman). The results of this study indicated that blood and lymphocyte samples stored in +4 °C, -20 °C and -80 °C up to 12 months can be used instead of fresh samples especially in human biomonitoring studies.


Assuntos
Ensaio Cometa/métodos , Dano ao DNA , DNA/sangue , Linfócitos/química , Manejo de Espécimes/métodos , Adulto , Temperatura Baixa , DNA/genética , Humanos , Masculino
9.
Genes (Basel) ; 12(4)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33924034

RESUMO

Mitochondrial diseases can be caused by pathogenic variants in nuclear or mitochondrial DNA-encoded genes that often lead to multisystemic symptoms and can have any mode of inheritance. Using a single test, Genome Sequencing (GS) can effectively identify variants in both genomes, but it has not yet been universally used as a first-line approach to diagnosing mitochondrial diseases due to related costs and challenges in data analysis. In this article, we report three patients with mitochondrial disease molecularly diagnosed through GS performed on DNA extracted from blood to demonstrate different diagnostic advantages of this technology, including the detection of a low-level heteroplasmic pathogenic variant, an intragenic nuclear DNA deletion, and a large mtDNA deletion. Current technical improvements and cost reductions are likely to lead to an expanded routine diagnostic usage of GS and of the complementary "Omic" technologies in mitochondrial diseases.


Assuntos
DNA/sangue , Variação Genética , Doenças Mitocondriais/diagnóstico , Sequenciamento Completo do Genoma/métodos , Adolescente , Pré-Escolar , Diagnóstico Precoce , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Doenças Mitocondriais/sangue , Doenças Mitocondriais/genética
10.
PLoS One ; 16(4): e0250265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33886636

RESUMO

Over the past years, neutrophil extracellular traps (NETs) were shown to contribute to states of acute and chronic inflammatory disease. They are composed of expelled chromatin and decorated by neutrophil-derived proteins. Therefore, the analysis of DNA complexes with myeloperoxidase (MPO) by ELISA has become an attractive tool to measure NET formation in in vitro and in vivo samples. When we used a published MPO-DNA ELISA protocol and included an isotype control for the anti-MPO coating antibody, we observed high assay specificity for in vitro prepared NET samples, whereas the specificity for in vivo plasma samples was low. In addition, the assay failed to detect in vitro generated MPO-DNA complexes when spiked into plasma. Therefore, we set out to improve the specificity of the MPO-DNA ELISA for plasma samples. We found that the use of Fab fragments or immunoglobulins from different species or reversal of the antibody pair led to either a high background or a low dynamic range of detection that did not improve the specificity for plasma samples. Also, the use of higher plasma dilutions or pre-clearing of plasma immunoglobulins were ineffective. Finally, we found that a commercial reagent designed to block human anti-mouse antibodies and multivalent substances increased the detection window between the MPO antibody and isotype control for highly diluted plasma. We applied this modified ELISA protocol to analyze MPO-DNA complexes in human blood samples of acute and chronic inflammatory conditions. While markers of neutrophil activation and NET formation such as MPO, elastase and citrullinated histone H3 correlated significantly, we observed no correlation with the levels of MPO-DNA complexes. Therefore, we conclude that ELISA measurements of MPO-DNA complexes in human plasma are highly questionable regarding specificity of NET detection. In general, plasma analyses by ELISA should more frequently include isotype controls for antibodies to demonstrate target specificity.


Assuntos
Proteínas de Ligação a DNA/sangue , Proteínas de Ligação a DNA/imunologia , DNA/sangue , DNA/imunologia , Armadilhas Extracelulares/imunologia , Peroxidase/sangue , Peroxidase/imunologia , Animais , Anticorpos Monoclonais/imunologia , Biomarcadores/sangue , Células Cultivadas , Ensaio de Imunoadsorção Enzimática/métodos , Histonas/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Camundongos , Ativação de Neutrófilo , Neutrófilos/imunologia , Plasma/imunologia
11.
Science ; 372(6538)2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833097

RESUMO

Liquid biopsies that analyze cell-free DNA in blood plasma are used for noninvasive prenatal testing, oncology, and monitoring of organ transplant recipients. DNA molecules are released into the plasma from various bodily tissues. Physical and molecular features of cell-free DNA fragments and their distribution over the genome bear information about their tissues of origin. Moreover, patterns of DNA methylation of these molecules reflect those of their tissue sources. The nucleosomal organization and nuclease content of the tissue of origin affect the fragmentation profile of plasma DNA molecules, such as fragment size and end motifs. Besides double-stranded linear fragments, other topological forms of cell-free DNA also exist-namely circular and single-stranded molecules. Enhanced by these features, liquid biopsies hold promise for the noninvasive detection of tissue-specific pathologies with a range of clinical applications.


Assuntos
Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Fragmentação do DNA , Metilação de DNA , DNA/sangue , DNA/genética , Biópsia Líquida , Animais , Biomarcadores/sangue , DNA Circular/sangue , DNA Mitocondrial/sangue , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Desoxirribonucleases/metabolismo , Epigênese Genética , Feminino , Feto , Humanos , Gravidez , Transplantes
12.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806359

RESUMO

In systemic mastocytosis (SM), qualitative and serial quantitative assessment of the KIT D816V mutation is of diagnostic and prognostic relevance. We investigated peripheral blood and bone marrow samples of 161 patients (indolent SM (ISM), n = 40; advanced SM, AdvSM, n = 121) at referral and during follow-up for the KIT D816V variant allele frequency (VAF) at the DNA-level and the KIT D816V expressed allele burden (EAB) at the RNA-level. A round robin test with four participating laboratories revealed an excellent correlation (r > 0.99, R2 > 0.98) between three different DNA-assays. VAF and EAB strongly correlated in ISM (r = 0.91, coefficient of determination, R2 = 0.84) but only to a lesser extent in AdvSM (r = 0.71; R2 = 0.5). However, as compared to an EAB/VAF ratio ≤2 (cohort A, 77/121 patients, 64%) receiver operating characteristic (ROC) analysis identified an EAB/VAF ratio of >2 (cohort B, 44/121 patients, 36%) as predictive for an advanced phenotype and a significantly inferior median survival (3.3 vs. 11.7 years; p = 0.005). In terms of overall survival, Cox-regression analysis was only significant for the EAB/VAF ratio >2 (p = 0.006) but not for VAF or EAB individually. This study demonstrates for the first time that the transcriptional activity of KIT D816V may play an important role in the pathophysiology of SM.


Assuntos
Mastocitose Sistêmica/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Medula Óssea/metabolismo , DNA/sangue , DNA/genética , DNA/metabolismo , Feminino , Frequência do Gene , Humanos , Masculino , Mastocitose Sistêmica/sangue , Mastocitose Sistêmica/metabolismo , Pessoa de Meia-Idade , Fenótipo , Prognóstico , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA/sangue , RNA/genética , RNA/metabolismo , Transcrição Genética
13.
Int J Legal Med ; 135(4): 1257-1265, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33754178

RESUMO

Evaluating the short tandem repeat (STR) in the field is important for the timely identification of a suspect. Several lines showed that the RapidHIT® ID system is reliable for DNA genotyping with buccal swabs and naked DNA. However, the application of this approach with blood samples has been poorly investigated. Because blood samples are among the most common forensic samples in our laboratory, further studies should be conducted. Here, we assessed the analytical performance of 19 STR loci with a newly developed RapidINTEL (RI) Sample Cartridge Kit by using the blood samples with known genotypes. Several commonly used substrates were included in the sensitivity study, and FTA cards proved to be the most promising sample carrier for blood storage and later identification. There was superior sensitivity and specificity with a 100% concordance rate for 0.5 µL of blood or 7 ng of genomic DNA. The performance for blood samples was comparable with that for the standard protocol. High success rate (90.57%) and high-concordance (100%) genotyping were automatically achieved over a wide range of operating conditions except for TH01. No contamination was observed throughout the study. Hematin, indigo, and humic acid had limited influence on the instrument system, while urea and melanin dramatically affected the genotyping results. Generally, the newly developed RI sample cartridge provided an alternative method for the STR genotyping of single-source blood samples over a wide range of operating conditions.


Assuntos
DNA/sangue , Técnicas de Genotipagem/instrumentação , Técnicas de Genotipagem/métodos , Repetições de Microssatélites , Feminino , Humanos , Masculino , Sensibilidade e Especificidade
14.
Crit Care Med ; 49(7): 1149-1158, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33729723

RESUMO

OBJECTIVES: Circulating nucleosomes and their component histones have been implicated as pathogenic in sepsis and acute respiratory distress syndrome in adults. However, their role in pediatric acute respiratory distress syndrome is unknown. DESIGN: We performed a prospective cohort study in children with acute respiratory distress syndrome, with plasma collection within 24 hours of acute respiratory distress syndrome onset. We associated nucleosome levels with severity of acute respiratory distress syndrome and with nonpulmonary organ failures and tested for association of nucleosomes with PICU mortality and ventilator-free days at 28 days in univariate and multivariable analyses. We also performed proteomics of DNA-bound plasma proteins in a matched case-control study of septic children with and without acute respiratory distress syndrome in order to identify specific histone proteins elevated in acute respiratory distress syndrome. SETTING: Large academic tertiary-care PICU. PATIENTS: Intubated children meeting Berlin criteria for acute respiratory distress syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We enrolled 333 children with acute respiratory distress syndrome, with 69 nonsurvivors (21%). Plasma nucleosomes were correlated with acute respiratory distress syndrome severity and with the number of nonpulmonary organ failures at acute respiratory distress syndrome onset. Nucleosomes were higher (p < 0.001) in nonsurvivors (0.40 [interquartile range, 0.20-0.71] arbitrary units) relative to survivors (0.10 [interquartile range, 0.04-0.25] arbitrary units). Nucleosomes were associated with PICU mortality in multivariable analysis (adjusted odds ratio 1.84 per 1 sd increase; 95% CI, 1.38-2.45; p < 0.001). Nucleosomes were also associated with a lower probability of being extubated alive by day 28 after multivariable adjustment (adjusted subdistribution hazard ratio, 0.74; 95% CI, 0.63-0.88; p = 0.001). Proteomic analysis demonstrated higher levels of the core nucleosome histones H2A, H2B, H3, and H4 in septic children with acute respiratory distress syndrome, relative to septic children without acute respiratory distress syndrome. CONCLUSIONS: Plasma nucleosomes are associated with acute respiratory distress syndrome severity, nonpulmonary organ failures, and worse outcomes in pediatric acute respiratory distress syndrome.


Assuntos
Histonas/sangue , Nucleossomos/metabolismo , Síndrome do Desconforto Respiratório/sangue , Síndrome do Desconforto Respiratório/mortalidade , Adolescente , Extubação , Estudos de Casos e Controles , Criança , Pré-Escolar , DNA/sangue , Feminino , Mortalidade Hospitalar , Humanos , Unidades de Terapia Intensiva Pediátrica , Masculino , Insuficiência de Múltiplos Órgãos/mortalidade , Prognóstico , Estudos Prospectivos , Proteômica , Respiração Artificial , Síndrome do Desconforto Respiratório/complicações , Sepse/sangue , Sepse/complicações , Índice de Gravidade de Doença , Taxa de Sobrevida
15.
Elife ; 102021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33752803

RESUMO

We developed genetic-epigenetic tissue mapping (GETMap) to determine the tissue composition of plasma DNA carrying genetic variants not present in the constitutional genome through comparing their methylation profiles with relevant tissues. We validated this approach by showing that, in pregnant women, circulating DNA carrying fetal-specific alleles was entirely placenta-derived. In lung transplant recipients, we showed that, at 72 hr after transplantation, the lung contributed only a median of 17% to the plasma DNA carrying donor-specific alleles, and hematopoietic cells contributed a median of 78%. In hepatocellular cancer patients, the liver was identified as the predominant source of plasma DNA carrying tumor-specific mutations. In a pregnant woman with lymphoma, plasma DNA molecules carrying cancer mutations and fetal-specific alleles were accurately shown to be derived from the lymphocytes and placenta, respectively. Analysis of tissue origin for plasma DNA carrying genetic variants is potentially useful for noninvasive prenatal testing, transplantation monitoring, and cancer screening.


Assuntos
DNA/sangue , Epigenômica/métodos , Neoplasias/genética , Transplante de Órgãos/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , DNA/genética , Metilação de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Epigênese Genética , Feminino , Feto/metabolismo , Variação Genética , Humanos , Neoplasias Hepáticas/genética , Linfoma/genética , Masculino , Pessoa de Meia-Idade , Neoplasias/sangue , Placenta/metabolismo , Gravidez , Análise de Sequência de DNA/métodos
16.
Innate Immun ; 27(3): 240-250, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33646058

RESUMO

Cell destruction results in plasma accumulation of cell-free DNA (cfDNA). Dynamic changes in circulating lymphocytes are features of COVID-19. We aimed to investigate if cfDNA level can serve in stratification of COVID-19 patients, and if cfDNA level is associated with alterations in lymphocyte subsets and neutrophil-to-lymphocyte ratio (NLR). This cross-sectional comparative study enrolled 64 SARS-CoV-2-positive patients. Patients were subdivided to severe and non-severe groups. Plasma cfDNA concentration was determined by real-time quantitative PCR. Lymphocyte subsets were assessed by flow cytometry. There was significant increase in cfDNA among severe cases when compared with non-severe cases. cfDNA showed positive correlation with NLR and inverse correlation with T cell percentage. cfDNA positively correlated with ferritin and C-reactive protein. The output data of performed ROC curves to differentiate severe from non-severe cases revealed that cfDNA at cut-off ≥17.31 ng/µl and AUC of 0.96 yielded (93%) sensitivity and (73%) specificity. In summary, excessive release of cfDNA can serve as sensitive COVID-19 severity predictor. There is an association between cfDNA up-regulation and NLR up-regulation and T cell percentage down-regulation. cfDNA level can be used in stratification and personalized monitoring strategies in COVID-19 patients.


Assuntos
COVID-19/diagnóstico , COVID-19/imunologia , DNA/sangue , Subpopulações de Linfócitos/patologia , Linfócitos/patologia , Neutrófilos/patologia , Adulto , Idoso , Proteína C-Reativa/análise , COVID-19/sangue , Estudos Transversais , Diagnóstico Diferencial , Feminino , Ferritinas/sangue , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Curva ROC , Sensibilidade e Especificidade , Linfócitos T/patologia , Adulto Jovem
17.
J Parasitol ; 107(2): 147-154, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33662113

RESUMO

Noting lipidomic changes following the parasitism of migrating birds, the metabolic needs of which are primarily fueled by lipids, can deepen our understanding of host-parasite interactions. We identified lipids of migrating Northern saw-whet owls (Aegolius acadicus) using collision-induced dissociation mass spectrometry, compared the lipidomic signatures of hemoparasite-infected and noninfected individuals, and performed cross-validation analyses to reveal associations between parasite infection and lipid levels. We found significantly lower levels of lipid classes phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingomyelin (SM) in infected Northern saw-whet owls than in the noninfected individuals. Conversely, we found higher levels for certain lysoPS and lysoPE species, and variable lipid level changes for free fatty acid (FFA) species. Reporting lipidomic changes observed between hemosporidian-infected and noninfected Northern saw-whet owls can strengthen our understanding of the mechanisms governing parasite proliferation in this species. Furthermore, our analysis indicated that lipidomic signatures are better predictors of parasite infection than the log-adjusted mass/wing chord body index, a metric commonly used to assess the influence of hemosporidia infection on the health of birds. Establishing a lipidomic profile for Northern saw-whet owls that provides baseline lipid levels during fall migration may assist future studies assessing causes of reductions in breeding brought about from subtle differences in behaviors such as delayed migration.


Assuntos
Doenças das Aves/parasitologia , Lipídeos/sangue , Infecções Protozoárias em Animais/sangue , Estrigiformes/parasitologia , Migração Animal , Animais , Doenças das Aves/sangue , Doenças das Aves/diagnóstico , Cromatografia Líquida/veterinária , DNA/sangue , Interações Hospedeiro-Parasita , Lipidômica , Espectrometria de Massas/métodos , Espectrometria de Massas/veterinária , América do Norte , Infecções Protozoárias em Animais/diagnóstico , Infecções Protozoárias em Animais/parasitologia , Espectrometria de Massas por Ionização por Electrospray/veterinária , Estrigiformes/sangue , Estrigiformes/fisiologia , Espectrometria de Massas em Tandem
18.
BMC Pregnancy Childbirth ; 21(1): 265, 2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33785011

RESUMO

BACKGROUND: Mounting evidence suggests that cesarean delivery may have a long-lasting effect on infant health. But the underlying mechanisms remain unclear. This study aims to examine whether cesarean delivery on maternal request without any medical indications (CDMR) impacts DNA methylation status in the umbilical cord blood of the infant. METHODS: A cross-sectional study was conducted in Shanghai, China. A total of 70 CDMR and 70 vaginal deliveries (VD) were recruited in 2012. The cord blood DNA methylation status was measured in 30 CDMR and 30 VD newborns using Illumina Infinium Human Methylation 450 K BeadChip. To validate the results, the cord blood DNA methylation status was measured in another 40 CDMR and 40 VD newborns using targeted bisulfite sequencing assay. A total of 497 CpG sites from 40 genes were included in the analysis. RESULTS: A total of 165 differentially methylated positions (DMPs) exhibited differences in DNA methylation by 10% or more between the CDMR and VD groups, many of which were related to the development of the immune system. Based on the targeted bisulfite sequencing assay, 16 genes (16/22, 72.7%) had higher methylation level in the CDMR group than the VD group. Among them, 5 genes were related to the immune system. After considering the estimation of cell type proportions, there was few significant differences in DNA methylation between CDMR and VD groups. CONCLUSIONS: The DMPs identified between CDMR and VD groups might be largely explained by the cell type proportions. Further studies are needed to examine DNA methylation in each cell type separately.


Assuntos
Cesárea/efeitos adversos , Metilação de DNA , Sangue Fetal/química , Adulto , Cesárea/estatística & dados numéricos , China , Ilhas de CpG/genética , Estudos Transversais , DNA/sangue , DNA/genética , Epigênese Genética , Feminino , Idade Gestacional , Humanos , Recém-Nascido , Masculino , Idade Materna , Gravidez
19.
Methods Mol Biol ; 2265: 235-245, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704719

RESUMO

Circulating tumor cells (CTCs) are cancer cells shed by the primary tumor or its metastases that circulate in the peripheral blood. CTCs are potential seeds for metastases, and their detection may allow early uncovering of metastatic dissemination and disease prognostication. To fully ascertain the biomarker potential of melanoma CTCs, sensitive and reliable methods are required. Melanoma-specific transcript analysis has been widely utilized as a standard approach for measuring the presence of CTCs. Here we describe a method for the analysis of CTCs through the detection of specific transcripts in CTC-enriched fractions.


Assuntos
Biomarcadores Tumorais/sangue , Melanoma/sangue , Células Neoplásicas Circulantes/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Biomarcadores Tumorais/genética , DNA/sangue , DNA/isolamento & purificação , Humanos , Melanoma/diagnóstico , Melanoma/genética , Melanoma/patologia , Células Neoplásicas Circulantes/patologia , RNA/sangue , RNA/isolamento & purificação
20.
Oxid Med Cell Longev ; 2021: 4034509, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33680283

RESUMO

Background: Parkinson's disease (PD) is a common neurodegenerative disease associated with accumulation of misfolding proteins and increased neuroinflammation, which may further impair the glymphatic system. The purpose of this study was to utilize diffusion tensor image analysis along the perivascular space (DTI-ALPS) to evaluate glymphatic system activity and its relationship with systemic oxidative stress status in PD patients. Methods: Magnetic resonance imaging and neuropsychological tests were conducted on 25 PD patients with normal cognition (PDN), 25 PD patients with mild cognitive impairment (PD-MCI), 38 PD patients with dementia (PDD), and 47 normal controls (NC). Oxidative stress status was assessed by plasma DNA level. Differences in ALPS-index among the subgroups were assessed and further correlated with cognitive functions and plasma DNA levels. Results: The PD-MCI and PDD groups showed significantly lower ALPS-index compared to normal controls. The ALPS-index was inversely correlated with plasma nuclear DNA, mitochondrial DNA levels, and cognitive scores. Conclusions: Lower diffusivity along the perivascular space, represented by lower ALPS-index, indicates impairment of the glymphatic system in PD patients. The correlation between elevated plasma nuclear DNA levels and lower ALPS-index supports the notion that PD patients may exhibit increased oxidative stress associated with glymphatic system microstructural alterations.


Assuntos
Cognição/fisiologia , DNA/sangue , Imagem de Tensor de Difusão , Sistema Glinfático/diagnóstico por imagem , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/fisiopatologia , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/sangue , Doença de Parkinson/psicologia , Índice de Gravidade de Doença
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